Coordination environment of Zn in foraminifera Elphidium aculeatum and Quinqueloculina seminula shells from a polluted site

Accepted Manuscript Coordination environment of Zn in foraminifera Elphidium aculeatum and Quinqueloculina seminula shells from a polluted site

Giovanni De Giudici, Carlo Meneghini, Daniela Medas, Carla Buosi, Pierpaolo Zuddas, Antonella Iadecola, Olivier Mathon, Antonietta Cherchi, Andrei Cristian Kuncser PII: DOI: Reference:

S0009-2541(17)30682-4 https://doi.org/10.1016/j.chemgeo.2017.12.009 CHEMGE 18580

To appear in:

Chemical Geology

Received date: Revised date: Accepted date:

3 August 2017 11 December 2017 12 December 2017

Please cite this article as: Giovanni De Giudici, Carlo Meneghini, Daniela Medas, Carla Buosi, Pierpaolo Zuddas, Antonella Iadecola, Olivier Mathon, Antonietta Cherchi, Andrei Cristian Kuncser , Coordination environment of Zn in foraminifera Elphidium aculeatum and Quinqueloculina seminula shells from a polluted site. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Chemge(2017), https://doi.org/10.1016/j.chemgeo.2017.12.009

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Title

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Coordination environment of Zn in foraminifera Elphidium aculeatum and Quinqueloculina seminula shells from a polluted site

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Authors

Giovanni De Giudicia*, Carlo Meneghinib, Daniela Medasa, Carla Buosia, Pierpaolo Zuddasc, Antonella

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Iadecolad,e, Olivier Mathond, Antonietta Cherchia, Andrei Cristian Kuncserf

Affiliations

Dipartimento di Scienze Chimiche e Geologiche, Università degli Studi di Cagliari, Via Trentino 51,

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a

I-09127 Cagliari, Italy.

Dipartimento di Scienze, Università di Roma Tre, 00146 Roma, Italy.

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Institut des Sciences de la Terre de Paris, UPMC- Sorbonne Universités, 4, place Jussieu 75252

Centre national de la recherche scientifique (CNRS), Réseau sur le stockage électrochimique de

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d

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ParisCédex05, France.

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b

l'énergie (RS2E), L’Orme des Merisiers Saint Aubin BP48 91192 Gif sur Yvette CEDEX. The European Synchrotron Radiation Facility (ESRF), 71 Avenue des Martyrs, 38000 Grenoble,

France. f

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e

National Institute of Materials Physics, Laboratory of Atomic Structures and Defects in Advanced

Materials, Atomistilor 105 bis, 077125 Magurele, Romania.

*Corresponding author: [email protected]

Keywords

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ACCEPTED MANUSCRIPT Foraminifera, zinc biogeochemical cycling, metal detoxification, synchrotron techniques

Abstract Foraminifera, unicellular organisms that are widespread throughout marine ecosystems, build Ca-

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carbonate shells that may incorporate trace metals present in the ocean waters because of natural or

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anthropogenic supply. In this study, we focussed on the trace element Zn, which is abundant in both

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contaminated and clean waters. We used X-ray and electron spectromicroscopy to investigate the Zn coordinative environment in individual shells of two species of benthic foraminifera, Elphidium

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aculeatum and Quinqueloculina seminula, that were sampled from a heavy-metal polluted area of Sardinia, Italy. These species synthesise the Ca-carbonate in extracellular and intracellular spaces,

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respectively, which implies some diversity in their physiologies and cation transport processes, and they can adapt and survive in metal-polluted environments. Our analyses of X-ray micro-fluorescence (μ-XRF) maps and Zn-K μ-X-ray absorption near-edge

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spectroscopy (XANES) reveal that although 50% of Zn occurs as a Ca substitute in calcite or as a

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tetracoordinate oxygen-adsorbed ion, detectable amounts can also be found in other Zn- independent mineral phases, particularly hydrozincite, whose formation is due to foraminiferal cellular processes.

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Other Zn phases, sphalerite (attributed to early diagenetic process) and Zn-phosphate, were recognised. Moreover, we found distinct differences in the Zn concentration, distribution and chemical speciation

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at the micro- and nano-metric scales of the investigated E. aculeatum and Q. seminula species. In the calcite needles of Q. seminula, Zn is uniformly distributed and recognised in a disordered local

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environment, which suggests that it is incorporated in the calcite phase during the intracellular calcification process. These findings offer insight into Zn incorporation in foraminifera and its potential application in biomonitoring and environmental studies.

1. Introduction Since the Middle Ordovician (485–444 Ma), foraminifera have been ubiquitous throughout all marine ecosystems that support eukaryotic life (Gradstein et al., 2012). These unicellular organisms build Cacarbonate shells that incorporate trace metals, whose concentrations specifically respond to variations

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ACCEPTED MANUSCRIPT in the water metal supply (Cherchi et al., 2009; Erez, 2003; Munsel et al., 2010; van Geen and Luoma, 1999), ocean evolution (Henderson, 2002) and global environmental and climate changes (Alve, 1991; Coccioni et al., 2009; Frontalini et al., 2009; Lea, 1999; Marchitto et al., 2000; Scott et al., 2005), thus making foraminifera valuable bioindicators. Trace metal incorporation in foraminiferal calcite shells is an interesting research topic that has a wide presence in biogeochemical scientific literature (Barker and Elderfield, 2002; Boyle, 1981; Elderfield,

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2000; Frontalini et al., 2009, 2015; Marchitto et al., 2000; Munsel et al., 2010; Rumolo et al., 2009; van

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Dijk et al., 2017). Zn, which is an abundant trace element and nutrient, is involved in marine

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biogeochemical reactions (de Nooijer et al., 2014; Erez, 2003; Marchitto et al., 2000; Morel and Price, 2003; Rauch and Pacyna, 2009; van Dijk et al., 2017; Weiner and Dove, 2003). The scientific literature

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describes the microscopic scale behaviour of Mg and B for some foraminifera (Branson et al., 2013, 2015; and references therein). Foraminiferal calcification has been used to calibrate Mg/Ca paleo-

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thermometers and as a B/Ca paleo-pH proxy to investigate the evolution of the carbonate saturation state in the oceans (Barker and Elderfield, 2002; Elderfield, 2000; Yu and Elderfield, 2007; Yu et al.

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2007a b).

