1Scientific RepoRts | 5:17793 | DOI: 10.1038/srep17793
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Effects of sample storage and shell 
orientation on LA-ICPMS trace 
element measurements on deep-
sea mussels
Luciana Génio1, Klaus Simon2, Steffen Kiel3,4 & Marina R. Cunha1
Geochemical markers are being increasingly applied to fundamental questions in population and 
community ecology in marine habitats because they allow inferences on individuals dispersal, but 
vital effects, small sample size and instrumental limitation are still challenging particularly in deep-
sea studies. Here we use shells of the deep-sea bivalve Idas modiolaeformis to assess potential effects 
of sample storage, mineralogy, and valve orientation on LA-ICPMS measurements. Trace element 
concentrations of 24Mg, 43Ca, 88Sr, 137Ba, 208Pb, and 238U are not affected by the two most commonly 
used storage methods of biologic deep-sea samples (frozen at –20°C and fixed in 95% ethanol); thus 
combined analysis of differently preserved specimens is possible when the number of individuals 
is insufficient and distinct sample fixation is needed for multiple purposes. Valve orientation had a 
strong impact on quantification of trace elements in the calcitic but not in the aragonitic layer of 
adult shells. Hence, to enable comparisons between adult shells and entirely aragonitic embryonic 
shells, a reference map of site-specific signatures can potentially be generated using the aragonitic 
layer of the adult shells. Understanding ontogenetic changes and environmental effects in trace 
element incorporation is critical before geochemical fingerprinting can be used as a tool for larval 
dispersal studies in the deep-sea.
The trace element and stable isotope compositions of biogenic carbonates formed by marine organisms 
are widely used to understand ocean biogeochemistry, climate history, ocean acidification, and anthro-
pogenic pollution, among a wide range of other topics1–7. A promising recent development are ‘natural 
tag’ or ‘fingerprinting’ studies to investigate dispersal pathways of marine larvae, contributing to the 
understanding of population connectivity patterns that are fundamental to biodiversity conservation 
and marine spatial management8,9. Tracing marine larval dispersal is challenging due to the small size of 
the larvae and the vastness of the ocean. The fingerprinting approach takes advantage of calcified struc-
tures, such as fish otoliths or bivalve shells that preserve the local geochemical signatures throughout 
the life cycle of the organism8. Bivalves form a calcified embryonic shell at or very close to their natal 
origin, then undergo a larval dispersal phase, and finally secrete a calcified shell as benthic adults, while 
retaining their embryonic and larval shells in the apical regions of the juvenile and adult shells. The trace 
element composition of the embryonic shell thus provides a ‘natural tag’ that allows inferences about the 
natal origin and hence larval dispersal distance of individual specimens8.
1Departamento de Biologia & CESAM – Centro de Estudos do Ambiente e do Mar, Universidade de Aveiro, 3810-
193 Aveiro, Portugal. 2Geoscience Center, Department of Geochemistry, University of Göttingen, Goldschmidtstr. 
5, 37077 Göttingen, Germany. 3Geoscience Center, Geobiology Group, University of Göttingen, Goldschmidtstr. 
3, 37077 Göttingen, Germany. 4Department of Palaeobiology, Naturhistoriska riksmuseet, Box 50007, 10405 
Stockholm, Sweden. Correspondence and requests for materials should be addressed to L.G. (email: luciana.
genio@gmail.com)
received: 19 May 2015
accepted: 26 October 2015
Published: 08 December 2015
OPEN
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2Scientific RepoRts | 5:17793 | DOI: 10.1038/srep17793
Challenges of geochemical marker applications include different biological effects on trace element 
incorporation between embryonic and adult shells10,11 as well as biases induced by preservation and stor-
age methods12–15. In this study, we explore the applicability of the elemental fingerprinting approach using 
chemosymbiotic deep-sea mussels (Bivalvia, Mytilidae, Bathymodiolinae). Similar to their shallow-water 
relatives, which are abundant and widely distributed ecosystem engineers within intertidal rocky shores, 
bathymodiolins are foundation species in deep-sea chemosynthesis-based communities worldwide16,17.
