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Time-Domain NMR study of Mediterranean scleractinian corals reveals skeletal-porosity sensitivity to environmental changes Paola Fantazzini, Stefano Mengoli, Stefania Evangelisti, Luca Pasquini, Manuel Mariani, Leonardo Brizi, Stefano Goffredo, Erik Caroselli, Fiorella Prada, Giuseppe Falini, Oren Levy, and Zvy Dubinsky Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 21 Oct 2013 Downloaded from http://pubs.acs.org on October 29, 2013
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Time-Domain NMR study of Mediterranean scleractinian corals reveals skeletal-porosity sensitivity to environmental changes Paola Fantazzinia,b*, Stefano Mengolic, Stefania Evangelistia, Luca Pasquinia, Manuel Mariania,b,
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Leonardo Brizia,b, Stefano Goffredod, Erik Carosellid, Fiorella Pradad, Giuseppe Falinie, Oren Levyf,
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Zvy Dubinskyf
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
a
Department of Physics and Astronomy, University of Bologna, Viale Berti Pichat 6/2, 40127 Bologna, Italy, b Centro Enrico Fermi, Roma, Italy, c Management Department, University of Bologna, Bologna, Italy, d Marine Science Group, Department of Biological, Geological and Environmental Sciences, University of Bologna, Bologna, Italy, e Department of Chemistry, University of Bologna, Bologna, Italy, f The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel. *
Corresponding author: Paola Fantazzini; tel: +390512095119; fax: +390512095047;
[email protected]
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Abstract
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Mediterranean corals are a natural model for studying global warming, as the Mediterranean
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basin is expected to be one of the most affected regions and the increase of temperature is
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one of the greatest threats for coral survival. We have analyzed for the first time with Time
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Domain Nuclear Magnetic Resonance (TD-NMR) porosity and pore-space structure,
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important aspects of coral skeletons, of two scleractinian corals, Balanophyllia europaea
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(zooxanthellate) and Leptopsammia pruvoti (non-zooxanthellate), taken from three different
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sites on the western Italian coast along a temperature gradient. Comparisons have been made
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with Mercury Intrusion Porosimetry and SEM images. TD-NMR parameters are sensitive to
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changes of the pore-structure of the two coral species. A parameter, related to the porosity,
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is larger for Leptopsammia pruvoti than for Balanophyllia europaea, confirming previous
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non-NMR results. Another parameter representing the fraction of the pore-volume with
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pore-sizes less than 10-20 µm is inversely related, with high statistical significance, to the
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mass of the specimen, and, for Balanophyllia europaea, to the temperature of the growing
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site. This effect in the zooxanthellate species, that could reduce its resistance to mechanical
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stresses, may depend on an inhibition of the photosynthetic process at elevated temperatures
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and could have particular consequences in determining the effects of global warming on
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these species.
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INTRODUCTION
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Corals and global warming. Global climate change is the defining environmental issue of
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our times and is expected to profoundly affect all levels of ecological hierarchies and a broad array
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of terrestrial and marine ecosystems.1-5 Marine communities are expected to be affected more than
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terrestrial ones by the effects of climate change,6 especially in temperate areas.7 Thus, the
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Mediterranean basin8 represents a natural focus of interest for researchers and at the same time a
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natural laboratory to model and predict climate change and its ecological effects. In particular, the
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increase of temperature is one of the greatest threats for corals, which can be considered as a probe
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of global warming effects, as it triggers bleaching events and widespread mortality.9-11 Several
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recent mass mortality events of Mediterranean corals have been reported as related to high
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temperatures.12-16
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This study focuses on two scleractinian species of the Mediterranean Sea, already studied as a
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model for climate change: Balanophyllia europaea (Risso, 1826) (B. europaea) and Leptopsammia
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pruvoti Lacaze-Duthiers (1897) (L. pruvoti)17 (Fig. 1). B. europaea is a solitary, zooxanthellate (i.e.
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symbiotic with unicellular algae named zooxanthellae) coral, endemic to the Mediterranean Sea. Its
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distribution is limited to depths of 0-50 m because of its symbiosis with zooxanthellae, which
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require light.17-19 L. pruvoti is a non-zooxanthellate and solitary scleractinian coral, distributed in the
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Mediterranean basin and along the European Atlantic coast from Portugal to Southern England and
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Ireland. Its distribution is limited to semi-enclosed rocky habitats, under overhangs, in caverns, and
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small crevices at 0–70 m depth.17,18 Corals were collected in three different Italian sites (see Fig. 2),
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where the porosity of the two species has been studied previously,17along a latitude and sea surface
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temperature (SST) gradient. Temperature, the variation of which is mainly influenced by latitude,20
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is linked to coral biometry, physiology, and demography.21-23
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Coral porosity and pore-size distribution. The porosity (pore-volume to sample-volume
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ratio) and the pore-space structure of mineralized tissues are of crucial importance in determining 3 ACS Paragon Plus Environment
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overall properties and biological functions, such as the coral skeleton resistance to natural and
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anthropogenic breakage. They are important parameters for studying scleractinian coral growth and
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the effects of abiotic and anthropogenic influences on coral reefs.24 Of additional interest is a good
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knowledge of the role of porosity in diagenesis.
