Chemical determination of silica in seagrass leaves reveals two operational silica pools in Zostera marina

Abstract

Silicon is a major driver of global primary productivity and CO2 sequestration, and is a beneficial element for the growth and environmental stress mitigation of many terrestrial and aquatic plants. However, only a few studies have examined the occurrence of silicon in seagrasses, and its function within seagrass ecosystems and the role of seagrasses in silicon cycling remain largely unexplored. This study uses for the first time two methods, the wet-alkaline digestion and the hydrofluoric acid digestion, to quantify silicon content in seagrass leaves using the species Zostera marina and elaborates on the potential role of silicon in seagrass biogeochemistry and ecology, as well as the role of seagrass ecosystems as a silicon reservoir. The results revealed that seagrass leaves contained 0.26% silicon:dry-weight, which is accumulated in two forms of silica: a labile form digested with the alkaline method and a resistant form digested only with acid digestion. These findings support chemical digestions for silicon quantification in seagrass leaves and provide new insights into the impact of seagrasses on the marine silicon cycle. Labile silica will be recycled upon leaf degradation, benefiting siliceous organisms, while refractory silica will contribute to the ecosystem’s buried silica stock and coupled carbon sequestration. In the Bay of Brest (France), the seagrass silicon reservoir was estimated at 0.18 ± 0.07 g Si m⁻², similar to that of benthic diatoms, underscoring the potential role of seagrasses in silicon biogeochemistry in the land–ocean continuum, where they might act as a buffer for silicon transport to the ocean.

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Biogeochemistry (2025) 168:3
https://doi.org/10.1007/s10533-024-01189-1
Chemical determination ofsilica inseagrass leaves reveals
two operational silica pools inZostera marina
JustineRoth · MorganeGallinari · JonasSchoelynck ·
GemaHernán · JuliaMáñez‑Crespo · AuroraM.Ricart ·
MaríaLópez‑Acosta
Received: 2 August 2024 / Accepted: 7 December 2024 / Published online: 19 December 2024
© The Author(s) 2024
Abstract Silicon is a major driver of global primary
productivity and CO2 sequestration, and is a benefi-
cial element for the growth and environmental stress
mitigation of many terrestrial and aquatic plants.
However, only a few studies have examined the
occurrence of silicon in seagrasses, and its function
within seagrass ecosystems and the role of seagrasses
in silicon cycling remain largely unexplored. This
study uses for the first time two methods, the wet-
alkaline digestion and the hydrofluoric acid digestion,
to quantify silicon content in seagrass leaves using
the species Zostera marina and elaborates on the
potential role of silicon in seagrass biogeochemistry
and ecology, as well as the role of seagrass ecosys-
tems as a silicon reservoir. The results revealed that
seagrass leaves contained 0.26% silicon:dry-weight,
which is accumulated in two forms of silica: a labile
form digested with the alkaline method and a resist-
ant form digested only with acid digestion. These
findings support chemical digestions for silicon quan-
tification in seagrass leaves and provide new insights
into the impact of seagrasses on the marine silicon
cycle. Labile silica will be recycled upon leaf degra-
dation, benefiting siliceous organisms, while refrac-
tory silica will contribute to the ecosystem’s buried
silica stock and coupled carbon sequestration. In the
Bay of Brest (France), the seagrass silicon reservoir
was estimated at 0.18 ± 0.07g Si m⁻2, similar to that
of benthic diatoms, underscoring the potential role
Responsible Editor: Christian Lønborg
Supplementary Information The online version
contains supplementary material available at https:// doi.
org/ 10. 1007/ s10533- 024- 01189-1.
J.Roth· M.Gallinari· M.López-Acosta(*)
Laboratoire Des Sciences de l’Environnement Marin,
UMR 6539, Institut Universitaire Européen de la Mer,
Technopôle Brest-Iroise, 29286Plouzané, France
e-mail: lopezacosta@iim.csic.es
J.Schoelynck
ECOSPHERE Research Group, University ofAntwerpen,
Universiteitsplein 1, 2610Wilrijk, Belgium
G.Hernán
Department ofMarine Ecology, Mediterranean Institute
forAdvanced Studies (IMEDEA, UIB-CSIC), Esporles,
Spain
J.Máñez-Crespo
Global Change Research Group, Mediterranean Institute
forAdvanced Studies (IMEDEA, UIB-CSIC), Esporles,
Spain
A.M.Ricart
Institut de Ciències del Mar (ICM), CSIC, Passeig Marítim
de La Barceloneta, 37-49, 08003Barcelona, Spain
A.M.Ricart
Bigelow Laboratory forOcean Sciences, 60 Bigelow Dr.,
EastBoothbay, ME04544, USA
M.López-Acosta
Instituto de Investigaciones Marinas (IIM), CSIC, C/
Eduardo Cabello 6, 36208Vigo, Spain
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of seagrasses in silicon biogeochemistry in the land–
ocean continuum, where they might act as a buffer for
silicon transport to the ocean.