On the basis of their physiological biosynthesis mechanism, foraminifera can synthesise Ca-carbonate

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in either extracellular or intracellular calcification sites (de Nooijer et al., 2014; Weiner and Dove, 2003), which implies some degree of diversity in their physiology and cation transport processes

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(Branson et al., 2013; Morel and Price, 2003; Weiner and Dove, 2003). The extracellular process driven by Rotaliida results in the formation of low-Mg calcite shells, and the intracellular process

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driven by Milioliida results in the formation of high-Mg calcite shells (Weiner and Dove, 2003, and references therein). The distributions and chemical speciations of elements in all foraminiferal shells

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represent the final product of the transportation of ions from ambient seawater to the calcification site. However, while recent literature has focussed on the microscopic mechanisms of biosynthesis, i.e. nanocrystal formation and aggregation (De Yoreo et al. 2015; Meldrum and Cölfen 2008), the direct observation and chemical speciation of trace elements in nanocrystal aggregates that build foraminiferal shells have been limited by instrumental sensitivity. As an abundant trace element and nutrient, Zn is involved in marine biogeochemical reactions and can accumulate in foraminiferal shells. Thus, Zn/Ca has been used as a proxy for understanding ocean water chemistry and paleocirculation (Bryan and Marchitto, 2010; Marchitto et al. 2003). The Zn partition coefficients for foraminiferal shells can depend on the species (Bryan and Marchitto, 2010). Van Dijk et al. (2017) found that Zn is likely to be taken up as aquo ion during foraminifera

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ACCEPTED MANUSCRIPT biomineralization. In this study, to investigate precisely the manner in which Zn is incorporated in foraminiferal shells that are collected from polluted sites from the micrometric to the nano-metric scale, we adopted a multitechnique/multi-scale approach that exploits the state-of-the-art complementary imaging techniques of X-ray absorption fine structure (XAFS) spectroscopy (Bunker, 2010; De Giudici et al, 2015; Rehr and Albers, 2000) and high-resolution transmission electron microscopy (HR-TEM) in the shells of the

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following two foraminiferal taxa: the Elphidium aculeatum (belonging to the Rotaliida order) and

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Quinqueloculina seminula (belonging to the Milioliida order). After an initial step of superficial

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cleaning to remove sediment grains while maintaining the shell shape, we proceeded from the macroto the nano-metric scale to determine both the metal speciation and location in individual shells. First,

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we investigated the average speciation of Zn by performing chemical analysis, X-ray diffraction and XAFS on bulk samples comprising hundreds of shells for each species. Then, we used µ-XRF (X-ray

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micro-fluorescence) and µ-XAFS (Bunker, 2010; Mathon et al., 2015) techniques to finely map at the micrometre scale the Zn distribution and its coordination environment in selected shells. Next, we

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identified, at the nano-scale, the chemical, structural and textural properties of nanophase biogenic Zn in individual shells of foraminifera (E. aculeatum and Q. seminula) by HR-TEM, energy dispersive

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spectroscopy (EDS) and selected area electron diffraction (SAED). In this study, we show that the incorporation of Zn in carbonate biomineralization can result in the formation of unexpected

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biosynthesised nanophases that, in turn, can be species-dependent.

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2. Materials and Methods

2.1. Sampling and benthic foraminiferal analysis In this study, we show results from samples collected in northern Sardinia at La Maddalena Harbour (a site where heavy-metal pollution occurred during several decades of military activity), as refined in preparation for the G8 meeting in 2009 (41°12'37.7196" N 09°25'55.4195" E; see Fig. A1). Results characterising the harbour sediments based on monitoring studies of the status of marine environments using benthic foraminifera as bioindicators are presented by Buosi et al. (2013), Salvi et al. (2015) and Schintu et al. (2015). In our study, we analysed, in detail, the Zn incorporation in foraminiferal shells

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ACCEPTED MANUSCRIPT from a short core with a length of 3 m (UC02bis, see Fig. 1 of Salvi et al. 2015), which we collected in the La Maddalena Harbour in October 2011. The analysis of superficial sediments by Salvi et al. (2015) revealed an enrichment in pollutants such as As, Hg, Zn, Cu, Pd, Cd and hydrocarbons (C > 12). The maximum concentration of Zn in sediments was found to be ca 1600 ppm (see Fig. 3 of Salvi et al. 2015). The aliquot used in monitoring the status of marine environments using benthic foraminifera as bioindicators was taken from the core-top surface interval of 0–2 cm of each half-core of the station

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and stored in polyethylene jars following the methodology protocol established by Schönfeld et al.

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(2012). The samples were then preserved in ethanol and rose bengal (2 g of rose bengal in 1000 ml of

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ethyl alcohol) to differentiate between living and dead foraminifera (Walton, 1952). For each sample, we dried and then weighed a constant volume of ~50 cm3 at 50 °C. After drying and sieving the

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residues in >63-µm meshes, we examined the foraminifera using a binocular microscope. In agreement with the protocol for statistical analysis and biomonitoring of foraminifera (Schönfeld et al., 2012), we

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selected at least 300 benthic foraminifera from each sample for micropalaeontological analysis. Owing to the scarcity of live populations (0.6% of the specimens), we included dead foraminiferal

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assemblages in this study. The lifetime of foraminifera is only few weeks. We interpreted all broken and discoloured shells and shell fragments as subfossils and excluded them from the count. We

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considered only relatively pristine foraminiferal shells to be recent. We can assume that the examined dead foraminiferal assemblages collected in the sediment interval of 0–2-cm accumulated in the last 20

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years. Thus, we can assume that the chemical compositions of foraminiferal shells reflect modern pollution. We identified benthic foraminifera according to the taxonomic works of Loeblich and

Zei (1993).

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Tappan (1987), Cimerman and Langer (1991), Hottinger et al. (1993) and Sgarrella and Moncharmont

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Boyle (1981) and Lea and Boyle (1991, and references therein) reported that to ensure the measurement of trace elements such as Cd and Ba owing to their cellular incorporation in foraminiferal shells, cleaning steps are necessary to avoid the contribution of small sediments grains. More recently, to calibrate the Mg/Ca ratio into a valuable paleothermometer, the cleaning procedures were revisited and developed for some proxy elements such as Mg, Sr, B, Cd and Zn. To extract the elements not hosted in the calcite structure but rather in the organic matter, clays and metal sulphides, or adsorbed onto the calcite surfaces, some authors began cleaning procedures by crushing the shells and subsequently following the following steps: i) removal of clays, ii) removal of organic matter, iii) removal of coarse-grained silicates, iv) removal of metals adsorbed and v) the eventual dissolution of the crushed and washed foraminifera for chemical measurements (Barker et al, 2003, and references 5