Like most mytilid mussels, bathymodiolins have two early ontogenetic phases; first a non-feeding 
embryo that secretes a D-shaped shell of 40–80 microns width (often called prodissoconch I, or PD1), 
which then develops into a plankton-feeding pelagic veliger larva that secretes an up to 500 micron 
wide prodissoconch II shell (PD2)18. After settlement, the mussel becomes sessile and forms the adult 
shell (dissoconch). While the prodissoconchs I and II are composed entirely of aragonite, the adult 
shell is bimineralic, consisting of a calcitic outer fibrous prismatic layer and a nacreous aragonitic inner 
layer19–21. Because there are differences in the incorporation of trace elements into the crystal lattices of 
aragonite and calcite22,23, and therefore ontogenetic differences between larval and adult mussel shells, 
Becker et al.24 pointed to the limited use of adult shells to generate a reference map of site-specific sig-
natures and assign natal origin of mussel larvae. Yet, distinguishing trace element signatures between 
mytilid shell layers has been overlooked, and previous studies focused on comparisons either between 
early larval shells or between post-settled juveniles and adults25–27.
Here we analyse the geochemical signatures of embryonic and adult shells of Idas modiolaeformis 
collected from a sunken wood log at the Gorringe Bank (NE Atlantic) with an overall focus on methodo-
logical issues. Our specific aims are (i) to assess the effect of sample orientation on the measured element 
concentrations of adult shell layers, (ii) to investigate potential biases induced by sample storage (frozen 
vs. ethanol), and (iii) to assess the potentiality of this species for larval tracking studies.
Materials and Methods
Specimen collection and preparation. The deep-sea mussel species Idas modiolaeformis was the 
dominant coloniser of a sunken wood log collected with the ROV Hercules (dive H1202, 36°38.57 N, 
11°36.19 W, 1296 m depth, 14/10/2011) during E/V Nautilus cruise NAO17 at the NW side of Gettysburg 
Seamount, Gorringe Bank (NE Atlantic). Onboard, the wooden log was divided into four segments, two 
of which were kept inside aseptic plastic bags at − 20 °C, and the other two were transferred to plastic 
containers filled with 95% ethanol. The samples (i.e. wooden logs and attached macrofauna) were kept in 
these conditions for a period of eight months. Prior to analyses, mussels were removed from the wood 
surface of both ethanol-preserved and frozen samples, photographed and measured under a binocular 
microscope. Valves were split open with a ceramic-coated razor blade and soft tissues were removed and 
kept for later molecular identification. Valves were transferred to Teflon beakers and immersed in 15% 
H2O2 (Rotipuran; P-LAB a.s.) buffered with 0.05 mol.L−1 NaOH (Tritipur; Merck Millipore) for approxi-
mately 10h to remove organic matter from the shell, including the periostracum. Valves were then rinsed 
three times in Milli-Q water and mounted on a petrographic slide against double sided tape using a 
wet paintbrush and Milli-Q water. Mounted valves were dried overnight in a C-100 laminar flow hood.
Elemental analysis of mussel shells. In order to assess the concentration and distribution of trace 
elements in the mussel shell and the potential effects of sample orientation, measurements were con-
ducted on inversely mounted valves of an adult mussel specimen (Fig. 1): the right valve was oriented 
with the outer surface facing upwards (so that the calcitic layer was analysed first), and the left valve 
inverted, with the inner surface upwards (so that the aragonitic layer was analysed first). On each valve, 
measurements were taken at nine spots along the entire length of the dissoconch shell (1.8 mm), and the 
shells were perforated (and analysed) through their entire thickness. For the assessment of the storage 
method effect, we compared the geochemical signatures of 10 ethanol-preserved (mean shell length 
3.36 ± 0.71 mm) and 10 frozen specimens (mean shell length 3.41 ± 0.64 mm). Valves were all mounted 
with the inner surface upwards, which also exposes the outer surface of the prodissoconch (PD1). 
Measurements were made on the PD1 (one spot) and dissoconch (three spots).