Keywords Biogenic silica· Silicon
biogeochemistry· Land–ocean continuum·
Seagrasses· Zostera marina
Introduction
Silicon, the second most abundant element in the
Earth’s crust after oxygen, exists in various forms in
terrestrial and aquatic ecosystems. It is present in a
dissolved state as silicic acid (dSi) and in a particulate
pool that includes lithogenic silica, which originates
from terrigenous rocks, and biogenic silica (bSi), the
hydrated amorphous form of silica (SiO2·nH2O) pro-
duced by organisms such as diatoms, plants, sponges,
and Rhizaria (Conley 2002; DeMaster 2003; Tréguer
et al. 2021). Accurately quantifying the silicon res-
ervoir in terrestrial and aquatic ecosystems is crucial
to understand the biogeochemical cycling of this ele-
ment at regional and global scales, which has sig-
nificant implications for global primary productivity,
carbon cycling, the functioning of marine food webs,
and the cycling of other major nutrients, including
nitrogen and phosphorus (Struyf etal. 2009; Tréguer
etal. 2018).
In plants, silicon is not considered an essential ele-
ment, but it is beneficial for the growth and develop-
ment of many terrestrial and aquatic species (Cooke
and Leishman 2011; Schoelynck and Struyf 2016;
Manivannan etal. 2023). Its accumulation increases
plant structural strength, mitigates metal toxicity,
and enhances resistance to pathogens and herbivores
(Liang et al. 2007; Ma and Yamaji 2008; Etesami
and Jeong 2018). Most plants accumulate silicon in
the form of solid amorphous silica bodies known as
phytoliths (Kameník etal. 2013; Koné et al. 2019),
which account for 0.1% to 10.0% of their dry weight
(Epstein 1999). Beyond providing structural, physi-
ological and protective benefits, phytoliths make
plant tissues more resistant to degradation after plant
death, thereby enhancing carbon sequestration (Parr
and Sullivan 2005; Song etal. 2016). Furthermore,
deposition and burial of phytoliths after plant death
constitute the main source of bSi in sediments of ter-
restrial and many aquatic habitats, making vegetated
ecosystems important silica sinks (Conley 2002;
Struyf etal. 2005).
Vegetated coastal habitats, such as those formed
by seagrass meadows, provide crucial ecosystem ser-
vices related to climate change mitigation, habitat
provision, and nutrient cycling (Barbier etal. 2011;
Duarte et al. 2013; Holmer 2019). Seagrass mead-
ows are among the most productive ecosystems in the
world and play an important role in biogeochemical
cycling (Marbà etal. 2006; Mateo etal. 2006). These
plants occur in coastal and estuarine ecosystems
under the influence of rivers and terrestrial streams,
which are the main input of silicon to the ocean
(Tréguer etal. 2021). Several studies have reported
a positive correlation between seagrass coverage and
dSi levels in the water column, but the causes are still
enigmatic (Herman et al. 1996; Kamermans et al.
1999). Moreover, other vegetated ecosystems in the
land–ocean aquatic continuum, such as salt marshes
and mangroves, sequester significant amounts of bSi
and control bSi exchange between terrestrial and
marine environments (Carey and Fulweiler 2014;
Elizondo etal. 2021), raising the question of whether
seagrasses might also interplay in the biogeochemical
cycling of silicon.
The elemental composition of seagrass tissues,
particularly leaves, has been extensively studied for
macronutrients such as nitrogen and phosphorus
as drivers of productivity and in relation to nutrient
limitation and disturbances like eutrophication (e.g.,
Duarte 1990; Pedersen and Borum 1992; Quigley
etal. 2020). However, information on silicon content
in seagrasses remains limited, with only few studies
quantifying the amount of silicon in seagrass leaves
to date (e.g., Herman etal. 1996; Vonk etal. 2018;
Rondevaldova et al. 2023). Silicon content in the
leaves of Zostera marina was measured along a dSi
gradient within an estuarine ecosystem in The Neth-
erlands to evaluate the effect of dSi concentrations
in the water column on seagrass bed depletion over a
20-year period (Herman etal. 1996). Further studies
have quantified silicon content in two seagrass species
(Enhalus acoroides and Halophila ovalis) in the Phil-
ippines (Rondevaldova et al. 2023) and across four
seagrass families as part of a global multi-elemental
database (Vonk etal. 2018). In these studies, silicon
content, in the form of silica, was determined using
acid digestion followed by atomic absorption spec-
trophotometry or inductively coupled plasma mass
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spectrometry, techniques that are relatively uncon-
ventional compared to those commonly employed in
plant silica research (Sauer etal. 2006). The limited
data on seagrass silicon content and the use of uncon-
ventional methods to determine it prevent an assess-
ment of the function of silicon in seagrass ecophysi-
ology and biogeochemistry.
