Introduction
As calcifiers, molluscs play an important role in the ocean carbonate cycle.
The calcium carbonate formed by marine organisms is a complex geochemical
source and sink of carbon, which controls total oceanic carbon content and
pCO2 (partial pressure of CO2) and more generally contributes to
ocean alkalinity (e.g. Gattuso and Hansson, 2011). The biomineralisation
process or, more precisely, the elemental and isotopic composition of biogenic
CaCO3, is affected by physiological processes (e.g. Freitas et al.,
2006; Gillikin et al., 2005b; Lowenstam and Weiner, 1989) and environmental
conditions at the time of deposition (e.g. Ferguson et al., 2011; Gazeau et
al., 2010; Hahn et al., 2014; Heinemann, 2011; Henkes et al., 2013; Lowenstam
and Weiner, 1989; Schöne et al., 2011). Molluscs are globally distributed
and demonstrate sequential growth, thus providing high-resolution seasonal
and sub-seasonal records of environmental conditions (e.g. Jones and
Quitmyer, 1996).
A number of bivalve species are extremely long lived, with lifetimes of many
decades or even centuries, e.g. fresh water pearl mussels (e.g. Schöne et
al., 2004), geoduck clams (e.g. Strom et al., 2004), ocean quahogs (e.g.
Schöne et al., 2005) and giant clams (e.g. Ayling et al., 2006; Bonham,
1965). Many oceanic conditions, such as seawater surface temperature,
productivity, circulation and carbon reservoir dynamics, have been
reconstructed successfully using bivalve shell records (e.g. Klein et al.,
1996; Lazareth et al., 2003; Richardson et al., 2004; Wanamaker et al.,
2011). However, an organism's physiological control on the chemical
composition of its shell can affect the reliability of the parameters
recorded (e.g. Gillikin et al., 2005b; Lazareth et al., 2013, 2003).
Differences between the measured isotopic and the elemental composition in carbonate
materials are typically attributed to a physiological or biological influence
commonly called the “vital effect” (Zeebe et al., 2008). Spero et
al. (1991) first attempted to describe the vital effect through the quantitative
modelling of stable carbon isotopes in foraminifera. The influence of the
vital effect is variable between species, which is problematic for the
development of a unique geochemical equilibrium model for seawater property
reconstructions.
The work of pioneers in the field of molluscan biomineralisation,
Bowerbank (1844) and Carpenter (1845, 1847) amongst others, shifted the focus
from fossil structures to “modern” biogenic CaCO3 structures (such as
coral skeleton and mollusc shells). Influential work by Urey et al. (1951) first used stable oxygen isotope values (δ18O)
in biogenic CaCO3 (belemnite rostrum) as a proxy for seasonal
temperature change. Certain limitations remained, such as separating
δ18Owater (which is linearly correlated to salinity) from
the temperature signals, leading to the development of other proxies
according to their temperature dependency, e.g. Sr / Ca and Mg / Ca
ratios (Gillikin et al., 2005a; Shirai et al., 2014). However, further work
showed that elemental composition is strongly controlled by biological
factors including growth rate (e.g. Gillikin et al., 2005b), calcification rate (e.g. Carré et
al., 2006), ontogenetic age (e.g. Purton et al., 1999), organic matrix (e.g.
Schöne et al., 2010; Takesue et al., 2008) and microstructure (overall
fabrics) (e.g. Schöne et al., 2013; Shirai et al., 2014).
Long-term records of seawater parameters (e.g. temperature, alkalinity and
pH) are essential for understanding past climatic and oceanic changes in the
current context of global climate change. Data from diverse geographical
locations are needed to develop a global understanding of those changes.
Records from bivalve shells have contributed considerably to our knowledge,
but environmental controls need to be well defined and differentiated from
biological and physiological factors (e.g. Jacob et al., 2008).
This study focuses on the microstructure and composition of shells from two
species of bivalves endemic to southwestern Australia to gain insight into the
physio-chemical processes involved in molluscan shell calcification. It
constitutes the first step towards monitoring global climate change through
biogenic carbonate in southwestern Australia. In general, limited geochemical
data are available for temperate Australian marine bivalves and, to our
knowledge, this work is the first multimodal (crystallographic, spectroscopic
and geochemical) study focusing on marine bivalve species from Australian
waters. The structure and mineral composition of shells from the Western
Australian bivalves F. tenuicostata and S. biradiata have
not been previously investigated as a potential proxy for future studies
using this integrated approach. Given the absence of corals along the
southern coast of Western Australia and the south coast of Australia as a
whole, bivalve shells could serve as an alternative proxy for generating
records of historical ocean chemistry provided that the overarching
mechanisms are well defined and understood (e.g. elemental equilibrium and the vital
effect). This study contributes to the global understanding of how marine
bivalves record environmental change through shell growth and the processes
governing marine biogenic carbonate production.
Materials and methods
Study area and marine setting
Samples were collected from the muddy sands (600–800 µm grain
size) of King George Sound at a depth of 3–5 m on the southern coast of
Western Australia near the town of Albany (Fig. 1). This area experiences
seasonal freshwater inputs, a temperate to Mediterranean climate, a mean annual
rainfall of 931.5 ± 0.2 mm (between 1877 and 2014), a maximum of
∼ 126.1 ± 0.2 mm of rainfall in August and a minimum of
∼ 30.1 ± 0.2 mm in December (Australia Government Bureau of
Meteorology; Brouwers et al., 2013; Klausmeyer and Shaw, 2009). The main
currents influencing the area are the Capes Current (coastal current) flowing
westward, the Leeuwin Current (shelf current) flowing eastward and the
Flinders Current (oceanic current) flowing westward (Cresswell and Golding,
1980). Further south, Australian waters meet the cold and nutrient-rich
Antarctic waters. The southwestern coastline of Australia is impacted by
strong winter storms influenced by pressure systems over the Great Australian
Bight, as well as the Leeuwin Current dynamics. Seawater surface temperatures
(SST) vary annually between 16 and 21 ∘C (August–September to
April–May) and bottom temperatures vary between 15 and 20.6 ∘C
(Australian Government Bureau of Meteorology) at 40–50 m of maximum depth.
