Journal of Structural Biology 185 (2014) 58–68

Contents lists available at ScienceDirect

Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi

Elemental and chemical characterization of dolphin enamel and dentine using X-ray and Raman microanalyzes (Cetacea: Delphinoidea and Inioidea) Carolina Loch a,b,⇑, Michael V. Swain b, Sara J. Fraser c, Keith C. Gordon c, Jules A. Kieser b, R. Ewan Fordyce a a b c

Department of Geology, University of Otago, Dunedin 9054, New Zealand Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin 9054, New Zealand MacDiarmid Institute of Advanced Materials and Nanotechnology, Department of Chemistry, University of Otago, Dunedin 9054, New Zealand

a r t i c l e

i n f o

Article history: Received 14 August 2013 Received in revised form 18 November 2013 Accepted 20 November 2013 Available online 25 November 2013 Keywords: Teeth Mammal Hydroxyapatite Raman spectroscopy EDX WDX

a b s t r a c t Dolphins show increased tooth number and simplified tooth shape compared to most mammals, together with a simpler ultrastructural organization and less demanding biomechanical function. However, it is unknown if these factors are also reflected in the chemical composition of their teeth. Here, the bulk chemical composition and elemental distribution in enamel and dentine of extant dolphins were characterized and interpreted using X-ray and spectroscopy techniques. Teeth of 10 species of Delphinida were analyzed by WDX, EDX and Raman spectroscopy. For most of the species sampled, the mineral content was higher in enamel than in dentine, increasing from inner towards outer enamel. The transition from dentine to enamel was marked by an increase in concentration of the major components Ca and P, but also in Na and Cl. Mg decreased from dentine to enamel. Concentrations of Sr and F were often low and below detection limits, but F peaked at the outer enamel region for some species. Raman spectroscopy analyzes showed characteristics similar to carbonated hydroxyapatite, with the strongest peak for 1 . Dentine samples revealed a higher diversity of the phosphate PO3 4 stretching mode at 960–961 cm peaks representative of organic components and proteins than enamel. The similar distribution pattern and small variation in average concentration of major and minor elements in dentine and enamel of dolphins suggest that they are subject to strong physiological control. A clear trend of the elemental variations for all dolphin species sampled suggests that the general pattern of tooth chemistry is conserved among the Mammalia. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Cetaceans (dolphins and whales) are singular mammals adapted to a fully aquatic life. The constraints of living in water dictate physiological and morphological modifications in basic functions such as respiration, circulation, feeding, and locomotion (Simpson, 1945). Profound anatomical modifications of the cetacean skull include adaptations for feeding in water (Flower, 1885; Werth, 2000), which in extant odontocetes has resulted in a feeding apparatus specialized for food acquisition, rather than reduction and processing of food in the oral cavity. Most odontocete species grasp and eat prey whole, and some others are able to perform suction feeding to catch and transport food items (Werth, 2000).

⇑ Corresponding author at: Department of Geology, University of Otago, Dunedin 9054, New Zealand. Fax: +64 03 479 7527. E-mail address: [email protected] (C. Loch). 1047-8477/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsb.2013.11.006

Dolphins have a unique configuration of their dentition when compared to most other mammals. In contrast with the eutherian dental standard, dolphins produce only one set of teeth that remains in place throughout their life (monophyodonty), the shape of their teeth is simplified (homodonty) and they have many more teeth (polydonty) than most terrestrial mammals (Flower, 1885; Myrick, 1991; Ungar, 2010). The evolutionary changes that marked the transition to water are not only reflected in the macroscopic shape and structure of the jaws and teeth of dolphins, but also in the finer organization and biomechanical demands of their dental tissues. In general, mammalian teeth are composed of three main structural tissues: enamel, dentine and cementum. Despite their common mineralogy of hydroxyapatite, different microstructures and chemical compositions are observed between these three tissues. While the combination of inorganic and organic content and water is common to all of them, the proportion of these components is variable. Enamel is highly mineralized, and in humans and some artiodactyls, the inorganic components account for about 96% of