Continuous ablation of shell material was performed with a 3 Hz pulsed ArF-Excimer Laser (Compex 
110, Lambda Physik, Göttingen, Germany) with 193 nm wavelength (5 J cm-2 energy, spot size 40 μ m). 
The ablated material was transported via an Ar-flow into the inductively coupled plasma (ICP) ion source 
of a quadrupole mass spectrometer (Perkin Elmer Elan DRC II, Canada). Eleven isotopes (24Mg, 27Al, 
29Si, 43Ca, 55Mn, 57Fe, 66Zn, 88Sr, 137Ba, 208Pb, 238U) were analysed for 10 ms each resulting in a complete 
mass spectrum of 1-second time; 200 spectra were recorded. The measured intensities were transformed 
into concentrations by applying an external calibration with NBS610 (NIST, USA) reference material, and 
an internal standardization using 43Ca as the internal standard isotope. The adhesive tape and the slide 
were analysed without mussel samples to assess the risk of contamination by the tape or slide material 
that might be ablated and included in the analysis.
Statistical analysis and data processing. Mass spectra were visualized with Iolite v2.1528, a 
non-commercial freeware solution implemented as a self-contained package running in the Igor Pro 
v6.34A (Wavemetrics Incorporated, Portland, Oregon, USA) environment. Concentration values were 
obtained by integrating the results in defined time intervals. On each dissoconch spot profile, two 
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3Scientific RepoRts | 5:17793 | DOI: 10.1038/srep17793
integration intervals were defined based on the switching intensities of Mg and Sr, corresponding to the 
transition between the calcitic and the aragonitic shell layers; namely, higher Mg and lower Sr quan-
tities for calcite, and vice-versa for aragonite (Fig.  2). One integration interval (20–70 seconds) was 
obtained from each PD1 isotope spectrum. Box and whiskers plots for each element ratio (X: 43Ca) 
Figure 1. Idas modiolaeformis shell valves mounted with inversed orientation. (A) Rv- outer surface 
facing upwards, and (B) Lv-inner surface facing upwards.
Figure 2. Mass spectra of six elements from Idas modiolaeformis shell valves with inversed orientation, (A) 
Rv-calcite facing upwards, and (B) Lv-aragonite facing upwards, showing integration intervals corresponding 
to calcitic (dc) and the aragonitic (da) shell layers.
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4Scientific RepoRts | 5:17793 | DOI: 10.1038/srep17793
were generated using the software GraphPad Prism v6.0 (GraphPad Software Inc., San Diego, California, 
USA). Comparisons of multi-element signatures between mineral layers (calcite and aragonite) of the 
two valves, and between storage methods (frozen vs ethanol) of the prodissoconch and dissoconch 
shells, were analysed with principal coordinates ordination (PCO) followed by permutational multivari-
ate analysis of variance (PERMANOVA), with “mineral layer”, “valve orientation”, “storage method” and 
ontogenetic “shell stage” as fixed factors, each with two levels. When significant differences were found 
(P(perm) < 0.05, number of permutations = 9999), a posteriori pairwise comparisons were also exam-
ined (within “valve orientation”, between “mineral layers”; between “valve orientation”, within “mineral 
layers”; between “valve orientation”, between “mineral layers”). Multivariate (all elements) and univariate 
(single elements) analyses were conducted on normalized data and resemblance matrices were calculated 
based on Euclidean distance, using PRIMER v6.1.1329 with the add-on PERMANOVA+ 30.
Results and Discussion
Effect of shell orientation. Out of the eleven analysed isotopes, six (24Mg, 43Ca, 88Sr, 137Ba, 208Pb, and 
238U) provided reliable measurements in the dissoconch shell of the deep-sea mussel Idas modiolaeformis. 