A map of King George Sound and the surrounding coastline in south Western
Australia.
The sampling site is indicated by the red star.
Species
The study uses two species of bivalves: Fulvia tenuicostata
(Lamarck, 1819) (the “thin-ribbed cockle”) and Soletellina biradiata
(Wood, 1815) (the “double-rayed sunset clam”; Fig. 2). Both species are
native and endemic to southwestern Australia (WoRMS, 2016). F. tenuicostata (Mollusca: Bivalvia: Veneroida: Cardiidae),
shown in Fig. 2a and b, is typically 50–55 mm in
length (anterior to posterior margin). The shells of this species are cream
coloured, becoming yellow towards the margin (the periostracum can show
staining from the surrounding mud). The shells are thin and delicate with
narrow ribs (Fig. 2a and b). This genus is found in moderately sheltered
areas of muddy sand (WoRMS, 2016). The shell structure of F. tenuicostata consists of a prismatic outer layer (OL), a simple
crossed-lamellar middle layer (ML) and a complex crossed-lamellar inner layer
(IL). S. biradiata (Mollusca: Bivalvia: Veneroida: Psammobiidae)
shells, shown in Fig. 2c and d, may measure up to 70 mm. The shells are thin
walled and have an elongated shape. With the thick periostracum removed, the
shells are cream coloured with tinges of pink and purple and two distinctive
pale radial rays extending from the umbo to the growing edge. S. biradiata is also found in moderately sheltered areas with muddy sand
substrates. The shell structure of S. biradiata consists of a
prismatic OL composed of acicular prismatic crystals directed toward the
outer surface and locally curved, a simple crossed-lamellar ML and a complex
crossed-lamellar IL. The OL may be absent, in which case the lamellae of the
ML extend to the outer surface as noted for other species of this subfamily
(Popov, 1986; Schneider and Carter, 2001).
Photographs of the external (left) and internal (right) views of the
bivalves Fulvia tenuicostata (a, b) and Soletellina biradiata (c, d). (e) A photo mosaic showing the ventral margin
(S. biradiata) as seen in a thin section. Powder was sampled from the
ventral margin for X-ray diffraction analysis (the white dashed lines and
arrows in
a, b, c and d). The area adjacent to the ventral margin was then
targeted for confocal Raman microscopy, LA-ICP-MS and EPMA (the red box in
e). The lower panel shows a representation of the laser spots along five
transects (T1–T5). T1–T3 are parallel to the shell edge, and T4 and T5 are
parallel to the external and internal shell margin (respectively).
Sample preparation
Shells (five F. tenuicostata and three S. biradiata) were
rinsed with fresh water, manually cleaned of any organic residues by
scrubbing, then soaked in sodium hypochlorite (12.3 vol%) for approximately 12 h. Shells were
thoroughly rinsed with Milli-Q water (EMD Millipore Corporation, Darmstadt, Germany) and then air dried. According to
Krause-Nehring et al. (2011), the treatment of biogenic calcium carbonate to
remove organic matter may have an impact on the elemental and crystallographic
composition in the bivalve Arctica islandica. The effects of sodium
hypochlorite (NaOCl) pretreatment (also used in our study) on A. islandica shell powder were insignificant compared to other treatments
tested in that study. Indeed, Sr / Ca and Mg / Ca ratios did not
change significantly, and very little to no dissolution or phase change was
observed. NaOCl treatment has also proved to be the most efficient method to remove
organic matter with the least effect on structure and composition in coral
cores (Nategaal et al., 2012). Krause-Nehring et al. (2011) performed their study on powdered,
pre-cleaned shells (manual removal of the periostracum) and found very little
effect. In our study, where intact shells were pretreated with NaOCl, we
suggest that the geochemical and crystallographic composition of the bivalves
studied here was not significantly altered despite having some hypochlorite
solution percolate through.
Shells were sampled along the growing edge using a hand-held dental drill for
X-ray diffraction analysis (Fig. 2). Fifty milligrams of very fine powder was
mixed with ∼ 10 mg of CaF2 (20 % by weight) and mounted on a
low-level background holder according to the settings described in
the Supplement S1. The bivalves were then cut along the axis of maximum growth (from
umbo to ventral margin), fixed in styrene polymer and mounted on a glass
slide with EPO-TEK 301 epoxy glue (Billerica, MA, USA). They were then
trimmed and polished to a thickness of 20–40 µm.
The thin-sectioned bivalves, thus prepared, were used for petrographic
observations, confocal Raman spectroscopy and laser ablation
mass spectrometry work. All the analyses described below targeted the ventral
margin of the shells, which is the youngest material. The growth increments of
F. tenuicostata are about twice those of S. biradiata at 1
and 0.5 mm, respectively. Each measurement was made to encapsulate
approximately the same growth period.
X-ray diffraction analysis
X-ray diffraction (XRD) analysis of the powdered shells was performed using a
PANalytical Empyrean diffractometer (Eindhoven, The Netherlands) operated at 40 kV and 40 mA (Cu
Kα radiation and Ni beta filter) and a PANalytical PIXcel position sensitive
detector. The particular settings are described in the Supplement S1. A corundum NIST
standard (676a; certificate number 382200) was used as a primary external
standard and calcium fluoride (CaF2) was used as a secondary internal
standard. The amount of the secondary standard was tested by analysing different
CaF2 : sample ratios. Since no major differences were observed between
the various ratios tested, a 20 % weight of CaF2 was chosen.
CaF2 (lattice parameter a=b=c=5.4662(2) Å and α=β=γ=90∘) is an ideal internal standard because its
diffraction peaks do not interfere with those of CaCO3.