We hypothesized that seagrasses may contain
a significant amount of silicon in their tissues and
thus could impact the silicon cycle of marine eco-
systems. To do a first estimate of whether seagrasses
are a silicon reservoir in the land–ocean continuum
and coastal ecosystems, we first assessed two com-
monly used methods for measuring bSi in marine
organisms, seawater, sediments, and terrestrial plants
―the wet-alkaline digestion with sodium hydrox-
ide (NaOH) or sodium carbonate (Na2CO3) and the
acid digestion with hydrofluoric acid (HF) (DeMaster
1981; Ragueneau et al. 2005b; Kraska and Breiten-
beck 2010; Maldonado et al. 2019)― to quantify
bSi content in seagrasses. Specifically, we focused on
quantifying bSi in the leaves of the species Z. marina,
a widespread circumpolar seagrass in the Northern
Hemisphere (Moore 2006), to compare these two
conventional methods in bSi studies with those used
previously (Herman et al. 1996; Vonk et al. 2018;
Rondevaldova etal. 2023). We evaluated whether it
exists differences between the results obtained with
each of these methods, which could suggest the exist-
ence of distinct functional groups of bSi in seagrass
tissues. We then did a first estimate of the silicon res-
ervoir of Z. marina in a temperate estuarine bay (Bay
of Brest, France), one of the best-studied coastal eco-
systems in terms of structure and ecosystem function-
ing, including silicon biogeochemistry (e.g., Chau-
vaud et al. 2000; Ragueneau et al. 2005a; Laruelle
etal. 2009). The silicon reservoir of Z. marina in the
bay was discussed within the regional silicon reser-
voirs and compared to those previously published for
other cohabiting silicifying organisms (i.e., benthic
diatoms and sponges; Leynaert et al. 2011; López-
Acosta etal. 2022) and riverine sources (Ragueneau
etal. 2005a). Ultimately, our findings aim to contrib-
ute to the ecological discussion regarding the impact
of bSi accumulation by seagrasses from the leaves to
the ecosystem scale.
Methods
Study site andsample preparation
Fieldwork was conducted in November 2021 in Lan-
véoc, France (48° 17′ 34.973’’ N, 4° 27′ 32.122’’
W), a sandy area located on the south coast of the
Bay of Brest (NE Atlantic; Fig.1). Seagrass beds of
Z. marina cover a total surface area of 1.00 km2 in
the Bay of Brest (Auby etal. 2018). At the sampling
location, the seagrass Z. marina forms a meadow of
0.23 km2 at water depth between 1.5 to 5m (Fig.1).
Lanvéoc’s meadow is representative of the seagrass
beds found in the Bay of Brest, which are very narrow
in the vertical dimension (from the top to the bottom
of the foreshore) but spread along the coastline with
a percentage coverage ranging from 5 to 25% (Auby
etal. 2018).
Fifty-eight shoots of Z. marina were randomly col-
lected by hand during low tide at a high-coefficient
tide (Fig.1) and immediately transported to the Labo-
ratory of Environmental Marine Sciences (LEMAR,
Plouzané, France) in a cooler. In the laboratory, the
seagrass shoots were cleaned, and the leaves were
sorted and measured for length and width to deter-
mine their biometric features, with data available in
Supplementary Information 1. For the determina-
tion of bSi, young leaves (2nd and 3rd newly formed
leaves) were selected, carefully cleaned of epiphytes
with a razor blade, rinsed with Milli-Q water, and
dried at 60 °C for 48h. The dried leaves were then
ground together to a fine powder using a ball mill
(MM400, Retsch), and the required amount of sam-
ple for the different analytical methods was weighed
directly into digestion tubes on a precision balance
(XS105 Analytical Balance, Mettler Toledo). This
methodological approach included technical repli-
cates to enable a comprehensive assessment of the
accuracy and performance of the analytical methods,
which constituted the main objective of this study.
Digestion protocols
Hydrofluoric acid digestion A 48-h digestion with
hydrofluoric acid (HF) is commonly used for the
digestion of bSi in siliceous organisms, including
plants (Saito etal. 2005; Ragueneau etal. 2005b; Mal-
donado etal. 2010). This acid dissolves the SiO2 com-
plex, whether of lithogenic or biogenic origin. This
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chemical property makes it an effective acid for the
measurement of resistant types of bSi, whose proper-
ties are similar to those of lithogenic silica.
Three masses of powder ofZ. marina leaves (5, 10,
and 15mg) were digested following the HF digestion
method (Ragueneau etal. 2005b) to (1) evaluate the
reproducibility of our measurements, and (2) deter-
mine the bSi content within Z. marina samples from
the Bay of Brest. This also allowed to establish the
optimal powder mass necessary for the wet-alkaline
digestion method. For this digestion, 5mL of 2.9N
HF (10%) were added into 15-mL polymethylpen-
tene centrifuge tubes (TPX, Nalgene) with Z. marina
leaves. Five replicates were used for each mass of
Z. marina evaluated. Three additional tubes without
leaves were considered as experimental blanks. The
tubes were tightly covered with a cap and kept under
a fume hood at room temperature for 48h to allow
the digestion of bSi. After 48h, each tube was centri-
fuged at 2500rpm for 5min. Then, 0.5mL of sample
was pipetted into a 15-mL polypropylene centrifuge
tube (Falcon) and neutralized by adding 14.5mL of a
saturated solution of boric acid (H3BO3, 60g/L). The
neutralized samples were kept at room temperature
until subsequent analyses for silicon determination
(see below), which were carried out no later than 48h
after acid neutralization.