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The measured values of these parameters depend on the measurement methods24,25 and can
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present spatial variations.26 This could be the reason why their dependence on environmental
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conditions remains largely unstudied, notwithstanding their importance and their variation with
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factors such as exposure, temperature, latitude depth and species.
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Recent investigations17,19,27 have shown that along the Italian coast porosity of B. europaea is
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positively correlated with SST. There is concern for the future of this species17,27,28 in relation to the
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current predictions of global warming by the Intergovernmental Panel on Climate Change.7 Instead,
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for L. pruvoti, both SST and solar radiation do not seem to influence significantly the porosity and
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the space colonization potential.17,19,29 An important aspect is the possible hierarchical structure of
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the porosity. Recently30 the flaw-tolerance in nacre has been ascribed to the nanoparticle-
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architecture of the aragonite platelet, which makes a crack propagate in an intergranular manner.
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The structure of porous media at different length scales is of great importance also in the use of
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corals as potential bone graft substitute material.31
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Porosity and pore-size distributions can be investigated by many methods.32-36 The results
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strongly depend on the physical principles adopted and on the assumptions on pore shape and
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connectivity (see Supporting Information). Porosity and pore size distributions by Mercury
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Intrusion Porosimetry (MIP) on eight different coral species36 showed large differences, with
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diameters ranging from 0.2 to 100 µm. However, it has been emphasized that particle compression
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and rupture can result from the high Hg pressure used. Time-Domain Nuclear Magnetic Resonance
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(TD-NMR)37 has the advantage of being non-destructive and non-invasive.
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particular Magnetic Resonance Relaxometry of 1H nuclei of water saturating the pore-space is an
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efficient tool to investigate pore-space structure. Known from the ’50s,38,39 and validated in the 4 ACS Paragon Plus Environment
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course of time by comparison with MIP and, for specific surface, with the Brunauer, Emmett, Teller
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(BET) method, it is now widely applied.40-52 It is particularly useful for porous media with wide
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pore-sizes distributions, as corals have. In this paper the distribution of the local transverse
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relaxation time (T2) of 1H of water saturating the pore-space of the cleaned coral skeletons,
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corresponding to distributions of “NMR pore-sizes”, are used (more details on NMR and surface
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effects in Supporting Information). To the best of our knowledge, this is the first time that this
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technique is applied for this kind of investigation of corals.
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MATERIALS AND METHODS
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The corals. Specimens of B. europaea and L. pruvoti (54 specimens in total) were randomly
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collected from three sites: Calafuria (CL), Palinuro (PL) and Pantelleria Isle (PN) (see Fig. 2). Coral
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tissue was totally removed and corals cleaned as described in Ref. 17. The skeletons were weighed
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to determine the mass (m). The total volume (VT) was determined19 including the volume of the oral
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cavity. Then the specimens were saturated with water for NMR measurements. Further details are
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reported in Supporting Information.
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Total NMR Signal, microporosity and cut-off definitions. The total NMR signal (SNMR),
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represented by the area below each T2 distribution, is proportional to the volume of water saturating
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the pore-space volume (VP). This signal divided by the total sample volume gives a value
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proportional to the total porosity of the specimen (see Supporting Information). The fraction of
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water with relaxation times over a given interval of the distribution corresponds to the pore volume
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fraction over a corresponding pore-size range. “NMR microporosity", “microporosity" for short,
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will indicate the fraction of VP where the smaller pores are weakly coupled by water diffusion to the
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large ones at the local relaxation time scale. This can be accomplished if the slope of the
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distribution shows a strong increase at a certain T2 value, to be chosen as the point of separation
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between “smaller” and “larger” pores. This relaxation time will be called the “cut-off”. The 5 ACS Paragon Plus Environment
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“microporosity” is then defined as the fraction of 1H signal with T2 smaller than the cut-off, divided
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by the total 1H signal. Operatively, it is the ratio of the area under the distribution for T2 smaller
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than the cut-off, to the total area under the distribution.