ACCEPTED MANUSCRIPT therein). While cleaning procedures are central to many research investigations as they can ensure satisfactory and reproducible results, they can also lead to the partial dissolution of foraminiferal shells (Yu et al, 2007a) and, as they are handled in bulk, some of the trace metal positions in the shells can be obliterated. In our study, to preserve the best elemental composition of the shells, we washed a number of foraminiferal specimens, which belonged to the UC02 bis-core collected during the sampling survey conducted by Salvi et al. (2015), with only ultrapure water in an ultrasonic bath for 3 min, and then

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rinsed them three times. All the foraminiferal shells had been previously investigated by ESEM

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(environmental scanning electron microscopy) in low-pressure mode for morphological and chemical

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composition analyses (without any Au or C surface coating). No Zn was detected in the foraminiferal shells through this technique and micrometric grains of seabed sediment were rarely found. After

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investigation by µ-beam techniques (X-ray), we re-investigated the shells using ESEM and TEM. We determined the Zn/Ca molar ratio values of our shell samples by laser ablation–inductively coupled

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plasma-mass spectrometry (LA-ICP-MS) and compared them with the values found in previous studies with high and low Zn contents (Table 1). We ablated the material with a 213-nm system laser (New

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Wave Research) connected to a quadrupole ICP-MS instrument (Perkin Elmer Sciex Elan DRC-e). We performed our calibration using an NIST SRM610 glass material and used 43Ca as the internal standard.

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With respect to Sardinia, we selected foraminifera isolated from sediments collected from the Bonifacio Strait (NE Sardinia; Buosi, 2010; Buosi et al., 2012) as a ‘blank’ sediment having a low Zn

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content (in the range of few tens of ppm). All analyses were performed at a frequency of 10 Hz and a spot size of 40 μm. We used He as a carrier gas that was mixed with Ar as a makeup gas before

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entering the ICP system. We performed quality control in the LA-ICP-MS analysis by analysing a natural standard glass, BCR-2, as certified by the USGS, which we considered to be an unknown

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sample (calculated precision < 10%). We processed the data using the GLITTER program. The data reported in Table 1 represents the mean values obtained from multiple locations on ~60 individual specimens each of E. aculeatum (hyaline, low-Mg calcite) and Q. seminula (porcelanaceous, high-Mg calcite).

2.2. X-ray absorption spectroscopy and X-ray fluorescence maps Zn K-edge XAFS and measurements on bulk samples were conducted to determine the fluorescence geometry on the ESRF-BM23 (Grenoble-France; Mathon et al., 2015) and the ELETTRA-XAFS

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ACCEPTED MANUSCRIPT (Trieste-Italy; Di Cicco et al., 2009) beamlines on two independent sample sets comprising ~50 mg of Elphidium spp. and Quinqueloculina spp. specimens belonging to different species (see Appendix Fig. A.1). The foraminifera were dried, ground, mixed with pure cellulose and pressed into solid pellets that were suitable for XAS measurements. We performed fluorescence geometry measurements while maintaining the samples at liquid nitrogen temperature. In addition, we performed transmission geometry measurements on an ample set of Zn-containing compounds and minerals to be used as

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references (Fig. 1). The main reference materials were as follows: a) Zn-calcite solid solution; b) Zn

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adsorbed on calcite; c) Zn adsorbed on hydroxyapatite; d) hydrozincite: Zn5(CO3)2(OH)6 Malfidano

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Mine, Sardinia (Medas et al., 2014a); e) ZnO Acros Organics 99%; f) hemimorphite: Zn4(Si2O7)(OH)2·H2O San Giovanni Mine, Sardinia (Medas et al., 2017); g) willemite: Zn2SiO4; h) Zn-

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sulphide: ZnS; i) Zn-ferrite: ZnFe2O4; l) Zn-phosphate: Zn3(PO4)2; m) Zn-Cysteine. We followed standard procedures in treating the XAS spectra for background removal and edge-jump normalisation

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(Meneghini et al., 2012; Medas et al., 2014b). We obtained quantitative local atomic structure details by refining the extended (EXAFS) regions using the standard EXAFS formula (Bunker, 2010). We conducted the µ-XRF maps and Zn K-edge µ-XANES measurements on selected individual specimens

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on the ESRF–BM23 beamline in which the X-ray spot was 4 × 4 µm2. The µ-XRF maps were

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measured at a fixed X-ray beam energy (12000 eV) using an ultrapure 13-element Ge multidetector for data collection while maintaining the detector deadtime to less than 10% to avoid nonlinear effects. We

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analysed the images using PyMCA software (Solé et al., 2007). The fluorescence signals from the Ca, Zn and Fe signals provide well-resolved peaks that are suitable for reliable element distribution

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analysis.

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2.3. Transmission electron microscopy Sample preparation: After SEM observations of individuals (see section results and Appendix A), we selected shells of E. aculetaum and Q. seminula. Then, we gently crushed the shells in agate mortar and dispersed them in ultrapure water in an ultrasonic bath for 3 min. We then deposited a droplet of this suspension onto a TEM Cu grid. For our analytical TEM/STEM investigations, we used a Cs-corrected JEM ARM 200F microscope equipped with a Schottky field emission gun (FEG), a Cs-corrector for the STEM mode, and a JEOL JED-2300T EDS unit. The acceleration voltage was 200 kV.

3. Results 7

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3.1. SEM imaging and element distribution across all shells. We compared the Zn/Ca ratios measured by laser ablation analysis of the La Maddalena samples with those of the Bonifacio samples (North Sardinia, formally an unpolluted site) and with data from the literature (Table 1). Based on the Zn/Ca ratios in our samples, we determined that La Maddalena

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Harbour was a slightly polluted site. Deep ocean samples showed the lowest content, while samples

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from industrial sites showed increased Zn/Ca ratios by one order of magnitude. In the foraminifera

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from La Maddalena, the Ca, Mg and Zn contents revealed that, besides the expected Mg content, the Zn levels are high in the high-Mg shells by a factor of ~4. In the samples we investigated in this study, Rotaliida from La Maddalena show a 2-fold increase in Zn with respect to Bonifacio, while Milioliida

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from La Maddalena show a 4–5-fold increase with respect to the Bonifacio Strait. We imaged the E. aculeatum and Q. seminula specimens with ESEM to reveal the presence of grains

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and materials of non-biosynthetic origin or those originating from post-mortem processes. We crushed some of the shells to investigate the inner surfaces of the chambers (Fig. 2). We found that most of the

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debris on the external surface and inside the chambers was made of calcium carbonate. We also found

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some framboids made of iron and sulphur in the shells of the two investigated taxa, which were attached to the external and inner surfaces; this was probably due to post-mortem processes (Figs. 2c and 2f). In addition, we found abundant remnants of diatoms inside the chambers of Q. seminula.