Wet‑alkaline digestion We also tested the wet-alka-
line digestion method, which is widely used to quan-
tify bSi of siliceous organisms (Meunier etal. 2014;
Maldonado et al. 2019) in different matrices, such
as marine sediments (DeMaster 1981; Conley 1998;
Kamatani and Oku 2000) or suspended particulate
matter in seawater (Krausse etal. 1983; Ragueneau
etal. 2005b), and has proved to completely dissolve
bSi particle types at the solid:solution ratio applied
(Saccone et al. 2007). This relies on the difference
between the rapid dissolution of bSi and the slower
release of silica from the coexisting clay minerals (i.e.,
lithogenic silica). The digestion time depends on the
reagent used (NaOH, Na2CO3) and the digested mate-
rials, which may consist of bSi that is more or less
resistant to the alkaline reagent (Kamatani and Oku
2000; Zhu et al. 2023). Wet-alkaline digestion has
never been used on seagrass tissues, so a kinetic assay
was conducted in a strong alkaline solution (i.e., 0.5M
NaOH) to determine the time necessary to completely
digest bSi, i.e., when the bSi concentration reaches a
plateau. Ten sampling times over 72h were used to
monitor the concentration of digested bSi in the sam-
ples.
For this digestion, 40 mL of 0.5 M NaOH was
added to 50-mL fluorinated ethylene propylene cen-
trifuge tubes (FEP, Nalgene), 5 tubes containing each
80mg of Z. marina powder and 3 tubes without pow-
der, which were considered as experimental blanks.
The samples were immediately placed in a shaking
water bath (Julabo SW22) preheated to 85°C with an
oscillation frequency of 52rpm. At each time inter-
val (1h, 2h, 3h, 4h, 5h, 6h, 8h, 24h, 48h, 72h),
an aliquot of 1 mL was taken from the FEP tubes
after centrifugation at 1,500rpm for 5min and neu-
tralized using 0.625mL of 1.0 M hydrochloric acid
solution into a 15-mL polypropylene centrifuge tube
(Falcon). The neutralized samples were then diluted
with 8.375 mL of Milli-Q water and stored in a
refrigerator (4°C) until subsequent analysis for sili-
con determination. The maximum bSi digested cor-
responded to the quantity of bSi retrieved when the
plateau was reached, indicating that all the bSi in the
sample was digested and released into the milieu. At
the end of the alkaline digestion, the digested mate-
rial was rinsed 3 times with Milli-Q water after cen-
trifugation at 1,500rpm for 5min before being dried
(60°C, > 48h) for subsequent HF digestion, follow-
ing the protocol described above. Subsequent HF
digestion was carried out to ensure that the wet-alka-
line solution recovered all bSi present in the samples.
Silicon determination
Dissolved silica in digested samples, either in 2.9N
HF or in 0.5 M NaOH, was analyzed according to
an improved analytical method of the molybdate
blue method (Aminot and Kerouel 2007) on an AA3
HR Autoanalyzer (SEAL Analytical) that allows the
automatic measurement of nutrient concentrations in
solution. Silicon calibration standards were prepared
using a Silicon Standard Solution of ammonium
fluorosilicate from Merck (traceable to SRM from
NIST acidic, (NH₄)₂SiF₆ in H₂O 1000 mg Si L−1
Certipur®) in the same matrices as the samples. For
samples digested with HF, the matrix consisted of 0.5
parts 2.9N HF and 14.5 parts 60 g L−1 H3BO3. For
samples digested using wet-alkaline digestion, the
matrix consisted of 1 part 0.5M NaOH, 0.6 parts 1M
HCl and 8.4 parts Milli-Q Water.
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The bSi content in the leaves of Z. marina was
expressed as dry weight content (Si%) using Eq.(1):
where 28.0855 g mol−1 is the silicon atomic mass.
Metadata and tracked calculations of bSi content in
leaves of Z. marina are available in Supplementary
Information 2.
Statistical analysis
The differences between the average Si% of Z. marina
obtained after acid digestion in 2.9N HF at each mass
(1)
Si
%=
(
[Si](𝜇M)∗10−6
)
∗Volume digestor(L)∗Dilution factor ∗28.0855 (g mol
−1
)∗
100
Mass sample
(
g
)
assessed (i.e., 5mg, 10mg, and 15 mg) were evalu-
ated with the one-way analysis of variation (ANOVA)
test using the R Stats package (R Core Team 2021).
The bSi extracted with the wet-alkaline digestion at
each sampling time were represented with Sigma-
plot 15.0 (Systat Software Inc.) and linear and non-
linear models were tested to determine the best fitting
kinetic model using the Kinetic Module of the Sig-
maplot software. One-way ANOVA and Tukey’s hon-
estly significant difference (Tukey’s HSD) tests were
then performed between each time point to compare
pairwise time points and analyze the curve tendency
Fig. 1 Distribution of Zostera marina beds in the Bay of Brest
(France). GPS data were collected on https:// cms. geobr etagne.
fr (2018) and computed in ArcMap (ArcGIS Desktop ver-
sion 10.8). The picture on the right shows a general view of
Lanvéoc’s meadow composed of Z. marina seagrass during a
high-coefficient low tide, where samples were collected for this
study
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using the Regression Wizard of Sigmaplot. Finally,
the differences between the average Si% in Z. marina
obtained at the end of each digestion (i.e., wet-alka-
line digestion, HF digestion, and the sum of wet-alka-
line digestion plus the subsequent HF digestion) were
also evaluated with a one-way ANOVA test and an
a posteriori pairwise Tukey’s HSD test. Before per-
forming any parametric test, data were assessed for
normality using the Shapiro–Wilk test and for homo-
scedasticity with Levene’s test, using the R Stats
package (R Core Team 2021).