The mineral phases were identified using the Inorganic Crystal Structure Database
(ICSD) and the International Centre for Diffraction Data (ICDD). The
Rietveld refinement technique was used to quantify the phases. The Scherrer
equation (Cullity and Weymouth, 1957) was used to calculate the coherently scattering domain
(CSD) size (i.e. crystallite size) using the dominant reflections of calcite
(104) and aragonite (111). Both the Rietveld refinement and the Scherrer
calculation were performed using the PANanlytical HighScore Plus software
(version 3.0e). The non-crystalline fraction, or amorphous material, in the
powder was typically calculated from the overestimation of the secondary
standard during the Rietveld refinement (Scarlett and Madsen, 2006).
Confocal Raman microscopy
Confocal Raman microscopy (CRM) uses intense monochromatic light (here, an
infrared laser) to irradiate samples. The subsequent molecular vibrations
produce a highly specific Raman spectrum that reflects the molecular
structure and the chemical identity of the samples. Changes in the intensity and
peak position indicate different vibrational modes of a molecule and
therefore
may reveal subtle differences in the crystalline form, especially between the
different polymorphs of CaCO3 (Nehrke and Nouet, 2011). CaCO3 peaks
measured using CRM are caused by the unit cell symmetry of the crystals and
the molecular carbonate ions (CO32-). Because of their shared
CO32--related vibrational modes, the differentiation between aragonite
and calcite was accomplished using the lower end of the spectrum, 50 to
1200 cm-1. In CRM, a spectroscopy system is coupled with a confocal
microscope, allowing for the mapping of samples with chemical sensitivity.
CRM was undertaken on thin sections of bivalve shells to confirm
XRD-determined mineral phases and to provide key information about the
crystallographic structure of CaCO3. To mitigate the high fluorescence
levels caused by a green excitation laser (wavelength 532 nm), a diode
infrared laser module (wavelength 785 nm) with 20 × objective and a 0.4 numerical aperture was
used, coupled with the Raman imaging system (WITec, Ulm, Germany; alpha300 RA+). CRM
was performed in reflection mode, whereby the scattered light is collected
through a 100 µm detection fibre connected to a UHTS 300
spectrometer equipped with a 300 lines/mm grating. The full Raman spectra at each imaging pixel
were acquired using a camera with a thermoelectrically cooled,
back-illuminated CCD chip. The first-order Raman peak of silicon
(520.2 cm-1) was used to optimise the alignment and signal intensity before
each analysis. Measurements were made as large area scans with the
accumulation of three to five spectra to minimise error (±1 cm-1) and
an integration time of 0.1 to 0.050 s. Two spectral ranges were measured: 0 to
1600 cm-1 and 1600 to 3000 cm-1; the second range was explored in
an attempt to detect organic functional groups (C-H or C=O) within the
crystalline structures. The CRM data were processed and analysed with the WITec
Project FOUR software.
Electron probe microanalysis
Quantitative chemical maps were acquired on petrographic thin sections using
a JEOL 8530F HyperProbe (JEOL Solutions for Innovation, Peabody, MA, USA) equipped with five tuneable wavelength dispersive
spectrometers. The operating conditions for the instrument calibration were comprised of a
40∘ take-off angle, a beam energy of 15 kV and a beam current of 20 nA.
The beam was defocussed to 2 µm. The elements were acquired using
analysing crystals: PET (pentaerythritol) for Ca Kα, S Kα
and Sr lα; TAP (thallium acid phthalate) for Na Kα and Mg
Kα. The standards used for the instrument calibration were barite for S
Kα, calcite for Ca Kα, celestite for Sr lα, periclase
for Mg Kα and jadeite for Na Kα. Peak counting times were
20 s and mean atomic number background corrections were used throughout
(Donovan and Tingle, 1996). The results are the average of three points and the detection
limits ranged from 0.008 wt % for S Kα to 0.010 wt % for Mg
Kα to 0.049 wt % for Sr lα. The Armstrong–Love/Scott
algorithm was used for data reduction (Armstrong, 1988). Quantitative element
maps were obtained using the Probe Image®
software (Probe Software, Inc., Eugene, OR, USA) for X-ray intensity acquisition. The beam current was 80 nA with a
40 ms per pixel dwell time and a 4 × 4 µm pixel
dimension. Image processing and quantification was performed off-line with
the CalcImage® software (Probe Software, Inc.) and output to
Surfer® (Golden Software, LLC, Golden, CO, USA).
The lattice parameters a, b and c (Å) of aragonite after
Rietveld refinement and other reference materials for comparison.
Perna canaliculus: marine bivalve, Acanthocardia tuberculata: marine bivalve and Strombus decorus persicus: marine
gastropod.
Origin
a
b
c
This study
G156
biogenic: Fulvia tenuicostata
5.7471
4.9634
7.9673
G158
biogenic: Fulvia tenuicostata
5.7481
4.9634
7.9678
G159
biogenic: Fulvia tenuicostata
5.7484
4.9624
7.9712
G160
biogenic: Fulvia tenuicostata
5.7480
4.9632
7.9692
G170
biogenic: Soletellina biradiata
5.7413
4.9580
7.9545
G171
biogenic: Soletellina biradiata
5.7519
4.9660
7.9629
G173
biogenic: Soletellina biradiata
5.7514
4.9602
7.9686
Pokroy et al. (2007)a
ICSD-98-015-7993
mineral: Morocco
5.7420
4.9630
7.9680
JCPDS-41-1475
mineral
5.7439
4.9623
7.9680
ICSD-98-015-7994
biogenic: P. canaliculus
5.7520
4.9670
7.9640
ICSD-98-015-7992
biogenic: A. tuberculata
5.7480
4.9650
7.9640
ICSD-98-015-7995
biogenic: S. decorus persicus
5.7530
4.9690
7.9590
ICSD-98-015-7996
biogenic: S. decorus persicus (bleached)
5.7430
4.9630
7.9640
ICSD-98-015-7997
biogenic: S. decorus persicus (annealed)
5.7530
4.9690
7.9610
De Villiers (1967)b
ICSD-98-001-5194
mineral: Nevada
5.7400
4.9670
7.9670
Bragg (1925)c
ICSD-98-005-6090
mineral: Hungary
5.7300
4.9500
7.9550
External standard
NIST 676a alumina
synthetic powder
4.7593
4.7593
12.992
Internal standard
CaF2
synthetic powder
5.4664
5.4664
5.4664
a JCPDS: data file 41-1475, Joint Committee on Powder Diffraction
Standards. Keller, L., Rask, J. and Buseck, P., 1989. JCPDS card no. 41-1475,
Arizona State Univ., Tempe, AZ, USA., ICDD Grant-in-Aid. b XRD from
single crystal CaCO3, Nevada. c XRD from single crystal CaCO3,
Hungary.