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Statistical Analysis. Statistical analysis was performed using Statistical Package STATA 9.0
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(StataCorp LP). To test the significance of the differences among species and growth site
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parametric and non-parametric tests were performed. Multivariate analyses were made using both
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Ordinary Least Squares (OLS) robust to outliers and non-parametric bootstrapping regression
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procedure following Efron,53 applied to check the robustness of the results, that could be affected by
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small sample bias. The models are described by the function:
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yi = a + b1 ⋅ mi + b2 ⋅ SSTi + ε i (1),
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the index i refers to the n-observations, yi is the value of the dependent variable and εi the
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corresponding error. The parameters a and bj (j=1,2) are the best fit parameters, to be determined by
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OLS referring to the independent variables m and SST. More details in Supporting Information.
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RESULTS AND DISCUSSION
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Figure 3 shows the T2 distributions of all B. europaea and L. pruvoti specimens. All the
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distributions show a main peak at long relaxation times and a long tail, of smaller amplitude, about
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three order of magnitudes wide. The major shape difference between the two species is the length of
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the tail. For B. europaea (a, c, e) the tails go down to T2 = (0.1 - 0.2) ms, values shorter than for L.
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pruvoti (b, d, f). The principal differences among each group are given by the total areas and are due
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to the wide range of masses and volumes of the specimens. In principle, 1H signal can be produced
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from other sources than water, namely the intraskeletal organic matrix, consisting of proteins,
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polysaccharides and lipids. In order to check this possibility, T2 distributions for a dry coral and the
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same after complete water saturation were obtained and are shown in Fig.4. The discussion reported
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for the fully water saturated sample there is no contribution to the signal from macromolecular
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nuclei and only a maximum on the order of 2% could be attributed to lipids.
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In order to discuss the distributions in Fig. 3 in terms of pore-sizes, one should get an
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approximate value for the radius of the pores inside the coral. The inset in Fig. 4 reports the MIP
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distribution for the same specimen. The major fraction of the pore-volume is given by pores whose
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entrance radii are on the order of tens of µm, while a minor fraction corresponds to a three order of
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magnitude long tail of very small pores, down to tens of nm. NMR and MIP are exceptionally
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similar and consistent. The two classes of pores are easily distinguished also in the distributions in
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Fig. 3, so that the two parameters “microporosity” and “cut-off” were determined for each
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distribution. The sharp boundary between the two classes, with a cut-off in the T2 range 200-400
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ms, suggests that the two classes of pores are not well connected by water diffusion during a local
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relaxation time. Also, the long tail indicates that these pores are poorly connected both to the other
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small pores and to the large ones in the major class. On the basis of the comparison with MIP,
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microporosity should correspond to pore sizes in the range from ∼10 nm to ∼10-20 µm. The
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existence of a wide class of pores with sizes less than 10-20 µm is well described in the SEM
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images of both species reported in Fig. 5.
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Table 1 shows means, standard errors and statistical significances of the differences between
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the two species by both parametric and non-parametric tests for all the variables considered:
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microporosity, cut-off, mass, total volume, SNMR, and SNMR/VT. The two species behave differently
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with high statistical significance (p< 0.01 for all variables, including NMR parameters). In
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particular mass and total volume for B. europaea are much larger than for L. pruvoti, and,
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viceversa, total porosity estimated by the ratio SNMR/VT is larger for L. pruvoti than for B. europaea.
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This result is consistent with the higher porosity obtained for L. pruvoti by previous non-NMR
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analysis,17 where the difference was considered as a likely consequence of the different habitat of
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the two species.
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Table 2 shows the differences of the means of the same species among growing sites. For B.
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europaea the differences have high statistical significance for almost all variables. Microporosity
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has the highest significance (microporosity, p < 0.01; cut-off, p < 0.05; mass and total volume, p <
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0.1; SNMR, p < 0.05 ). For L. pruvoti, only NMR parameters cut-off and microporosity show
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significant differences among sites (p< 0.05-0.1).
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Table S1 of Supporting Information shows the results of the correlation among variables
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performed separately for B. europaea (Panel A) and L. pruvoti (Panel B). For both species, the pairs
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of variables mass and total volume, mass and SNMR, total volume and SNMR are significantly
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correlated (p < 0.01). The correlations between microporosity and cut-off, and microporosity and
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mass are statistically significant for both species (p < 0.01 for B. europaea, p < 0.05 for L. pruvoti).