Seagrass silicon stock at the regional level
At the Bay of Brest, Z. marina occupies a total sur-
face of 1.00 km2, of which 95% is located in three
seagrass meadows: Kernizi, Roscanvel and Lanvéoc
(Fig.1). To estimate the silicon stock of the meadows
of Z. marina in the Bay of Brest, we used data of bio-
mass of Z. marina leaves per m2 from the three main
meadows of the bay. Data for the Kernizi and Roscan-
vel meadows were obtained from existing literature
(Auby etal. 2018; Boyé etal. 2022) and the REBENT
monitoring programme (http:// www. rebent. org/), and
that for the Lanvéoc meadow was determined in this
study using the same methodology as for the other
meadows (Auby et al. 2018). Shoots were collected
using 0.1m2 quadrats randomly positioned within the
seagrass meadows (n = 5). Shoots within each quadrat
were sampled to determine the biomass of epiphyte-
cleaned leaves after 48h of desiccation at 60°C. This
data, along with the silicon content (Si%) measured in
this study, were used to estimate the silicon reservoir
in leaves of Z. marina at the different meadows and at
the bay as a whole. Note that our approach to estimat-
ing the silicon stock in Z. marina for the bay does not
account for potential intra-annual variability.
Results
Assessment of silica extraction methods on seagrass
leaves
Prior to evaluating the performance of hydrofluo-
ric acid digestion and wet-alkaline digestion meth-
ods in the extraction of bSi from seagrass samples,
preliminary tests were conducted to determine the
applicability of these methods to seagrass leaves and
whether any adjustments were necessary.
The evaluation of the reproducibility of bSi
extraction using the acid digestion method with
15 mL of 2.9N HF showed no significant differ-
ences between the three masses examined (F = 1.6,
df = 2, p-value = 0.24; Fig.2). This finding indicates
that small dry weight masses of seagrass, as low as
5 mg, are suitable for determining bSi content in
leaves of Z. marina.
The time required for complete digestion of bSi
using wet-alkaline digestion varies depending on
the origin and structural complexity of the bSi. To
determine the necessary digestion time for seagrass
bSi in 0.5M NaOH, a kinetic assay was conducted
to identify when the dSi concentration reached a
plateau, i.e., the digestion was completed. Experi-
mental data showed that dSi concentration within
the digestion tubes (n = 5) increased linearly during
the first 8h, then reaching a plateau at 24h, with a
mean dSi concentration of 11.81 ± 0.06µmol Si L−1,
beyond which no further increased was observed
(Fig.3). This was corroborated by the kinetic model
describing the digestion process, which followed a
saturable sigmoidal sigmoid function of 3-param-
eter (R2 = 0.989, p-value < 0.01; Fig.3). This model
also confirmed that the plateau was reached at 24h,
with a maximum modelled dSi concentration of
11.63 ± 0.19 µmol Si L−1. These results indicated
that digestion of Z. marina bSiin 0.5M NaOH was
complete at 24h.
Fig. 2 Average (± SD) silicon content (Si%) in Z. marina
leaves. Three different masses (5, 10, 15mg) of Z. marina with
five replicates were digested using 2.9N HF
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Hydrofluoric acid and wet-alkaline digestion
comparison
The bSi content extracted using HF digestion and
wet-alkaline digestion showed significant differences
(F = 11.6, df = 2, p-value < 0.01; Fig. 4). A pairwise
comparison further confirmed significant differences
between the bSi content obtained with these two
methods (p-value < 0.01; Fig. 4). The average bSi
content of Z. marina leaves measured with the HF
digestion method was 0.26 ± 0.01 Si% (mean ± SD;
n = 15), whereas the amount of silica extracted with
0.5M NaOH after 24h of digestion was 0.15 ± 0.001
Si% (n = 5). This amount corresponded to only
57.70 ± 0.38% of the bSi extracted using 2.9N HF.
Samples digested in 0.5M NaOH for bSi extrac-
tion were afterwards digested in 2.9N HF using the
same method as before. This additional HF diges-
tion continued to extract bSi from Z. marina leaf
samples (Fig.4). The bSi content retrieved with the
HF digestion conducted after wet-alkaline diges-
tion was 0.10 ± 0.01 Si% (n = 5). The total bSi con-
tent retrieved with the wet-alkaline digestion and
the subsequent HF digestion was 0.26 ± 0.01 Si%
(n = 5), showing no significant differences when
compared to the bSi content obtained with only HF
digestion (p-value = 0.95). These results suggest
that Z. marina contain two operational silica pools:
a labile one, digested by the alkaline method, and a
more resistant one, digested only by HF.