The X-ray diffraction patterns of specimens of Fulvia tenuicostata (a) and Soletellina biradiata (b).
Each XRD pattern is offset from the next (along the y axis) to show the
consistency of the diffraction peak positions. The sample ID is indicated to the
right of each diffraction pattern. Intensity is relative. Note: G154, G157
and G172 were not analysed using XRD because the sample weight was too low.
The main diffraction peaks indicated for each phase identified aragonite,
calcite and calcium fluorite CaF2.
Laser ablation ICP-MS
The analysis of carbonate material was undertaken using a Resonetics RESOlution
M-50A-LR (Australian Scientific Instruments, Fyshwick, Australia) incorporating a Compex 102 excimer laser (Coherent Inc., Santa Clara, CA, USA) coupled with an
Agilent (Santa Clara, CA, USA)
7700s quadrupole ICP-MS on shell material that had already been thin
sectioned. Following two cleaning pulses and a 20 s period of background
analysis, samples were spot ablated for 30 s at a 7 Hz repetition rate
using a 75 µm beam and a laser energy of 5 J cm-2. Oxide
polyatomic interferences were minimised by tuning flow rates for a
ThO / Th ratio of < 0.5 %. The sample cell was flushed with ultra-high-purity He (350 mL min-1) and N2 (3.8 mL min-1), and
high-purity Ar was employed as the plasma carrier gas. The international glass
standard NIST 612 and the coral standard MACS-3 were used as the primary
reference materials to calculate the elemental concentrations (using
stoichiometric 43Ca as the internal standard element) and to correct for
instrument drift on all elements. Standard blocks were run after every 20th
unknown sample. The mass spectra were reduced using iolite (Armstrong, 1988).
Data were collected on a total of 16 elements measured as 7Li,
24Mg, 28Si, 31P, 34S, 44Ca, 52Cr, 55Mn,
56Fe, 63Cu, 66Zn, 75As, 88Sr, 111Cd, 138Ba
and 208Pb. The Si concentration was used to determine if or when the laser hit
the glass slide. The measurements taken when the laser hit the glass were
discarded.
The aragonite lattice distortion presented as Δa/a, Δb/b
and Δc/c compared to the mineral standard (Pokroy et al.,
2006) for four specimens of
F. tenuicostata and three of S. biradiata. The y axis
indicates the amount of distortion compared to ICSD-98-015-7993 mentioned
above. Negative distortions are indicative of shrinking, and positive distortions
are indicative of stretching.
Trace element concentration profiles were measured in four shells of
F. tenuicostata and two of S. biradiata. Five transects
were measured on each shell: three parallel transects of seven to eight
spots (T1–T3) oblique to the shell surface, one transect along the outer
portion of the outer shell layer (T4; 10 spots) and one along the inner
portion of the outer shell surface (T5; 10 spots; Fig. 2). Transects T1–T3
were measured to assess the elemental variation towards the inner shell surface
and the potential thickness of the last growth layer deposited. T4 and T5
were measured to compare trace element concentrations in the innermost layer
of the shell to the outermost layer.
Results
Shell mineral composition
The XRD patterns indicate that the shells are composed of calcite and aragonite
based on matching diffraction peaks (Fig. 3). The third identifiable
diffraction pattern represents the internal standard, CaF2 (20 %
weight of the total sample). The Rietveld refinement was applied in order to
obtain an
accurate phase quantification for each sample; however, the values produced
by the software corresponding to the weight ratio of CaF2 added were
lower than the actual amount. Consequently, the results are not
considered useful. Quantification errors in XRD are commonly caused by
residual moisture, large organic content, amorphous crystallites or, in this
case, errors due to small sample sizes and insufficient particle size
reduction (Scarlett and Madsen, 2006). In the present case, moisture, organic
material and amorphous crystals could all cause an overestimation (Scarlett
and Madsen, 2006), but the underestimation was likely caused by
the small sample size (∼ 50 mg). Accurate quantification on small
sample sizes can be obtained by using synchrotron technology. Although the
phases cannot be quantified, the lattice parameters and crystallite sizes
(i.e. the coherently scattering domain or CSD) produced post-refinement and
after the Scherrer calculation can be used because of the addition of the
pre-calibrated CaF2 standard.
The typical Raman spectra from S. biradiata and F. tenuicostata (a). The blue boxes correspond to the filters applied to the
data: filter 1 (Σ peak area) centred at 209 cm-1
(width = 36.6 cm-1), filter 2 (Σ peak area) centred at
282 cm-1 (width = 66.3 cm-1), filter 3 (centre of mass,
weighted width) centred at 1085 cm-1 (width = 36.6 cm-1) and
filter 4 (Σ peak area) centred at 1450 cm-1
(width = 600 cm-1). The blue dashed box, filter 5, encapsulates
filter 1 and a filter centred at 152 cm-1 (Σ peak area,
width = 36.6 cm-1) to represent their calculated peak intensity
ratio. The blue and red spectra (b) show the peak intensity
difference seen at 152 and 209 cm-1. Each filter measures the peak area,
except filter 3, which measures the full width at half maximum and produces a
qualitative map. Filter 1 targeted the aragonitic phase, and filter 2 targeted
calcite. Filter 3, by measuring variation in FWHM, represents crystallinity
and crystallite order. Filter 4, by targeting the tail of the spectrum, shows
fluorescence, since the peaks related to organics tend to show best in the tail of
the spectrum. And finally, the peak intensity ratios (filter 5) show changes
in the crystallite orientation (aragonite crystallites).