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Figure 6 reveals a counter-intuitive behavior: the longer the cut-offs, the smaller the
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microporosities, while, at the first glance, one would expect the contrary. The pore-space
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architecture differs between samples with higher or lower cut-offs (between smaller microporosity
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or higher microporosity). The scatterplots in Figure S2 of Supporting Information and in Fig. 7,
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showing cut-off versus mass and microporosity versus mass, respectively, suggest that the observed
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correlation between cut-off and mass are governed by the mass: the smaller the mass, the higher the
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microporosity and the shorter the cut-off. As the corals increase their mass, both the cut-off (which
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separates the two main pore classes) and the ratio between the fraction of the two pore classes
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change, with larger pores becoming more abundant. This effect is shown also by L. pruvoti, but it is
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not as marked as in B. europaea due to the smaller range of masses of the corals. This is consistent
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with the gradual “filling up” of the smaller pores with the growth of the coral. A secondary infilling
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of skeletal pores in the older portion of the skeleton is a consistent characteristic of the skeletal
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density of branches of tall branching corals, in which growing tips are very porous, while basal
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regions are extremely dense.62
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In order to study how microporosity, considered as a dependent variable, is affected by mass
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and SST, multiple regression analysis was performed (Eq. 1) both for all the specimens and for the 8 ACS Paragon Plus Environment
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two species separately. Panel A of Table 3 summarizes the results. First of all, it is important to
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observe that from a statistical point of view a potential correlation among the variables m and SST
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does not invalidate the results, as all the values of the variance inflation factor (VIF) are 0.1). Overall, it emerges that the mass significantly affects microporosity at least at the 5
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percent level (p< 0.05 for L. pruvoti and p< 0.01 for B. europaea). Results for microporosity vs
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SST show that a significant relationship exists for B. europaea (p< 0.05) but not for L. pruvoti (p>
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0.1).
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In a previous study17 it has been shown that porosity depends on temperature for B. europaea,
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but not for L. pruvoti. It has been hypothesized that the increase in porosity with temperature in the
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zooxanthellate species could depend on an inhibition of the photosynthetic process at elevated
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temperatures,23,63 causing an attenuation of calcification64 with possible negative consequences also
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on space colonization and population density.19,27 The NMR results point in the same direction and
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seem to indicate also that this effect could be accompanied by a reduction of microporosity,
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meaning an increase of the fraction of the largest pores in the pore-space. 9 ACS Paragon Plus Environment
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TD-NMR is a quick, non-invasive, non-destructive method that does not use ionizing radiation,
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that can be applied to gain insight regarding the pore-space architecture of scleractinian corals,
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showing differences between species and growing sites, and sensitivity to environmental changes.
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Of course, this method, as well as MIP, BET and the hydrostatic balance method, gives information
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on the connected porosity only and, as such, can be applied in systems with low fractions of isolated
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pores. This method can give information not attainable in other ways, like changes of the internal
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architecture of corals described by microporosity and cut-off with increasing mass and growing
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temperature. Even if this method cannot spatially locate the heterogeneity of the pore space, the
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existence of a clear cut-off in almost all the distributions (a very high slope at a certain point of the
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distribution) means that the smallest pores are not well connected by diffusion at the NMR time
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scale (corresponding to the local value of T2) to the largest ones. Moreover the NMR defined
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parameter “microporosity” can quantify the ratio between the volume of the smallest pores (sizes
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less than 10-20 µm) and the total pore-volume.
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The increased fraction of larger pores in the zooxanthellate corals with increasing SST, that could
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reduce their resistance to mechanical stresses, could have particular consequences in determining
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effects of global warming on these species.65 The described method will be applied in future work
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to the effects of ocean acidification on the skeletal properties of corals66 and other calcifying
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organisms.
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ACKNOWLEDGEMENTS
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This research has received funding from the European Research Council under the European
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Union's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement n° 249930 –
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CoralWarm: Corals and global warming: the Mediterranean versus the Red Sea. We also thank F.
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Gizzi, C. Marchini and S. Prantoni for help in collecting the samples, the diving centers Centro
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Immersioni Pantelleria and Il Pesciolino for logistic assistance in the field, the Scientific Diving 10 ACS Paragon Plus Environment
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School for collaboration in the underwater activities, Fausto Peddis for MIP measurements, Gianni
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Neto for pictures of living specimens and Robert James Sidford Brown for carefully reading the
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manuscript.
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Supporting Information Available
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All references numbers in Supporting Information refer to the Reference list in the main text.
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This information is available free of charge via the Internet at http://pubs.acs.org/.
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B. europaea
Parameter
microporosity cut-off m VT SNMR SNMR/ VT
268 269 270 271 272 273 274 275
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L. pruvoti
n
mean
se
n
mean
se
27 27 27 27 27 27
31.3 337 0.88 0.92 4060 4800
1.6 15 0.15 0.17 820 275
26 26 26 27 27 27
38.6 249 0.25 0.20 1233 6993
2.1 20 0.04 0.03 165 349
t
Z
2.79*** 3.59*** 3.92*** 4.13*** 3.38*** 4.94***
264 6.66*** 12.92*** 265 11.19*** 17.46*** 266 11.21*** 17.60*** 267
Table 1. Descriptive and test statistics split by species *** p