Fig. 3 Extraction of dis-
solved silica (dSi; in µmol
Si L-1) through time (in
hours). Error bars represent
standard deviation calcu-
lated from five replicates
digested using 0.5M
NaOH. The statistics of
model’s goodness of fit are
available in Supplementary
Information 3
Fig. 4 Average (± SD)
silicon content (Si%) of
Zostera marina extracted
using 2.9N HF, 0.5M NaOH,
and the sum of wet-alkaline
digestion using 0.5M NaOH
and a subsequent 2.9N HF
digestion. Significant differ-
ences (p < 0.05) are indicated
with letters according to the
results of a one-way ANOVA
analysis and the a posteriori
pairwise Tukey’s HSD test
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Seagrass silicon reservoir at the regional level
Based on the bSi content measured in the leaves of
Z. marina in this study (0.26 Si%), we estimated the
silicon pool in the Z. marina meadows in the Bay
of Brest (Table1). The average (± SD) silicon stock
within leaves of Z. marina ranged from 0.12 ± 0.04
to 0.18 ± 0.08 g Si m−2, depending on the meadow
(Table1). The Roscanvel meadow, which constitutes
65% of the total distribution area of Z. marina in the
Bay of Brest and exhibited the highest leaf biomass,
accounted for the largest silicon reservoir in the bay
(118.9 ± 50.3 × 103g Si). At the bay level, the silicon
reservoir in the leaves of Z. marina was estimated at
167.4 ± 70.0 × 103g Si (Table1).
Discussion
After examining the performance of digestion with
acid and alkaline reagents, chemical digestion with
2.9N HF emerged as the optimal method for deter-
mining the total amount of bSi in Z. marina leaves.
Our results showed that the commonly used wet-alka-
line digestion failed to recover all the bSi content in
seagrass leaves, unlike HF digestion. Notably, after
24h, bSi was no longer released from the leaves of
Z. marina during the wet-alkaline digestion, sug-
gesting the presence of a fraction of bSi within sea-
grass leaves that is resistant to extraction by 0.5 M
NaOH and is only extracted with aggressive HF
digestion. This is supported by the results of the HF
digestion conducted after the wet-alkaline diges-
tion, which allowed the retrieval of 100% of the bSi
content. These outcomes suggested the existence of
two operational pools of bSi in Z. marina leaves: a
more labile and digestible bSi that is released by wet-
alkaline digestion and a more resistant bSi that is only
released with 2.9N HF.
While the HF digestion method has demonstrated
efficacy in determining the total bSi content in Z.
marina leaves, a sequential digestion involving 24h
in 0.5M NaOH followed by 48h in 2.9N HF emerge
as a powerful combination of methods to measure
the labile and refractory fractions of bSi in seagrass
leaves, which is neither possible with the HF diges-
tion method nor with the methods used in previous
studies (Herman etal. 1996; Vonk etal. 2018; Ron-
devaldova et al. 2023). The quantification of these
two bSi fractions is crucial for evaluating the impact
of seagrass meadows on the marine silicon cycle, as
these distinct forms of silica are likely to undergo dif-
ferent incorporations into the biogeochemical cycle
of silicon. Presumably, the labile bSi within seagrass
leaves will be recycled and become available for uti-
lization by other silicified organisms (e.g., diatoms,
Rhizaria, sponges) upon seagrass leaf degradation,
whereas the refractory seagrass bSi will merely con-
tribute to the standing stock of silica buried in sedi-
ments underneath seagrass meadows. This process
would be similar to that of other amorphous silica
sources, which have varying degrees of solubility and
reactivity to dissolution (Saccone et al. 2007). Fur-
ther studies are needed to evaluate the role of seagrass
ecosystems as either sources or sinks of silicon in the
biogeochemical cycle of this element.
The mechanisms of silica deposition and the
exact location of silicon accumulation in Z. marina
Table 1 Silicon reservoir in leaves of Zostera marina seagrass
in the Bay of Brest (France). Area (106 m2) of each meadow
and the total distribution of Z. marina for the Bay of Brest was
estimated from the latest distribution mapping of Z. marina in
this habitat (OFB-TBM environment 2021). Average (± SD)
leaf biomass (g Z. marina m−2) of the Kernizi and the Ros-
canvel meadows were calculated elsewhere (Auby etal. 2018;
Boyé etal. 2022) and that of Lanvéoc was calculated in this
study, together with the silicon content of Z. marina leaves
(0.26 Si%). See Supplementary Information 2 for raw data and
detailed calculations
Seagrass meadow Meadow surface (106
m2)
Leaf biomass (g Z. marina
m−2)
Silicon reservoir (g Si
m−2)
Total silicon
reservoir (103g
Si)
Kernizi 0.07 46.99 (± 14.37) 0.12 (± 0.04) 8.6 (± 2.6)
Roscanvel 0.65 70.37 (± 29.75) 0.18 (± 0.08) 118.9 (± 50.3)
Lanvéoc 0.23 66.76 (± 28.66) 0.17 (± 0.07) 39.9 (± 17.1)
Bay of Brest 1.00 65.19 (± 27.28) 0.18 (± 0.07) 167.4 (± 70.0)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Biogeochemistry (2025) 168:3 Page 9 of 13 3
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remain unknown, but several lines of evidence pro-
vide insights. Recent studies have documented the
presence of phytoliths in the roots, stems, and leaves
of Z. marina (Rong etal. 2024). If these phytoliths
are analogous to those found in terrestrial and fresh-
water plants, they would completely dissolve dur-
ing wet alkaline digestion (Saccone et al. 2007;
Meunier et al. 2014), consistent with the fraction
digested in our study with 0.5M NaOH. Further-
more, Z. marina encodes Slp1, a protein that medi-
ates vesicular transport of silica into the apoplastic
space (Nawaz et al. 2020). The presence of Slp1
suggests that Z. marina may use a silica deposition
mechanism similar to that observed in the monocot
angiosperm Sorghum bicolor, in which Slp1 facili-
tates silica incorporation through vesicular trans-
port rather than through the Lsi1/2 pathway, which
is absent in Z. marina (Kumar et al. 2020). This
alternative pathway likely results in the deposition
of silica in amorphous forms within the cell wall or
extracellular matrix. Such silica deposits, integrated
within the organic matrix and reported as small
phytoliths in other plant species, are often resistant
to extraction with alkaline digestion (Prychid etal.