All of the samples analysed were indexed to the orthorhombic symmetry system
for aragonite and the hexagonal system for calcite. The lattice parameters
(a,b,c) generated by the Rietveld refinement differ from the geological
reference materials for both calcite and aragonite (Table 1). All samples of
F. tenuicostata manifest expansion in aragonite and calcite along
the a axis (Fig. 4). All samples of S. biradiata, except one
(G170),
show the same phenomenon. In contrast, G170 shows contraction along all axes.
The b axis in both phases and both species also generally shows a positive
distortion (stretching) with a few exceptions (F. tenuicostata,
G159; S. biradiata, G170 and G173; all three in aragonite only). The
calcite c axis shows shrinking in both species. The distortion along the
c axis in aragonite is variable (two out of four F. tenuicostata
show contraction and two out of three for S. biradiata). The largest
distortion was found in calcite, particularly for a and b (+0.12 to
+0.02 %); c only shows small distortions of -0.03 to
-0.004 %. Aragonite is distorted along the a axis between +0.03 and
+0.017 %; the b and c axes manifest smaller distortions of +0.014 and
-0.008 % at a maximum, respectively. The main difference between the two
species considered here lies in the amount of distortion, whereby
F. tenuicostata manifests a smaller distortion compared to
S. biradiata (Fig. 4). The maximum distortion in aragonite is 0.02 %. It is 0.066 % in calcite for F. tenuicostata (both Δa/a)
and 0.03 and ∼ 0.13 %, respectively, in S. biradiata (also
Δa/a). Furthermore, although minimal, F. tenuicostata shows
generalised stretching (0.004 % maximum) along the c axis in aragonite,
whereas S. biradiata shows shrinking (-0.008 % maximum).
The CSD sizes estimated using the Scherrer equation (with K=0.9) for the
four dominant aragonite diffraction peaks corresponding to reflections (111),
(102), (201) and (122) reveal aragonite crystals ranging from ∼ 34 to
∼ 144 nm long (Table 2). The majority of the CSD values fall in the
nanocrystal category (< 100 nm), but some, especially those for the (122)
plane, belong to the larger ultra-fine category (100–500 nm). The CSD sizes
estimated for calcite used one dominant diffraction peak corresponding to
reflection (104) to reveal smaller crystallites than in aragonite (∼ 23
to ∼ 29 nm long).
The aragonite and calcite crystallite size (coherently scattering
domain, or CSD) in nm, estimated using the Scherrer equation (Cullity and Weymouth, 1957)
with K=0.9 on four main diffraction peaks of aragonite and the peak (104) of
calcite.
Sample ID
CSD (nm)
Aragonite
Calcite
peak (hkl)
peak (hkl)
(111)
(102)
(201)
(122)
(104)
F. tenuicostata
G155
68.5
58.8
41.6
105.2
25.5
G156
64.2
69.9
–
58.3
26.2
G158
69.1
64.4
42.3
–
28.3
G159
45.6
50.1
35.6
34.4
29.1
G160
66.3
73.0
–
–
26.2
S. biradiata
G170
69.1
68.1
51.2
–
29.0
G171
111.7
72.3
37.2
143.8
23.3
G173
93.7
93.9
46.8
99.1
24.6
Different phases in the samples were identified using a combination of
lattice modes and internal modes. Calcite and aragonite can be identified
using the librational modes Lc (282 cm-1) and La
(209 cm-1), respectively, and the in-plane band v4 (713 cm-1
for calcite and 702, 706 and 717 cm-1 for aragonite); the shared peaks
(translational mode, T, ∼ 152 cm-1 and symmetry stretch,
v1, 1085 cm-1) represent the CO32- ion motion and the
C-O bond stretching common to both CaCO3 polymorphs (Cuif et al.,
2012, Fig. 5). Although very small and broad compared to aragonite-specific
peaks, only the Lc peak identified
calcite. The most pronounced aragonite peak is La. The scan range
of the spectrometer was increased to 3000 cm-1 in order to detect the
potential presence of organic compounds (e.g. Nehrke and Nouet, 2011);
however, no features characteristic of these groups were found. As a
consequence, the spectra presented here only extend to 2000 cm-1.
Four filters were applied to each Raman spectrum (Fig. 5) to highlight
different phases or reveal particular features of the shell: (1) Σ
peak area of aragonite (centred at 209 cm-1); (2) Σ peak area
of calcite (centred at 282 cm-1); (3) centre of mass and weighted width
(full width at half maximum) of v1 (centred at 1085 cm-1); and
(4) Σ peak area of the tail of the spectrum targeting fluorescence
(centred at 1450 cm-1). Peak intensities at 152 cm-1
(translational mode T) and 209 cm-1 were also ratioed (PIR) to
distinguish the crystal orientation variations (Figs. 5 and 6).
Images extracted from the filters applied to the Raman spectra (see
Fig. 5) from samples G171 (S. biradiata) (a–e) and G155
(F. tenuicostata) (f–j). Scale bar:
7000 µm (a–e) and 1000 µm (f–j). Colour
scale: the bright colours correspond to high intensities (high CCD counts)
and the
dark colours correspond to low intensities (low CCD counts). The sub-horizontal
and curved lines, most visible in (a–c) and (f–h), are
structural features of the shells. Scale bar: 500 µm.
Aragonite and calcite were both found in all samples analysed using CRM. The
small intensity of the calcite peak indicates that it is a minor phase. Two
distinct types of spectra are visible for aragonite (Fig. 5), both with
identical peak positions but different peak intensity ratios (PIR) (Figs. 5,
6e and j) between the translational mode T
and the other peaks characteristic of aragonite (filter 5, Figs. 5 and 6).
The full width at half maximum (FWHM) of the symmetric stretch
v1(CaCO32-) reveals sequential variations or, more precisely,
alternating high and low peak widths, especially in sample G154 (Fig. 6c and h).