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While focusing the 5-micron-sized electron beam on the shell cuts and on other areas not affected by encrustation or not polluted by grain sediments (Figs. 2d, 2e, 2g, 2h), we found the presence of S, Cl,

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Si, Al and P traces along with Mg on the shells of E. aculeatum and Q. seminula. The presence of these elements inside the shells (beam spot on shell cuts in Fig. 2) indicates that they are not exclusively

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contained in grain sediments adhering to the surface or post-mortem framboids. On the contrary, they are likely to be embedded into the shell wall. We found the encrustation comprised Fe, S, Cl, Al, Si, Na and K (Fig. 2e). As expected, we detected Mg in both taxa, with a peak of higher intensity in high-Mg shells (Q. seminula). To gain deep insight into the Zn distribution and chemical Zn species in the calcite shells, we investigated the foraminiferal specimens by synchrotron-based µ-XRF and Zn K-edge µ-XANES (Fig. 3). Figure 3 shows the µ-XRF maps measured with 4 × 4-m2 pixel resolution on four selected

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ACCEPTED MANUSCRIPT investigated samples, i.e. two E. aculeatum and two Q. seminula, which highlight the Ca, Zn and Fe distributions. The Zn distribution not only resembles that of Ca but also depicts more intense spots, which are more evident in E. aculeatum than in Q. seminula. We found a significant amount of Fe with a less regular distribution than that of Zn and which displayed spots and inhomogeneity (Fig. 3). The Pearson linear correlation coefficient ρ, which we calculated to determine the Zn–Ca, Zn–Fe and Fe– Ca fluorescence intensity distributions, clearly indicates that the Zn and Ca correlation in E. aculeatum

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is weak (ρZnCa < 30%), while it is significantly high for Q. seminula (ρZnCa > 50%). This finding

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quantifies the less homogeneous dispersion of Zn in the calcite of low-Mg shells. Moreover, the Fe and

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Zn distributions appear to be strongly correlated (ρZnFe > 50%) in all the samples (slightly high for E. aculeatum), which suggests that Zn and Fe trace elements tend to be co-located. We also noticed a

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negligible correlation between Fe and Ca (ρCaFe < 10%), which points to the spotty distribution of Fe in both foraminiferal taxa.

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We obtained further details by analysing the distribution of Zn with respect to Ca. The fluorescence intensity ratio RZnCa = IZn/ICa is roughly proportional to the Zn/Ca concentration ratio. For the sake of

RZnCa

R ZnCa

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RN 

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comparison in Fig. 3, we report the distribution of the normalised intensity ratio as follows:

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where R ZnCa is the average Zn/Ca fluorescence intensity ratio in the sample. The apparent values of the RN cumulative distribution function (CDF) and the differential distribution function (DDF) allow a

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comparison of the Zn and Ca distribution in the shells and highlight the differences between them in the two foraminiferal taxa. The Q. seminula RN distributions are very similar, with 95% of the data

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falling within 0.3 < RN < 2. However, the RN distributions for the E. aculeatum data show a wide variability between the two samples, with long tails in the high-RN region. This finding, which is in accordance with the correlation analysis, indicates a less homogeneous distribution of Zn in E. aculeatum shells than in the Q. seminula. 3.2 Analysis of Zn coordination environment in bulk samples. To understand the chemical speciation of Zn, we used the XAFS technique because it is a well-ascribed chemically selective local atomic probe (Bunker, 2010) that is sensitive to the average atomic structure and coordination chemistry around the absorber. We quantitatively analysed the near-edge (XANES) and extended (EXAFS) regions of the Zn K-edge XAFS spectra as they provide valuable 9

ACCEPTED MANUSCRIPT complementary information (Benfatto and Meneghini, 2014; Monesi et al., 2005). In the EXAFS region, we performed data analysis by fitting the experimental data to a model curve obtained by the standard EXAFS formula (Bunker, 2010; Meneghini et al., 2012), which enables us to understand the local structure around the absorber of the coordination shells and provides for each absorber the average coordination numbers (N), interatomic distances (R) and variances (2) of the interatomic distributions (mean square relative displacement). The near-edge region of the absorption spectra

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contains details about the electronic state (valence) and local structure (coordination chemistry and

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local symmetry) around the absorbing atoms and can be used as a fingerprint to precisely identify

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chemical species in mixtures and complex materials. Linear combination analysis (LCA; Benfatto and Meneghini, 2014) is widely used for quick and reliable XANES interpretation of natural materials as it

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is a powerful method for quantifying the relative amounts of constituent chemical species. Initially, to obtain a signal mediated over hundreds of individual shells, we investigated bulk samples

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of the two foraminiferal taxa collected at La Maddalena Harbour. Figure 4 shows a summary of the results of our Zn K-edge XAFS (near-edge, XANES, and extended, EXAFS, regions) measurements

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(Di Cicco et al., 2009; Mathon et al., 2015) (see Fig. 1). The Zn XANES spectra of the foraminifera samples show significant differences between the Elphidium spp. and Quinqueloculina spp. data (Fig.

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4A), which indicates different average Zn coordination environments that are related to the different natures of the Zn chemical and mineralogical speciation of the two taxa.

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We quantitatively analysed the XANES spectra by fitting the normalised spectra to a linear combination (LCA) of the Zn XANES spectra of reference compounds. Of the several reference

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compounds (Fig. 1), the main contributions required for best fit are hydrozincite Zn5(CO3)2(OH)6 and a Zn-calcite solid solution (Zn/calcite). An additional contribution is necessary to improve the best fit,

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which we identified as a 4-fold coordinated Zn that matches the Zn adsorbed onto calcite surfaces or the Zn in the hydroxyapatite (Zn/Hap; Tang et al., 2009) references, as these two references provide similar fit agreements in the refinement. We refer to this component as Zn4C in the following discussion and figures. Based on the EXAFS analysis as well, this contribution to the LCA represents a phase in which Zn has a preferential 4-fold coordination that is more distorted with respect to the Zn/calcite solid solution but more ordered than the Zn adsorbed onto the calcite surfaces. Figure 4B shows the phase composition resulting from the LCA, which shows the uncertainties in the calculation while considering the dispersion of the results among the samples. The LCA quantifies the compositional differences of Zn-containing phases between the two taxa; the Elphidium spp. samples depict a systematically high hydrozincite-like phase and Zn/adsorbed and Zn/Ca solid-solution contents than the

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ACCEPTED MANUSCRIPT Quinqueloculina spp. data. The fine structure and oscillations in the Elphidium spp. Zn XANES spectra suggest a fairly well ordered Zn-neighbouring atomic structure. The features observed in the Quinqueloculina spp. XANES are smooth, which indicates a great structural disorder at the atomic scale. An analysis of the extended regions (EXAFS) of the spectra confirms this trend (Fig. 4C); the Elphidium spp. data depict a structured spectrum with features similar to those of the Zn-hydrozincite EXAFS spectrum, while the

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spectrum of Quinqueloculina spp. depicts the smooth features characteristic of a disordered structure.