2003; Kameník et al. 2013). The tight binding of
silica to organic components may explain why some
of the bSi in Z. marina resists alkaline digestion
and requires digestion with 2.9N HF for complete
extraction.
Seagrass distribution is globally prevalent in
coastal environments, extending from intertidal to
subtidal depths in both estuarine and marine ecosys-
tems (Green and Short 2003). In the Bay of Brest, a
semi-enclosed coastal ecosystem in the NE Atlantic
Ocean with tidal ranges of 2 to 8m, Z. marina mainly
occurs in discontinuous patches in the intertidal zone
(Auby etal. 2018). This study provides the first esti-
mate of the silicon content of Z. marina meadows in
the bay, offering a basis for comparison with other
silicifiers in the ecosystem. However, this initial esti-
mate does not account for potential variability, such
as, for example, changes in silicon content as a func-
tion of leaf age. If silica accumulation in seagrass fol-
lows patterns observed in terrestrial and freshwater
plants, where silicon accumulation tends to increase
with leaf age (e.g., Motomura et al. 2008; Querné
et al. 2012), it is likely that our estimate represents
a conservative or underestimated value of the silicon
content in these meadows.
When comparing the average silicon reservoir of
seagrasses in the bay (0.18 ± 0.07g Si m⁻2) with that
of benthic diatoms at subtidal depths (0.11 ± 0.09 g
Si m−2; Grossteffan etal. 2024), both are quite simi-
lar. However, intertidal sponges have a significantly
higher average silicon reservoir (4.3 ± 4.2g Si m−2;
López-Acosta et al. 2022). This large difference is
due to the much higher silicon content in sponges
(22.34—26.88 Si%, depending on the species present
in the intertidal zone of the bay; López-Acosta etal.
2022) compared to Z. marina (0.26 Si%). Despite
this, sponges and Z. marina generally occupy dif-
ferent areas. Sponges primarily grow on rocky sub-
strates, while Z. marina grow on sandy and muddy
bottoms (Boyé etal. 2022; López-Acosta etal. 2022).
Therefore, each likely contributes to silicon cycling
in separate zones within the land–ocean continuum
of the bay. The potential impact of seagrass distribu-
tion on silicon input to the ocean via rivers and ter-
restrial streams remains to be studied. In the bay,
rivers and terrestrial streams are the main source of
dSi, supporting about half of the diatom production in
this diatom-rich ecosystem (Ragueneau etal. 2005a),
and, globally, account for 52% of dSi entering the
ocean (Tréguer etal. 2021). As some of the seagrass
bSi showed to be reluctant to dissolution, seagrasses
might act as a buffer in the transport of dSi to the
ocean realm.
To date, only few studies have quantified the bSi
content in seagrass tissues. Herman et al. (1996)
determined bSi in Z. marina leaves from the Rhine-
Meuse Estuary (SW Netherlands), finding values
ranging from 0.02 to 0.66 Si% depending on loca-
tion within the estuary, and hypothesizing dSi uptake
and leaf bSi content correlate with ambient dSi con-
centrations. Vonk etal. (2018) measured bSi in sea-
grass species across four families (Posidoniaceae,
Hydrocharitaceae, Cymodoceaceae and Zosteraceae),
reporting no significant differences between families
and an average bSi content of 0.08 Si%. Within the
family Zosteraceae, Z. marina exhibited the highest
bSi content (0.27 Si%). These values are in agree-
ment with those measured in this study in leaves
of Z. marina of the Bay of Brest (0.26 Si%), show-
ing that alkaline and acid digestions are suitable for
measuring bSi in seagrasses. The most comprehen-
sive study evaluating bSi content of freshwater veg-
etation, based on 83 different species analyzed with
wet-alkaline digestion, showed a bSi content of 0.45
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Biogeochemistry (2025) 168:3
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Si% (Schoelynck and Struyf 2016), which is about 5
times higher than the mean content described for sea-
grasses (0.08 Si%; Vonk et al. 2018) and 1.5 times
higher than the mean content measured in Z. marina
(0.26 Si%; Herman etal. 1996; Vonk etal. 2018, this
study).
Silica accumulation is widely recognized as a
functional trait in freshwater vegetation, with plants
showing adaptations to the prevailing environmental
conditions and dominant mechanical forces in their
ecosystems (Schoelynck and Struyf 2016). The func-
tional roles of dSi uptake in plants include responses
to both biotic (e.g., herbivory) and abiotic (e.g., heavy
metals) stresses (Bhat et al. 2019; Acevedo et al.
2021). Additionally, substantial evidence supports
a connection between silicon and carbon uptake in
freshwater vegetation. In these plants, dSi is assimi-
lated and deposited in the form of solid amorphous
silica bodies, known as phytoliths (Kameník et al.