Two elements, 27Al and 57Fe gave clearly erroneous readings (e.g. negative values) and were eliminated 
from the analysis31. Because the amounts of 66Zn, 55Mn and 29Si in the tape and the slide were twice as 
high as in the shell samples, these isotopes were also removed from further analyses. Figure 3 compares 
the concentration values of the elements in the calcitic and aragonitic layers of two valves in relation 
to “valve orientation”. When the shell was oriented with the outer shell upwards (calcite ablated first, 
Fig. 2A) the trace element compositions of the calcitic and aragonitic layers were significantly different 
(Table 1). The calcitic layer is significantly enriched in Mg and Pb and depleted in Sr, U and Ba when 
compared to the aragonitic layer (Fig. 4). However, in the valve with the inner shell upwards (aragonite 
ablated first, Fig. 2B), element signatures of the calcitic and aragonitic layers could not be distinguished, 
except for Mg concentration. In this case, measurements in the calcitic layer differed with “valve orien-
tation”, often significantly, while measurements in the aragonitic layer were similar irrespective of “valve 
orientation” (Fig. 3, Table 1).
Distinguishing calcitic and aragonitic layers in mytilid adult shells is crucial when trace element data 
are being compared between adult and embryonic or larval shells because the latter are entirely arag-
onitic. Our study indicates that bias can potentially be induced by the orientation of the shell during 
Figure 3. Element concentrations (X:Ca, parts per million) in calcitic (dc, black diagonal strikes) and 
aragonitic (da, grey brick pattern) layers of the dissoconch shell, on inversely mounted valves: Rv-calcite 
facing upwards, Lv-aragonite facing upwards. Bottom left: sketch showing the different shell layers in 
different shell orientations. Vertical lines: median value; boxes: interquartile range (IQR); whiskers: values 
< 1.5 × IQR. Asterisks denote outliers (> 1.5 × IQR).
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5Scientific RepoRts | 5:17793 | DOI: 10.1038/srep17793
measurements. In agreement with previous studies22,23, magnesium concentrations were always higher in 
the calcitic layer, independent of shell orientation, thus allowing easy distinction of the two mineralogies 
in the mass spectra obtained by LA-ICPMS. However, significant differences in the concentrations of all 
other trace elements were detected when the calcitic (outer) layer was ablated and measured first, but not 
when the nacreous (inner) layer was measured first. This suggests that the ablation of aragonitic nacreous 
layer caused contamination on measurements of the calcitic layer, while there was little or no contami-
nation from the ablation of calcite on measurements of the aragonitic layer. Further work is needed to 
clarify if this is due to the different mineralogies of the two shell layers, but this may be explained by the 
particular ultrastructure of nacre. Nacre is composed of stacks of minute (ca. 1 × 10 × 10 micron) plates 
enveloped in an organic matrix32. These tiny plates, or fragments of them, may be more easily eroded 
during ablation, thus contaminating subsequent measurements of the calcitic layer. On the other hand, 
the more densely packed fibrous prisms composing the calcitic layer are unlikely to contaminate subse-
quent ablation of the nacre. As a result, the magnesium spectrum is a reliable indicator of mineralogy 
when selecting the intervals for measurement integration, but quantification of other elements of distinct 
mineral layers must take into account the orientation of the sample.
All Elements Mg:Ca Sr:Ca Pb:Ca U:Ca Ba:Ca
Within “valve orientation”, between “mineral layers”
Rv_da, 
Rv_dc 6.404**** 7.537**** 8.433**** 4.779**** 8.792**** 2.118*
Lv_da, 
Lv_dc 1.363
ns 4.676*** 0.650ns 1.948ns 0.359ns 0.993ns
Between “valve orientation”, within “mineral layers”
Rv_da, 
Lv_da 0.520
ns 2.599* 0.304ns 0.473ns 0.407ns 0.053ns
Rv_dc, 
Lv_dc 3.737**** 5.651**** 8.332**** 1.167
ns 8.184**** 1.884*
Between “valve orientation”, between “mineral layers”
Rv_dc, 
Lv_da 5.078**** 8.662**** 8.152**** 3.410** 4.423**** 1.594
ns
Rv_da, 
Lv_dc 1.608* 2.671* 0.357
ns 2.769** 0.0800ns 1.095ns
Table 1.  Multivariate and univariate results of PERMANOVA a posteriori pairwise comparisons of 
element concentrations between shell “mineral layers” (da - dissoconch aragonite, dc – dissoconch 
calcite) in relation to “valve orientation” (Rv - calcite facing upwards (outer surface), Lv – aragonite 
facing upwards (inner surface), refer to sketch on Fig. 3). ****P(perm) < 0.0001; ***P(perm) < 0.001; 
**P(perm) < 0.01; *P(perm) < 0.05; ns: not significant.
Figure 4. Principal coordinate (PCO) analysis of multi-element signatures in calcitic (dc) and aragonitic 
(da) layers of the dissoconch shell, on inversely mounted valves: RV-calcite facing upwards, LV-aragonite 
facing upwards (see sketch in Fig. 2).