The other samples show more discrete changes in FWHM. Filter 4 (the filter
showing fluorescence) reveals clear banding throughout the samples (Fig. 6e and
j).
Elemental composition
The EPMA results show that the total elemental compositions are consistently between 98
and 100 wt % (Figs. 7a and 8a). Calcium is
generally consistent across the samples with an average concentration of
∼ 39.5 wt % (Figs. 8b, 9b). S and Na comprise up to 0.16 and
0.66 wt %, respectively, but importantly show variations in concentration
with opposing trends; i.e. high Na corresponds to low S and vice versa
(Fig. 6). The concentrations of S and Na correspond to growth lines with
overall Na content higher towards the external surface of the shell and S
towards the internal surface. Low S and high Na correspond to higher
fluorescence levels detected using CRM (1450 cm-1 filter; Fig. 6d and
i). These results are consistent for both S. biradiata (Fig. 7) and
F. tenuicostata (Fig. 8). Ca is typically uniformly distributed
throughout each shell; nevertheless, the patterns revealed by CRM correspond
to the
patterns on the Ca distribution maps. The patterns revealed by filters 4 and 5
(crystallinity and crystal orientation, respectively), when applied to the Raman
spectrum, correspond to those visible on the Ca wt % map. Mg and Sr are
below the instrumental detection limit (Table 3) and are not presented here.
The EPMA elemental map of sample G171, S. biradiata, for
totals (a), Ca concentration (b), S
concentration (c) and Na concentration (d). The Mg and Sr
concentrations were below the instrumental detection limit and are therefore not
presented
here. The arrow (a) indicates the direction of growth. Note: this shell
shows a thick edge compared to the adjacent shell section.
The S. biradiata shell thickness is variable along the cross section.
The EPMA detection limits (3σ) on bivalve samples. The Mg and Sr
contents in both S. biradiata and F. tenuicostata were
below the limits of detection (see Figs. 8 and 9).
Element (symbol)
Fulvia tenuicostata
Soletellina biradiata
Sodium (Na)
0.1355
0.17
Magnesium (Mg)
0.102
0.127
Sulfur (S)
0.01
0.085
Calcium (Ca)
0.115
0.1425
Strontium (Sr)
0.47
0.595
The trace element concentration profiles measured using LA-ICP-MS showed that the NIST160
and MACS standard reproducibility is in the range of acceptable values (between
0.5 and 4 % on most elements; Supplement S2). Very little reproducibility
was observed between T1, T2 and T3 (Figs. 9 and 10) for all six shells
tested;
however, compositionally distinct layers were identified, as were differences
between species (Figs. 11 and S3). In F. tenuicostata, the ratios of
Sr / Ca, S / Ca and Pb / Ca along transects T1–T3 generally
increase from the external surface inwards (maximum +1 mmol mol-1,
+3 mmol mol-1 and +0.05 µmol mol-1, respectively;
Supplement S3). Only Sr / Ca in G158 decreases by 2 mmol mol-1
inwards (Supplement S3). Mg / Ca, P / Ca
and Ba / Ca (Figs. 11 and S3) follow the opposite trend, with a maximum
decrease of 1.5 mmol mol-1, 0.06 mmol mol-1 and
3 µmol mol-1, respectively. The elemental ratios in
S. biradiata all decrease along the T1–T3 transects from the outer
to the inner layer (Mg / Ca -0.15 mmol mol-1, P / Ca
-0.04 mmol mol-1, Li / Ca -4 µmol mol-1,
Ba / Ca -2 µmol mol-1), except Pb / Ca
(+0.06 µmol mol-1) and S / Ca, which is relatively
stable at 1.3 mmol mol-1. Sr / Ca increases inwards
(+1.5 mmol mol-1) but decreases to its original level at the edge of
the internal layer (∼ 2 mmol mol-1). Li, Ca, Mn and Ba
concentrations in F. tenuicostata are not linearly correlated to the
other trace elements measured, but a statistically significant (paired t test)
linear correlation was found between [P] / [Sr] (R2=0.70, p<2.2×10-16), [P] / [S] (R2=0.74, p<2.2×10-16),
[P] / [Mg] (R2=0.55, p=1.283×10-7) and [S] / [Mg]
(R2=0.55, p<2.2×10-16). Li, Mg, Ca and Mn in
S. biradiata are not linearly correlated to the other trace elements
measured, but a statistically significant (paired t test) linear
correlation was found between [Sr] / [S] (R2=0.74, p<2.2×10-16), [Sr] / [Pb] (R2=0.60, p<2.2×10-16),
[Sr] / [Ba] (R2=0.92, p<2.2×10-16), [Ba] / [S]
(R2=0.66, p<2.2×10-16) and [Ba] / [Pb] (R2=0.55,
p<2.2×10-16).
The EPMA elemental map of sample G155, F. tenuicostata, for
totals (a), Ca concentration (b), S
concentration (c) and Na concentration (d). Mg and Sr were
below the instrumental detection limit and are therefore not presented. The dashed line
indicates scan overlap, since the sample was scanned twice to optimise
precision and quality. Scale bar: 500 µm. The arrow indicates the
direction of growth.
The location of the laser spots along the five transects. T1–T3 transect
towards the inner shell surface and parallel to each other. T4 transects along the
outer portion of the outer shell layer and T5 along the inner portion of the
outer shell layer. The inner and outer portions of the outer shell layer are
indicated
by “Int.” and “Ext.”, respectively. The shell layers are indicated by OL for the outer
layer,
ML for the middle layer and IL for the internal layer. Note the absence of the outer shell layer
and the inner shell layer in these sections of S. biradiata and
F. tenuicostata, respectively. T4 and T5 were sampled from different
shell layers as labelled.
The LA-ICP-MS results for transects T1, T2 and T3. The blue lines represent
the results for S. biradiata (G171) and the red lines represent the results for
F. tenuicostata (G155). The five laser spots or transects were done on
S.biradiata.