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The Fourier transform moduli (|FT|) provide an intuitive understanding of the average Zn local

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structure (Fig. 4D), with main peaks signalling a neighbour coordination shell. By comparing the |FT|, we can confirm the close similarity of the Elphidium spp. and hydrozincite data, and we note that the

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weak next-neighbour shells observed in the Elphidium spp. data can very likely be ascribed to great structural disorder in the biosynthesised mineral than in the hydrozincite standard (Medas et al. 2014a).

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The Quinqueloculina spp. EXAFS spectrum is characterised by a single nearest-neighbour shell and very weak signals coming from next-neighbour shells, which definitively confirm our observation of a

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disordered structural environment of Zn in these Miliolida shells. We performed a quantitative analysis of the EXAFS spectra in the reciprocal space by considering

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relevant next-neighbour shells up to ~4 Å (multi-shell approach; Medas et al., 2014), and Table 2 summarises the local structural parameters. An analysis of the Zn-EXAFS spectrum of hydrozincite,

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which we used to tailor the analysis procedure, reveals that it matches the structure described in the literature (Ghose, 1964; ICSD, 2011). The Elphidium spp. data reveals a first Zn–O shell at RZnO = 2.01

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±0.01 Å with an average coordination number of NZnO = 5.4 ±0.5. We found two Zn–Zn nextneighbour shells at 3.15 Å and 3.55 Å, which were in agreement with the hydrozincite model. This

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agreement definitively confirms that a fraction of Zn is embedded in an ordered local environment that is similar to that of the hydrozincite crystallographic phase. The analysis of Quinqueloculina spp. EXAFS spectrum indicates that, except for the first shell (Zn-O), the next-neighbour coordination shells are weak and are definitively attenuated by structural disorder. Attempts to include next-neighbour coordination shells do not statistically improve the fitting even if the formation of a coordination shell at ~3.9–4.0 Å cannot be excluded. These findings are consistent with the formation of an inner-sphere Zn2+ sorption complex, which is likely adsorbed onto the surface of calcite grains. Interestingly, the hydrozincite fingerprint observed in the Quinqueloculina spp. XANES fitting (LCA) is missing in the extended region (Zn–Zn next-neighbour shells around 3.15 Å and 3.55 Å), which suggests the absence of a medium-range order, probably because of a highly

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ACCEPTED MANUSCRIPT disordered and/or nanosized hydrozincite-like phase in the Miliolida samples.

3.3 Variability of Zn coordination environment across all shells Since the µ-XRF data indicate that the Zn distribution is not uniform across all shells, the question arises whether the different RN values could be related to different Zn phases resulting in different

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coordination chemistries. We were given access to the µ-XAS facility at Beamline 23 (ESRF-

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Grenoble) to collect the Zn K-edge XAFS spectra at selected points in the mapped areas with 4 × 4-µm

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resolution. We selected µ-XAS points in low (L), medium (M) and high (H) RN regions to identify any differences in the Zn coordination chemistries. At each point, we collected several (5–10) energy scans

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in the XANES region and then averaged the spectra results to ensure statistical validity. Then, we used LCA to quantitatively analyse the normalised Zn K-edge µ-XANES spectra (Figs. 5 and 6). We note that the distribution of Zn in different phases changes as a function of RN and also differs in E.

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aculeatum and Q. seminula. According to our LCA results of the bulk Zn XANES spectra, the main contributions that are necessary to fit the µ-XANES spectra are Zn-hydrozincite, Zn/calcite and Zn4C,

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the last of which indicates either Zn adsorbed in calcite or hydroxyapatite, as discussed above. Notably,

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the E. aculeatum XANES spectra measured in high-RN regions also exhibit a large fraction of other Znrich phases that have spectral features similar to the ZnS reference (up to 60%), Zn-phosphate

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Zn3(PO4)2 (up to 30%) and ZnFe2O4 (up to 10%) (see Fig. 6). We observed these Zn-rich phases in E. aculeatum and not in Q. seminula. We also note that the LCA analysis cannot distinguish rare Zn-

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containing phases (i.e. if the fraction of Zn in the sample is less than 3–5%). Thus, the possibility of minority-phase contributions cannot be excluded. Before exploring the origin of these unexpected phases, we stress that in addition to the already-

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mentioned difference between the E. aculeatum and Q. seminula specimens, the µ-XANES spectra collected at different points also exhibited significant variability within the Elphidium and Quinqueloculina specimens (Figs. 5 and 6). The LCA fitting results of the µ-XANES spectra indicate that hydrozincite is present in all the investigated micro-areas, while the Zn XANES features also indicate the presence of other components such as Zn-calcite, 4-fold-coordinated Zn (Zn4C), Znsulphide (ZnS) and Zn-phosphate Zn3(PO4)2. Our analysis of µ-XANES spectra suggests an intraspecimen variability related to the local Zn concentration. Particularly, high values of the hydrozincite component correspond to high RN values, while Zn/calcite and Zn4C phases are favoured in regions having low and medium RN values.

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3.4. Recognition of Zn and Fe nanophases. We used HR-TEM to analyse fragments of E. aculeatum shells to determine the distributions and

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structural states of trace elements incorporated within the calcite matrix of plate-like nanocrystals (Figs.

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7 and 8). We found a 50 × 20-nm crystal rich in Fe and O neighbouring an aggregate of calcite nanocrystals from the shell and an aggregate of Zn carbonate nanocrystals, each of which had an

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individual size of ~5 nm. Based on the SAED pattern, we identified the main reflections of hydrozincite. In other areas of the E. aculeatum shell (Fig. 8), we found Zn to be co-located with S. In

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this case, the SAED pattern indicates the presence of nanocrystalline sphalerite, ZnS. Q. seminula shell fragments comprise needles of calcite-like single crystals (Fig. 9). We indexed the

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electron diffraction patterns on the selected area shown in Fig. 9 as forms of hydrated calcium carbonate (X-ray analysis indicates a Mg-calcite composition for the investigated Miliolida, see also

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Appendix Fig. A.1). The distributions of Fe and Zn generally indicate their co-location with Ca and Mg in the calcite needles, with some patches of Fe, Zn and S. Noticeably, the Zn distribution is much more

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homogeneous than that in the Elphidium samples. Also, the Fe distribution shows few intense spots. We observed no independent phases of Fe and Zn by SAED, which agrees with the µ-XRF elemental

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maps, CDF and DDF curves and the µ-XAS identification of the Zn–O shell alone. EDS data from the

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4. Discussion

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TEM analysis are in good agreement with the EDS data from the ESEM analysis.