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6Scientific RepoRts | 5:17793 | DOI: 10.1038/srep17793
However, measurements of trace element concentrations in the aragonitic layer (nacre) of the dis-
soconch shells were nearly identical regardless of which layer was measured first. This is useful for 
comparisons between adult (aragonitic layer) and embryonic shells because laser ablation measurements 
of the prodissoconch I of Idas modiolaeformis (and likely other mytilid species due to similar shell mor-
phologies) is facilitated by positioning the shell with the inner (aragonitic) side facing upwards; this ori-
entation exposes the umbonal region allowing vertical laser shots on the outer surface of the embryonic 
shell. Measurements can thus be taken from both prodissoconch and dissoconch shells without the need 
to re-orient the specimen.
All Elements Mg:Ca Sr:Ca Pb:Ca U:Ca Ba:Ca
Source df MS Pseudo-F MS Pseudo-F MS Pseudo-F MS Pseudo-F MS Pseudo-F MS Pseudo-F
 Shell stage 1 125.2 66.05**** 28.73 104.3**** 32.49 196.0**** 4.086 4.253* 30.98 141.8**** 28.93 105.1****
 Stor. method 1 0.333 0.175ns 0.070 0.255ns 0.068 0.413ns 0.002 0.002ns 0.073 0.332ns 0.12 0.434ns
 Stage x Stor. 1 1.2 0.633ns 0.28 1.016ns 0.475 2.868ns 0.327 0.340ns 0.084 0.383ns 0.035 0.127ns
 Residuals 36 1.896 0.275 0.166 0.961 0.218 0.275
Prodissoconch
 Stor. method 1 3.606 0.710ns 0.907 0.902ns 1.872 1.967ns 0.150 0.143ns 0.374 0.362ns 0.304 0.292ns
 Residuals 18 5.077 1.005 0.952 1.047 1.035 1.038
Dissoconch
 Stor. method 1 1.536 0.296ns 0.180 0.172ns 0.906 0.901ns 0.209 0.200ns 0.038 0.036ns 0.202 0.194ns
 Residuals 18 5.192 1.046 1.005 1.044 1.053 1.044
Table 2.  Effect of storage method (−20 °C versus 95% ethanol) on the geochemical compositions 
of ontogenetic shell stages (prodissoconch and dissoconch) based on PERMANOVA. df = degrees 
of freedom; MS = mean sum of squares; Pseudo-F = F value by permutation; ****P(perm) < 0.0001; 
*P(perm) < 0.05; ns: not significant.
Figure 5. Element concentrations (X:Ca, parts per million) in prodissoconch (pd) and dissoconch (da) 
shell regions (sketch of valve cross section in bottom left corner of figure) in specimens preserved in 
ethanol (Et, grey brick pattern) and frozen (Fr, black brick pattern). Vertical lines: median value; boxes: 
interquartile range (IQR); whiskers: values < 1.5 × IQR. Asterisks denote outliers (> 1.5 × IQR).
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7Scientific RepoRts | 5:17793 | DOI: 10.1038/srep17793
Storage method. The storage method (frozen at − 20 °C vs 95% ethanol) had no significant effect on 
the quantification of Mg, Sr, Pb, U and Ba in prodissoconch and dissoconch shells of Idas modiolaeformis 
(Table 2), either considering individual elements analysed separately (Fig. 5) or multi-element signatures 
(Fig. 6). Measurements obtained from differently preserved specimens can thus be analysed together with 
a negligible risk of biasing the results. This is promising and particularly relevant in deep-sea organisms 
because sampling is often limited to a small number of individuals, and splitting of samples into various 
fixatives is commonly required for multiple methodological applications (e.g. molecular and morpholog-
ical studies). Previous reports on the effects of sample handling and storage on fish otoliths and deep-sea 
coral microchemistry identified small but measurable effects on the concentrations of some trace ele-
ments including Ba, in frozen and ethanol preserved samples, respectively12,15. However, in both cases 
the differences were small or negligible relative to instrument precision, or to the variability between 
the otolith’s core and edge. Swan et al.14 also observed small differences between pairs of treated otoliths 
but the variation attributable to storage and handing effects was smaller than between-geographic area 
differences.