The LA-ICP-MS results for the T4 and T5 transects. The blue lines represent
the results for S. biradiata (G171) and the red lines represent the results for
F. tenuicostata (G155).
Graphs showing the significant linear correlations of the LA-ICP-MS
measurements for F. tenuicostata (Mg / P, Mg / S and
Sr / P) and S. biradiata (Sr / S, Sr / Pb and
Sr / Ba).
T4–T5 measurements confirm the compositional differences between the external and
internal layers (Figs. 12 and S3). Overall, the external layer of the S. biradiata shells studied demonstrates higher ratios of Li / Ca,
Mg / Ca, P / Ca, Sr / Ca and Ba / Ca compared to the internal
layer. Only S / Ca and Pb / Ca do not follow this pattern, where
S / Ca is the same in both layers and Pb / Ca is higher in the
internal layer (Figs. 12 and S3). In comparison, F. tenuicostata
shells show higher ratios of Mg / Ca, Sr / Ca, S / Ca,
Ba / Ca and Pb / Ca in their internal layer compared to their
external layer. P / Ca decreases inwards and Li / Ca is essentially
consistent.
S and P are indicators of organic content, and their presence in the bivalves
reflects a variation in macromolecule content throughout the shells.
F. tenuicostata has a higher S-containing macromolecule content in
the organic matrix of its internal layer, whereas a higher P / Ca ratio in
the outer layer suggests that the organic matrix is richer in P-containing
macromolecules in that section of the shell. The shells of S. biradiata
are visibly more complex. They also show high S / Ca and low P / Ca
towards the innermost layer, but for most of the shell, variable S / Ca
and P / Ca ratios suggest that the external layers are characterised by
alternating P- and S-containing macromolecules in organic components
(Figs. 7–8 and 10–11).
Discussion
Mineralogy and microstructure
Although S. biradiata and F. tenuicostata belong to
subfamilies typically described as aragonitic (Schneider and Carter, 2001),
the XRD analysis and CRM results reveal a dominant aragonitic composition and
a minor calcite fraction. The Rietveld refinement did not allow for the
quantification of the different phases; however, CRM confirmed the presence
of calcite by measuring small intensities of the calcite librational mode
Lc compared to that of aragonite
La. The Raman spectra of S. biradiata have a slightly
more intense calcite peak at 282 cm-1 in comparison to
F. tenuicostata, and the XRD pattern shows that the main calcite peak in
F. tenuicostata is generally sharper and offset towards
30∘ 2θ compared to that of S. biradiata. According to Schroeder
et al. (1969), this peak shift is indicative of the presence of Mg in
carbonate, which suggests that some Mg2+ ions have beeb substituted for
Ca2+ in the CaCO3 crystal lattice in F. tenuicostata.
Considering the preferential incorporation of Mg2+ into calcite as
opposed to aragonite (Mucci and Morse, 1982), we conclude that although both
species appear to have similar amounts of calcite incorporated within their
aragonitic shell, in F. tenuicostata, Ca2+ ions have been
partially substituted by Mg2+ in the calcite lattice. This is not the
case in S. biradiata. Sample G170 (S. biradiata), which
shows shrinking in its aragonite phase according to the XRD results, has a
negative peak shift (CRM) indicative of Sr2+ substitution in aragonite.
The LA-ICP-MS analysis of G170 was not undertaken because the thin section
was too cracked towards the ventral margin; therefore, the higher Sr / Ca
in G170 is assumed from the distortion measurements and CRM.
Aragonite is present in two different orientations in most of the F. tenuicostata samples. The combination of these two orientations (e.g.
Fig. 6) clearly corresponds to the crossed-lamellar microstructure
characteristic of many bivalve shells. The microstructure of S. biradiata shells consists of a prismatic outer shell layer, a
crossed-lamellar middle layer and a complex crossed-lamellar inner layer (not
present in the portion of the shell in Fig. 6). Both species precipitate
their shells differently from a structural point of view. The precipitation
of microstructural units in a crossed-lamellar pattern is complex. The
structural complexity of F. tenuicostata, with its crossed-lamellar
pattern associated with marked ribs, suggests a higher level of biological
control on CaCO3 precipitation, which may influence the overall
elemental composition of the shell. For example, Paquette and Reeder (1995)
suggested that crystal surface structure has an effect on trace element
incorporation. As such, the different microstructures present in the two
species studied here may be expected to yield different elemental
compositions.
The measured lattice parameters reveal lattice distortion in both F. tenuicostata and S. biradiata compared to mineral CaCO3.
Pokroy et al. (2006) also reported lattice distortions (Perna canaliculus, Acanthocardia tuberculata and Strombus decorus). After lattice parameter permutation, stretching was found along
the a and b axes in both species, and shrinking was found along the
c axis in S. biradiata but not in F. tenuicostata.
The distortion was not as pronounced in S. biradiatata and F. tenuicostata compared to the species studied by Pokroy et al. (2006), which
can be explained by the use of annealing to relax the CaCO3 structure.
The maximum anisotropic distortion in aragonite is Δa/a 1.1×10-3, Δb/b 2.5×10-4 and Δc/c 3×10-4 for F. tenuicostata, and Δa/a 1.7×10-3,
Δb/b 7×10-4 and Δc/c -6×10-4 for
S. biradiata. According to Pokroy et al. (2006), the
organic molecules could be the source of these structural distortions.
However, given that the F. tenuicostata lattice is more stretched than that
of S. biradiata, the substitution of Ca2+ ions by Mg2+ ions
described previously could also be a source of distortions. Ion substitution,
which has been measured here using XRD, could also be a cause of this
distortion because crystal structures accommodate trace elements through dilation
or contraction of the ionic site (structural relaxation), which is a factor
in the flexibility and stability of the surrounding structure (Finch and Allison,
2007; Loste et al., 2003).