Zn is a nutrient that becomes toxic to aquatic life when its bioavailability is too high. The bioavailability of Zn depends on both natural and industrial processes. In the geological record, owing to volcanism and hydrothermal activity, the anomalous background Zn concentration can be detected in sedimentary rocks far away from the source (see, for instance, Larson 1991; Larson and Erba, 1999). At the regional or local scale, Zn geochemical anomalies can be due to the weathering and erosion of rocks or industrial emissions. In Sardinia, owing to mine and industrial activities, heavy metal content in marine sediments can increase by more than one order of magnitude (Romano et al. 2017; Salvi et al. 2015). Trace metal speciation in foraminifera has been debated by many researchers. Boyle (1981) pointed out

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ACCEPTED MANUSCRIPT that trace metals can be incorporated into the structure of calcite, but they can also be associated with silicates and other minerals that pollute the shells, and in organic matter and post-mortem phases. For this reason, foraminifera have often been studied after careful cleaning using procedures that avoid any contamination that would limit their paleothermometers, paleochemical and environmental use and exploitation. In this study, we are mostly interested in gaining deep insight into the speciation and distribution of Zn from the bulk to molecular scale. Based on the phase and elemental sensitivities of

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synchrotron radiation and electron microscopy techniques, we can distinguish the fate of Zn in different

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areas, while deep cleaning of the shells may have obscured specific relevant details. In this manner,

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based on our understanding of Zn incorporation into the lattice of nanocrystals or other phases produced or incorporated during shell construction, we can properly use Zn as a proxy for trace-metal

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toxicity in water.

Biomineralisation processes involve complex sequences of different cellular and molecular reaction

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steps and mechanisms. Rotaliida synthesise calcium carbonate by modifying their water chemistry and pH by physiological and biochemical machinery comprising vacuoles, endo-exocystosis, ion pumping

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and membranes (de Nooijer et al., 2014; Weiner and Dove, 2003 and references therein). However, the biomineralisation pathway of Milioliida, to date, is poorly understood. The most significant difference

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is that Rotaliida synthesise calcite crystals and construct their shells extracellularly, while Milioliida synthesise calcite needles in vacuoles and eventually transport them in the extracellular space to

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construct their shells (Robbins et al., 2016). According to recent studies (De Yoreo et al, 2015; Meldrum and Cölfen, 2008; Weiner and Dove, 2003; and references therein), biominerals are shown to

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be an assemblage of smaller units that range in size from nanometres to micrometres and are glued by carboxylic groups, in which organic molecules and trace metals can be adsorbed onto grain surfaces

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and trace elements can enter the biosynthesised calcite lattice and modify its cellular volume (Han et al., 2013; Medas et al. 2014a). These gluing phases could be prone to dissolution by deep washing, thus hindering the observation of relevant details. In this study, we provide an accurate structural investigation of low-Mg calcite, which is made extracellularly by E. aculeatum, and high-Mg calcite, which is made intracellularly by Q. seminula. Results of both the -XRF and HR-TEM analyses indicate that Zn is more uniformly distributed in calcite needles made of micrometric crystals (Q. seminula) than it is in calcite nanocrystals (E. aculeatum), and the uniformity of distribution and Zn/Ca ratio are related to the coordination environment. We had soft-cleaned the investigated foraminifera to avoid any potential hindering of biomineralization processes; thus, contamination from sediments or post-mortem processes must be

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ACCEPTED MANUSCRIPT considered and discussed. The presence of Zn in silicates can be ruled out (or considered to be rare, below the detection limit of the techniques) in the investigated samples. Framboids of Fe and S and encrustation on the inner surfaces of the chambers (Fig. 2) are very likely due to post-mortem processes. Our ESEM analysis results show detectable traces of Fe, S, P, Al, Na and Si in the side-cut shell and in other microscopically clean shell areas. Our synchrotron-based µ-XRF and µ-XAS analyses results provide the following useful insights into the mineralogy and coordination

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environment for interpreting the observed Zn speciation.

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i) The areas with low RN, where µ-XAS analysis indicates the occurrence of Zn in Zn/Ca solid

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solution or the adsorption of Zn onto calcite surfaces and inter-grain spaces, are ascribed to foraminiferal cellular biosynthesis. However, within these areas, other Zn phases were also

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investigated by X-rays and TEM; therefore, hydrozincite found along with a Zn/Ca solid solution is most likely produced during the primary biomineralization process. To the best

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of our knowledge, this is the first time Zn has been shown to occur in a different form than Zn/Ca solid solution or as surface complexes in foraminiferal calcite. Hydrozincite is a

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mineral that is not commonly found in shallow seawater even very close to hydrozinciterich calamine deposits (Romano et al., 2017). As a post-mortem precipitation process of

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hydrozincite would result in noticeable precipitation of this mineral in sediments, we can

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safely exclude a post-mortem origin for this phase.

ii) Among the phases recognised in E. aculeatum shells, ferric oxide commonly occurs in seabed

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sediments. Thus, we cannot actually distinguish between ferric oxide originating from pollution and that originating from biosynthesis. However, observations of nanocrystals

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with a size of 20 × 50 nm (TEM probe) indicate that these nanoparticles can be easily incorporated or extracellularly synthesised during the assembly of the shell-building units. However, the micrometric Fe encrustation of the inner surfaces of foraminifera is likely due to post-mortem processes.

iii) Sphalerites, which are framboids of Fe and S, seem to result from post-mortem processes driven by microbes (sulphate-reducing bacteria). However, the location of nanometre-sized ZnS crystals close to calcite aggregates (Fig. 8) and the presence of detectable amounts of S within the shell (Fig. 2) suggest that ZnS could be the product of extracellular biosynthesis processes. Sulphide ions can be embodied in organic molecules involved in cellular

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ACCEPTED MANUSCRIPT physiology. Metallothioneines have been found close to the Golgi apparatus and can be used by foraminifera for metal detoxification (Carney et al., 2007; Le Cadre and Debenay, 2006;). They contain SH− thiol groups that strongly bind Zn2+, thus forming cellular biochemical complexes that comprise foraminifera (Frontalini et al., 2015; Ono et al., 1989). Our XAS analyses results clearly revealed the presence of ZnS in E. aculeatum shells but not in Q. seminula shells. Further studies are required to disentangle the ambiguities

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characterising the interpretation of ZnS occurrence.