2013; Koné et al. 2019). During phytolith forma-
tion, some organic carbon becomes occluded within
the phytolith structure (referred to as PhytOC), mak-
ing it highly resistant to decomposition and thereby
facilitating the long-term biogeochemical sequestra-
tion of atmospheric CO2 (Jones and Milne 1963; Parr
and Sullivan 2005; Song etal. 2016). Recent findings
suggest that this mechanism also occurs in seagrasses
(Rong etal. 2024), indicating that the quantification
of bSi in seagrass ecosystems could serve as a proxy
for carbon sequestration via PhytOC accumulation
within seagrass tissues.
Seagrass habitats are being lost at an estimated
rate of 7% per year worldwide (Dunic etal. 2021),
which has led to conservation and restoration initia-
tives aimed at mitigating this trend (De Los Santos
et al. 2019). In the implementation of these initia-
tives, the concentration of dSi in seawater is often not
measured but can be critical, as low dSi concentra-
tion (< 15µM Si) may be a factor in the decline of
estuarine seagrass populations (Herman etal. 1996).
In the current context of a changing ocean, the global
decrease in dSi loads caused by eutrophic conditions
and human use of terrestrial waters may lead to dSi
depletion in many coastal ecosystems (Ittekkot etal.
2006; Zhang etal. 2020; Taucher etal. 2022). There-
fore, understanding the silicon cycle in relation to
seagrasses will be essential for addressing the effects
of climate change in coastal areas and for the conser-
vation of seagrass ecosystems.
Conclusion
The findings reported here indicate that the wet-
alkaline digestion and hydrofluoric acid digestion
methods are valid for seagrass bSi quantification.
Additionally, our study highlights the presence of two
operational pools of bSi in seagrass leaves: a labile
form released by wet-alkaline digestion, and a resist-
ant form that can only be extracted with hydrofluoric
acid digestion. These results provide insights into the
impact of seagrasses on the biogeochemical cycle of
silicon in the land–ocean continuum, as labile silica
will be recycled upon leaf degradation, benefiting
siliceous organisms living in the coastal environment,
while refractory silica will contribute to the buried
silica standing stock in the ecosystem. Our study
provides a methodological basis for further research
on the ecological significance of silicon in seagrass
physiological fitness, functional ecology, and biogeo-
chemical dynamics in seagrass ecosystems.
Acknowledgements The authors thank Jacques Grall for
his assistance during fieldwork, the staff of the PACHIDERM
analytical platform for providing analytical tools, and the
staff maintaining the SOMLIT-Lanvéoc database for making
public their information on the stock of benthic diatoms. The
authors also thank the staff of the Marine Observatory of the
IUEM for their dedicated efforts in monitoring seagrass bio-
mass in the Bay of Brest over the past 20 years, whose data
have been instrumental in quantifying the silicon reservoir in
Zostera marina for this study. Jean-Dominique Meunier and
Eva Scozzina are also thanked for their input during method
determination in the early stages of this study. This research
was supported by the ISblue project, Interdisciplinary graduate
school for the blue planet (ANR-17-EURE-0015), co-funded
by a grant from the French government under the program
‘Investissements d’Avenir’ (grant SiSea, Research themes 1 &
4), and by Campus France Spain (grant FanMa-Si) to ML-A.
GH was supported by a postdoctoral ‘Vicenç Mut’ contract
co-funded by the Council of European Funds, University, and
Culture of the Government of the Balearic Islands, ARM was
supported by project PCI2021-122040-2B funded by MCIN/
AEI/10.13039/501100011033 and the European Union – Next-
Generation EU/ Recovery, Transformation and Resilience Plan,
and ML-A was supported by two postdoctoral fellowships
(IN606B-2019/002 and IN606C-2023/001) funded by ‘Xunta
de Galicia’.
Author contributions Justine Roth: Investigation, Methodol-
ogy, Formal analysis, Data curation, Visualization, Writing—
original draft, Writing—review & editing. Morgane Gallinari:
Investigation, Methodology, Visualization, Resources, Writ-
ing—review & editing. Jonas Schoelynck: Visualization, Writ-
ing—review & editing. Gema Hernán: Visualization, Writ-
ing—review & editing. Julia Mañez-Crespo: Visualization,
Writing—review & editing. Aurora M. Ricart: Visualization,
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Biogeochemistry (2025) 168:3 Page 11 of 13 3
Vol.: (0123456789)
Writing—review & editing. María López-Acosta: Conceptual-
ization, Investigation, Methodology, Data curation, Visualiza-
tion, Funding acquisition, Project administration, Resources,
Supervision, Validation, Writing—original draft, Writing—
review & editing.
Funding Open Access funding provided thanks to the
CRUE-CSIC agreement with Springer Nature. ISblue,SiSea
(ANR-17-EURE-0015,Maria Lopez Acosta,"Investissements
d’Avenir"),Maria Lopez Acosta,Campus France,FanMa-
Si,Maria Lopez Acosta
Data availability All data of this study are available within
this article and at Supplementary Information 1–3.
Declarations
Competing interests The authors declare that they have no
known competing financial interests or personal relationships
that could have appeared to influence the work reported in this
paper.
Open Access This article is licensed under a Creative Com-
mons Attribution 4.0 International License, which permits
use, sharing, adaptation, distribution and reproduction in any
medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Crea-
tive Commons licence, and indicate if changes were made. The
images or other third party material in this article are included
in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your
intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/.
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