Applicability to larval tracking studies. The same five elements (Mg, Sr, Pb, U and Ba) were meas-
ured in the embryonic (aragonitic) shells (prodissoconch I) of I. modiolaeformis and showed significantly 
higher concentrations than in the aragonitic layer of dissoconch shells (Figs  5 and 6). This may either 
reflect different physicochemical conditions in the environment where the embryonic and adult shells 
precipitated, or indicate that ontogenetic changes in vital effects influence the incorporation of trace 
elements into the shell. While the simple enrichment in all five trace elements intuitively suggests chang-
ing vital effects - possibly in adults there is an increased discrimination ability against ions that are not 
calcium or carbonate - these relationships are not straightforward for all elements11. This has implica-
tions on the use of dissoconch shells to generate site-specific elemental signatures because putative vital 
effects interfere with abiotic conditions limiting the assignment of other recruited individuals to their 
natal origin. An alternative is to cultivate larvae in situ to generate site-specific larval signatures that can 
be compared with larval shells of naturally settled juveniles24. However, transplanting larvae in deep-sea 
habitats is extremely challenging owing to time and financial constraints and technical difficulties of 
in vitro larval rearing18. Moreover, empirical evidence points towards ontogenetic vertical migrations 
of planktotrophic bathymodiolin larvae, allowing long-distance dispersal in surface waters for periods 
as long as one year33, but it is unknown how far the developing embryos drift from their natal source 
during the ~8 days-long embryonic stage, when they secrete the prodissoconch I shell. This poses addi-
tional challenges to determine the natal origin of deep-sea mussel larvae. Further research is needed to 
elucidate the influence of abiotic factors and biologically mediated processes controlling trace element 
incorporation in larval and adult shells of deep-sea mussels.
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Acknowledgements
We are grateful to Ocean Exploration Trust and the chief-scientist, scientific team, ROV technical teams, 
captains and crews of the oceanographic cruise (E/V Nautilus NAO17) that allowed sample collection 
for this work. This manuscript benefited from the criticisms and insights of Henry Carson and one 
anonymous reviewer. Financial support was provided by the HERMIONE project, Grant Agreement 
no. 226354 funded by the European Community’s Seventh Framework Programme (FP7/2007-2013), by 
European Funds through COMPETE, and by National Funds through the Portuguese Science Foundation 
(FCT) within project PEst-C/MAR/LA0017/2013 and UID/AMB/50017/2013. SK was supported by the 
Deutsche Forschungsgemeinschaft through grant Ki802/7, and LG received support from FCT through 
post-doctoral fellowships (SFRH/BPD/51858/2012 and SFRH/BPD/96142/2013) funded by the Human 
Potential Operational Programme (POPH), inscribed in the National Strategic Reference Framework 
(QREN) and partially subsidized by the European Social Fund.
Author Contributions
L.G. conceived the study, prepared the samples and guided LA-ICPMS measurements, performed the 
statistical analyses, drafted the manuscript and prepared the figures. K.S. performed the LA-ICPMS 
www.nature.com/scientificreports/
9Scientific RepoRts | 5:17793 | DOI: 10.1038/srep17793
measurements. S.K. helped to conceive the study and to draft the manuscript. M.R.C. collected the 
samples and contributed to draft the manuscript. All authors read and approved the final manuscript.
Additional Information
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Génio, L. et al. Effects of sample storage and shell orientation on LA-ICPMS 
trace element measurements on deep-sea mussels. Sci. Rep. 5, 17793; doi: 10.1038/srep17793 (2015).
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