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Intra-species and inter-species variability of minor and trace elements distributions in foraminiferal shells have been well documented by a fairly large number of researchers and are attributed to the

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variability of mineralisation mechanisms in the bioconstruction of calcite shells (Barker and Elderfield, 2002; Branson et al., 2013, 2015; Hathorne et al., 2003; Munsel et al., 2010; Sadekov et al., 2005; van

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Dijk et al., 2017; Weiner and Dove, 2003). The results of this investigation reveal that Zn variability in two genera of Miliolida and Rotaliida can be selectively caused by cellular mechanisms and the process

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of calcite shell construction, thus demonstrating the following: 1) the presence of hydrozincite as an independent biosynthesised Zn-phase; 2) a relationship between the local concentration and

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coordination environment of Zn.

This metal is emerging as a proxy for environmental and geological scientific analyses (de Nooijer et

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al., 2014; Katz et al., 2010; van Dijk et al., 2017). Zn can probably be incorporated as an aquo ion from seawater (van Dijk et al., 2017), which undergoes physiological and cellular transport and chemical

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reactions, and can be subsequently hosted in several micro-environments in which its co-location with S, Fe, P and C neighbouring ions indicates different possible coexisting molecular incorporation

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mechanisms. Our findings offer a key to understand the processes ruling the behaviour of Zn in biomineralization processes participating in biogeochemical cycles in detail. In this study, we investigated the speciation of Zn in foraminiferal shells from a polluted site in detail and found that the amount of Zn incorporated in a calcite shell can exceed the amounts of adsorbed Zn and ZnS. However, other Zn species can be abundant, depending on the local concentration of Zn. Other experiments are being conducted to disentangle ambiguities regarding the incorporation of Zn and other trace elements ions and the Zn speciation during biomineralization processes in marine nonpolluted environments.

5. Conclusions

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ACCEPTED MANUSCRIPT Based on the results of our study, we can draw the following conclusions: (1) Our chemical analysis of clean areas of foraminiferal shell reveals the presence of traces of Fe, S, P, Si and Al. (2) Combining μ-XRF and μ-XAS techniques enables the identification of shell areas where Zn occurs as a solid calcite solution or is adsorbed onto calcite surfaces owing to foraminiferal cellular processes.

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(3) In the above-mentioned spatial locations, Zn also occurs in an independent and unexpected

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phase, which is hydrozincite. We argue that this phase is also due to foraminiferal cellular

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processes.

(4) Nanometre-size sphalerite and iron oxide were found by XAS and TEM, which may be formed

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by cellular synthesis, while framboids of Fe and S are certainly the result of post-mortem processes.

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(5) Zn speciation, size of crystal domains and distributions over all shells differ for the two investigated species.

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(6) In calcite needles of Q. seminula, Zn is uniformly distributed. Also, because it occurs in a less ordered environment, Zn seems to be incorporated more easily during the biologically mediated

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carbonation process.

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All the detected Zn species are involved in biogeochemical cycles of Zn and in early diagenesis and should be considered while using the Zn/Ca ratio or Zn isotopes as proxies. Further studies are required

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to investigate the observed features of Zn speciation and distribution in shells that may also be common to other foraminiferal species and other localities. These insights have significant implications and

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provide a preliminary basis for the development of tools to properly understand and use foraminifera in biomonitoring, environmental studies and paleoceanography.

Acknowledgements We from

acknowledge RAS

and

CESA RAS/FBS

(grant (grant

number: number:

E58C16000080003)

F72F16003080002)

grants

for funding. F. Podda provided LA-ICP-MS analysis of foraminiferal shells. P. Lattanzi provided useful comments and suggestions on XAS experiments and the text of this paper. The ESRF EV-94 and

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ACCEPTED MANUSCRIPT ES-540 proposals provided access to the BM 23 micro-focus experiment. The CERIC–ERIC program (proposal 2016 2061, 2017 7041) provided access to XAFS and HR-TEM (NIMP). C. B. and A. C. collected, analysed and interpreted data from foraminiferal tests from the cores. D. M., C. M. and G. De G. collected, analysed and interpreted XAS spectra at BM 23 (ESRF) and XAFS (Elettra Spa), and HR-TEM data in Bucharest (NIMP). P. Z. contributed to the discussion and writing of this work. O. M. and A. I. contributed to the set-up of Beamline BM 23 at ESRF as well as data collection and

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interpretation. A. C. K. contributed HR-TEM expertise in the collection of structural and chemical data.

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The editorial roles and input of Editor Jeremy Fein and two anonymous referees are appreciated.

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systematics and autoecology. Boll. Soc. Paleontol. Ital. 32, 145–264.

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Figure captions Figure 1. Normalised Zn K-edge XANES. Spectra collected in reference compounds and used for analysis (LCA) of foraminifera bulk- and micro-XANES data: a) Zn/calcite solid solution; b) Zn adsorbed on calcite; c) Zn adsorbed on hydroxyapatite; d) hydrozincite: Zn5(CO3)2(OH)6; e) ZnO; f) hemimorphite: Zn4(Si2O7)(OH)2·H2O; g) willemite: Zn2SiO4; h) Zn-sulphide: ZnS; i)

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Zn-ferrite: ZnFe2O4; l) Zn-phosphate: Zn3(PO4)2; m) Zn-cysteine.

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Figure 2. ESEM images of Elphidium aculeatum and Quinqueloculina seminula. Images were acquired with an acceleration voltage of 20 kV under low-pressure conditions with no surface coating. EDS spectra in Figures c, d, g and h were collected with a

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spot size of ca 5 μm.

Figure 3. Microbeam X-ray fluorescence analysis of individual foraminiferal shells. Panels A.a–A.f and B.a–B.f: X-ray

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fluorescence micro-maps (4 × 4 µm2) on two Elphidium aculeatum. Samples (top left panels) and two Quinqueloculina seminula. Samples (bottom-left panels). To highlight the distribution of each element, only the Ca (a, d) or Zn (b, e) or Fe (c, f) fluorescence contributions are shown in each panel. Intensities are proportional to the element concentration. Both Zn and Fe distributions

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depict spots and inhomogeneities, Zn appears more homogeneously diffused on the Quinqueloculina samples. The linear correlation coefficients (Pearson correlation) reported in panel C quantify the relationship between the Ca, Zn and Fe intensity distributions on Elphidium and Quinqueloculina spectroscopic images (see text). Panels D–E depict the cumulative (CDF) and

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differential (DDF) distribution functions of the normalised Zn/Ca fluorescence intensity ratios (RN) in each map; the Quinqueloculina maps depict very similar distributions, while the Elphidium data show wide variability and asymmetric

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distributions with long tails in the high-RN region. Zn K-edge µ-XANES (see Fig. 3) were measured at selected points in the low (L), medium (M) and high (H) regions of RN. The arrows in Fig. 3 indicate the areas selected for collecting the µ-XANES spectra

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shown in Fig. 5.