Chemical composition
The LA-ICP-MS results show that Mg is positively correlated to both S and P,
as is
Sr to P in F. tenuicostata. These Mg correlations are not evident
for S. biradiata, but Sr is positively correlated to S, Ba and Pb
(Fig. 12), as is Ba to S in this latter species.
The strong correlations found in the present study seem to indicate that Mg
incorporation is influenced by both phosphorus- and sulfur-containing
macromolecules, i.e. both soluble and insoluble fractions of the organic
shell matrix in F. tenuicostata but not in S. biradiata.
The incorporation of Sr appears to be influenced by the insoluble fraction of
the organic matrix in S. biradiata and the soluble fraction in
F. tenuicostata. The correlation found between Ba and S also
suggests that the insoluble organic matrix influences the incorporation of Ba
in S. biradiata. Shirai et al. (2014) suggested that the incorporation
of Sr is highly biologically mediated based on the strong correlation between
Sr / Ca, S / Ca and the shell microstructure and that the elemental
composition varies at a scale comparable to that of crystallites, i.e. the
micrometre scale. The relatively coarse resolution used for LA-ICP-MS
(75 µm) does not allow us to verify the spatial scale
(< 10 µm) at which the elemental composition of F. tenuicostata and S. biradiata varies, but we can differentiate
between the types of organic macromolecules influencing the shell geochemical
composition, i.e. S-dominated or P-dominated. Our findings agree with those of
Shirai et al. (2014) in that changes in the microstructure and the S / Ca ratio
reflect changes in the organic composition at the mineralisation front. Our
findings further show that P / Ca also reflects compositional changes at
the mineralisation front. Consequently, we can say that not only do the
organic matrices at the calcification front impact the chemical composition
of the shell, but its composition also impacts the incorporation of trace
elements in the shell, regardless of whether it is S- or P-dominated. This
result is consistent with a number of previous studies showing that
S-containing and P-containing macromolecules of the shell matrix, insoluble
and soluble, respectively, influence nucleation, growth and crystal structure
(e.g. Borbas et al., 1991; Crenshaw, 1982; Reddy, 1977; Wheeler and Sikes,
1984). In the context of geochemical proxies, it becomes evident that the
combined effects of the shell organic matrix composition and the environmental
parameters (e.g. SST) may cause variation in the Mg / Ca and Sr / Ca
ratios, two of the most common proxies used for reconstructing paleo-SST.
Additional complexity is also evident. P and S concentrations are consistent
(compare the transects of T1, T2 and T3) in F. tenuicostata but highly
variable in S. biradiata. In the latter, low Mg corresponds to high
P and S, and high Sr to high S and low P. This suggests that the
incorporation of these elements within the shell of F. tenuicostata
is predominantly influenced by seawater, particularly Mg. In
S. biradiata, it is predominantly influenced by the organic matrix
composition, as demonstrated by the strong correlations in the elements related to the organic
matrix. The fact that S. biradiata is visibly richer
in the organic matrix and owes its strength to its organic fraction, whereas
F. tenuicostata shells have a thicker and more complex structure
(e.g. ridges), might be one explanation for this finding. Another remarkable
point of comparison that might help in understanding the differences between the
two species of interest lies in their taxonomy. Indeed, both species belong
to the Heterodonta subclass and the Cardiida order, but they belong to different
superfamilies: Cardioidea and Tellinoidea for F. tenuicostata and
S. biradiata, respectively (Ponder and Lindberg, 2008). Different
organic content and shell organic matrix composition might be explained by
evolved differences in the mineralisation processes (Cuif et al., 2011). However,
without quantifying organic matrix components (total, soluble and insoluble)
and identifying its composition, this remains speculative.
These results not only reinforce the knowledge that the shell organic matrix
of bivalves is involved in biomineralisation, but also that its level of
influence depends on its composition (phosphorus-containing or
sulfur-containing macromolecules) and the specific element being
incorporated into the CaCO3. Furthermore, these results show that this
phenomenon is species specific. The incorporation of the main elements (e.g.
Mg, Sr and Ba) used as proxies from marine properties (e.g. SST) is highly
mediated by the shell organic matrix in general.
Conclusions
The combination of geochemical, spectroscopic and crystallographic analyses
on specimens of F. tenuicostata and S. biradiata from King
George Sound (south Western Australia) has revealed compositional and
microstructural differences. Although the specimens were sampled from the
same location and experienced the same marine conditions during their growth,
they display different characteristics. Both species have been described as
purely aragonitic, but low levels of calcite and Mg-calcite are present in
S. biradiata and F. tenuicostata, respectively. The LA-ICP-MS
measurements revealed correlations between levels of Mg, S and P (F. tenuicostata) and Sr, S and Ba (S. biradiata). Although analysing
the composition of the shell organic matrix was beyond the scope of this
study, the organic matrices of both bivalve species differ in composition and
consequently in the way trace elements are incorporated into the CaCO3.
The incorporation of Sr and Ba is affected by the S-containing matrix of
S. biradiata, and the incorporation of Mg and Sr in F. tenuicostata is more influenced by S- and P-containing organic
macromolecules. The substitution of Ca2+ by Mg2+ in calcite in
F. tenuicostata appears to be influenced by the composition of the
organic matrix. The combination of S- and P-containing organics could be
promoting ionic substitution in the shells of F. tenuicostata.
The differences between these two species are clear and raise questions about
genetic determinism, considering they belong to different taxonomic
superfamilies. This has consequences for environmental proxy applications.
The present study shows that the elemental composition of marine bivalve
shells is species specific and influenced by multiple factors, such as
crystallographic structures, organic macromolecule composition and
environmental setting. These factors complicate the use of bivalve shells as
a proxy for reconstructing the past and present properties of seawater.
Mg / Ca and Sr / Ca ratios are amongst the most common proxies used
for SST reconstructions; however, this study shows that the elemental changes are not
solely caused by SST variation. The high definition multimodal and multidisciplinary
characterisation of both the organic composition and the microstructure are central to
understanding calcification and elemental incorporation. Only then can
geochemical proxies be used to reconstruct true time series and the “vital
effect” accurately described.