Figure 4. XAFS analyses of bulk samples of Elphidium spp. and Quinqueloculina spp. Panel A shows the Zn K-edge XANES of two

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spectra of Elphidium spp., two spectra of Quinqueloculina spp. and four standards: a) hydrozincite: Zn5(CO3)2(OH)6 Malfidano Mine; b) Zn/calcite solid solution; c) Zn adsorbed on calcite; d)Zn adsorbed on hydroxyapatite, see Fig. 1). Panel B shows the linear combination analyses of Zn K-edge XANES spectra on Elphidium spp. and Quinqueloculina spp.; the main phase

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contributions have been individuated as hydrozincite (Hy), Zn–Ca solid solution (Calc) and 4-fold-coordinated Zn adsorbed (Zn4c). In panels C and D, the fine structures depict a difference in the crystal local order of the hydrozincite Zn position in Elphidium spp. and Quinqueloculina spp.

Figure 5. Analyses of local coordination environment of Zn in foraminiferal shell, arrows in Fig. 3 indicate the areas selected for collecting µ-XANES. Linear combination analyses of Zn K-edge µ-XANES spectra on Elphidium aculeatum (panel A) and Quinqueloculina seminula (panel B). Data are labelled according to the region of the normalised Zn/Ca intensity ratio RN (see Fig. 3). The main phase contributions have been individuated as hydrozincite (Hy), Zn–Ca solid solution (Calc) and 4-fold-coordinated Zn adsorbed (Zn4c). The phase fractions for LCA for selected µ-XANES spectra presented in panels C. The panels D resume the phase fractions for the spectra corresponding to different RN regions. The amount of hydrozincite is weak in the Quinqueloculina data (M and L) and in Elphidium (L) data. Fitting the µ-XANES of Elphidium collected on high Zn/Ca ratio regions (H) requires additional contributions as, for example, Zn-sulphide: ZnS (top panel on the right). For the sake of comparison, the amount hydrozincite + Zn/calcite + Zn4c is always normalised to 1 in panel D.

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Figure 6. µ-XANES LCA analysis. µ-XANES LCA analysis reveals the presence of mineral-like Zn phases as Zn-phosphate: Zn3(PO4)2 (ZnP) (top panel) and ZnS (middle panel). The Elphidium (M) LCA analysis is reported for the sake of completeness.

Figure 7. Zn and Fe nanophases in foraminiferal shells. (A) Top: TEM image of an Elphidium aculeatum shell fragment and SAED pattern of hydrozincite and calcite nanocrystal aggregates neighbouring an iron-rich nanocrystal marked with *. Bottom:

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STEM image with EDS maps of Ca, O, Zn, S and Fe.

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Figure 8. ZnS nanocrystals in foraminiferal shells. Top: STEM image of an Elphidium aculeatum shell fragment and SAED

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pattern of sphalerite and calcite nanocrystal aggregates. Bottom: STEM-EDS maps of Ca, Zn and S.

Figure 9. Structure of needle-like and chemical distributions of elements in foraminiferal shells. Top: TEM images of

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Quinqueloculina seminula shell fragments and inserted SAED patterns of crystals pointing to the formation of hydrated calcite forms like monohydrocalcite (upper-left image) and ikaite CaCO3(H2O)6 (upper-right and bottom-left images); STEM images on

M

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the right bottom side with the EDS maps of Ca, Zn and Fe.

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Table legends

Table 1. Laser ablation inductively coupled plasma-mass spectrometry (LA-ICPMS) data. LA-ICPMS analysis of foraminiferal

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shells and literature data.

Table 2. Best-fit structural parameters from EXAFS data analysis. (*) indicates fixed coordination numbers, constrained

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according to crystallographic models; the standard errors on the refined parameters are in parenthesis, calculated using the

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MINOS commend of MINUT package (Robbins et al., 2016).

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Hyaline shells

Porcelanaceous shells

Location

Description

References

Zn/Ca (µmol/mol)

-

from 1000 to 1800

PortovesmePortoscuso

Industrial area (2 m depth)

Cherchi et al., 2009

Zn/Ca (µmol/mol)

-

from 465 to 5217

Santa Gilla lagoon

Near industrial complex and industrial port

Frontalini et al., 2009

Zn/Ca (µmol/mol)

~ 1000*

-

Harbour of Naples

Commercial and tourist port

Rumolo et al., 2009

87

271

La Maddalena

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Table 1.

Close to an harbour

This study

Zn/Ca (µmol/mol)

37

60

Bonifacio

Non-industrially-polluted area

This study

Zn/Ca (µmol/mol)

-

38

Capitana

from 1.22 to 5.55

-

World's deep ocean Deep ocean (>1500 m depth)

Marchitto et al., 2000

from 0.76 to 2.26

-

North Atlantic Ocean

Deep ocean

Yu et al., 2005

37.3

3

Lamont-Doherty South Atlantic core V22174

Boyle, 1981

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Polluted sites

Zn/Ca (µmol/mol)

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Slightly polluted sites

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Zn/Ca (µmol/mol)

Non-industrially-polluted area

M

Zn/Ca (µmol/mol) Zn/Ca (µmol/mol)

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Low Zn sites

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*from Figure 2, Rumolo et al., 2009. Bonifacio is an unpolluted site geographically neighboring to La Maddalena.

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Cherchi et al., 2009

ACCEPTED MANUSCRIPT Table 2.

Sample

Shell

CN

R(Å)

σ2(x102Å)

ZnO

5.2*

2.013(8)

1.0 (1)

ZnZn

2*

3.13(2)

0.8(2)

ZnZn

5.2*

3.53(1)

0.6(1)

Zn-O

5.4(5)

Zn-Zn

2*

Zn-Zn

5.2*

Zn-O

5.7(6)

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R2=0.05

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Hydrozincite

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Quinqueloculina spp. R2=0.03

2.01(1)

1.1(2)

3.15(3)

1.0 (3)

3.55(2)

1.2(2)

2.01(2)

1.5(3)

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R2=0.03

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Elphidium spp.

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M

CN = coordination number; R = interatomic distance; σ2 =Debye-Waller factor

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Graphics Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9