59

C. Loch et al. / Journal of Structural Biology 185 (2014) 58–68

its dry weight, in contrast to about 1% of organic material (Cuy et al., 2002; Hillson, 2005; Brügmann et al., 2012). Dentine and cementum, on the other hand, are much less mineralized. Dentine is composed of about 72% of inorganic material and 20% organic components, predominantly collagen fibers. Cementum has a chemical composition similar to bone, composed of approximately 70% of mineral and 22% organic substances. The remaining percentage in enamel, dentine, and cementum represents water content (Wentrup-Byrne et al., 1997; Dorozhkin and Epple, 2002; Hillson, 2005). In teeth, the inorganic components consist mostly of calcium phosphate minerals, notably in the form of apatite (Dorozhkin and Epple, 2002). While their main constituents are calcium and phosphate, other ions, such as hydroxyl and fluoride, may also be present. Most of the constituent biological apatite in bones and teeth is in the form of hydroxyapatite Ca10(PO4)6(OH)2, but continuous variation between hydroxyapatite and fluorapatite Ca10(PO4)6F2 is often common (Hillson, 2005). Pure hydroxyapatite never occurs in biological systems, as other ions are always incorporated into the apatite structure. Most common substitutions include sodium (Na+) or strontium (Sr2+) replacing calcium, carbonates ðCO2 3 Þ instead of phosphates, and chloride (Cl) or carbonates ðCO2 3 Þ replacing hydroxyl (Dorozhkin and Epple, 2002; Hillson, 2005; Brügmann et al., 2012; Peek and Clementz, 2012). Organic components are an important and often underappreciated part of dental tissues. Dentine and cementum are rich in collagen, a fibrous protein organized in banded spiral chains. Ground substance, an amorphous organic material composed of proteins other than collagen, is also part of fresh dentine and cementum. Enamel, despite being heavily mineralized, contains a small fraction of proteins in its composition. Newly formed enamel is composed of approximately 30% protein, mainly amelogenins, tuftelins, and ameloblastins. During maturation, enamel proteins are broken down and removed, remaining mainly in the enamel– dentine junction, where they form enamel tufts, and also around prism sheaths. These proteins and peptides represent about 1% of the composition of mature enamel (Wentrup-Byrne et al., 1997; Hillson, 2005), and are directly related to the mechanical properties and stress–strain response of enamel (He and Swain, 2008). The physical, structural, physiological, functional, and phylogenetic characteristics of dental biominerals are strongly dependent on both their chemical constituents and ultrastructure (WentrupByrne et al., 1997). Understanding these properties is important for elucidating tooth function and biomechanics. The ratios of Ca/ P and minor inorganic components have a marked influence on the mechanical properties of biological hydroxyapatite (Cuy et al., 2002). Also, the presence and distribution of trace elements in dental tissues is fundamental to a broader knowledge of life history and environmental conditions for both individuals and species (Cruwys et al., 1994). Many analytical approaches may be used to determine the chemical composition of dental tissues. Microprobe analysis involves irradiating a sample by a focused electron beam, causing interactions between the electrons in the beam and in the sample. This technique allows the elements at a specific point on the surface of a sample to be measured, either using energy-dispersive (EDX) or wavelength-dispersive (WDX) X-ray techniques. While EDX allows the qualitative determination of the elemental composition of the specimen, WDX counts only the X-rays of a single wavelength for a period of time, providing more accurate quantification (Cruwys et al., 1994). In addition to mineral components, it is also necessary to analyze and characterize the organic constituents of dental tissues. Until recently, studies of the organic matrix in dental tissues have required destructive chemical removal of the mineral phase first (Veis, 1984; Wentrup-Byrne et al., 1997), by using mineral acids.

Raman spectroscopy, on the other hand, is a rapid and nondestructive technique that exploits molecular vibrations induced by laser irradiation (McGoverin et al., 2008; Thomas et al., 2011). Raman spectroscopy can elucidate compositional differences of the organic and inorganic components based in the vibrational modes of specific protein and phosphate molecules (Wentrup-Byrne et al., 1997; Kirchner et al., 1997). The limited food processing and reduction executed by most dolphins suggest that their teeth might not be subject to the same evolutionary pressures as those in most other mammals. Previous studies have shown that the increase in tooth number and subsequent simplification in tooth shape of most dolphins was also reflected in the ultrastructural organization and biomechanical response of enamel and dentine (Loch et al., 2013a,b). It is unknown, however, if the simplified dental structure and less constrained biomechanical demands are also reflected in the chemical composition of dental tissues. The aim of this paper is to characterize and interpret the bulk chemical composition and elemental distribution in enamel and dentine of extant dolphins using multiple complementary techniques. 2. Material and methods 2.1. Material examined Teeth of 10 odontocete species were used in this study (Table 1). They represent a range of tooth sizes and feeding strategies among Delphinida and were selected because their mechanical properties have been estimated in a previous study (Loch et al., 2013b). Teeth were obtained from deceased-stranded animals currently deposited in museums, and were prepared normally by water maceration. Previous to the study, these teeth were stored dry or immersed in ethanol. All species in this study were represented by a single individual, and normally 1–2 teeth were analyzed per species. Low sample sizes are typical in cetacean studies, because of logistic, ethical and legal restrictions. For the Amazon River dolphin, only a bunodont posterior tooth was analyzed. Although it is believed that the elemental and compositional features described in these specimens were typical of each species, the conclusions are provisional and were made with some caution. Tooth surfaces were cleaned with ethanol and were embedded in epoxy resin (Epofix™ Cold-Setting Embedding Resin, Struers, Copenhagen, Denmark). After setting for 24 h, specimens were longitudinally sectioned parallel to the buccal-lingual plane (Fig. 1) using a MOD13 diamond grit blade (Struers, Copenhagen, Denmark) with a high-speed saw (Accutom-50, Struers, Copenhagen, Denmark) under water irrigation. Mounted specimens were polished on a TegraPol-21 polisher (Struers, Copenhagen, Denmark) with 1200 grit silicon carbide

Table 1 Species analyzed. Names follow the ‘‘List of marine mammal species and subspecies of the Society for Marine Mammalogy’’ (Committee on Taxonomy, 2012). Species

Common name

Specimen number

Delphinoidea Stenella coeruleoalba Tursiops truncatus Sotalia guianensis Steno bredanensis Cephalorhynchus hectori Globicephala melas Orcinus orca Phocoena spinipinnis

Striped dolphin Bottlenose dolphin Guiana dolphin Rough-toothed dolphin Hector’s dolphin Long-finned pilot whale Killer whale Burmeister’s porpoise

UFSC 1344 UFSC 1349 UFSC 1203 UFSC 1234 H 179 UFSC 1093 UFSC 1127 UFSC 1025

Inioidea Pontoporia blainvillei Inia geoffrensis

Franciscana dolphin Amazon River dolphin

UFSC 1310 IEPA 1899

60

C. Loch et al. / Journal of Structural Biology 185 (2014) 58–68

sampled on dentine (crown dentine and dentine near the EDJ (enamel–dentine junction) – Spots 1 and 2) and three on enamel (enamel near the EDJ, mid and outer enamel – Spots 3–5) (Fig. 2). Three transect lines consisting of five sampling spots were taken at about 50 lm from each other. Data obtained were reduced using ZAF corrections. These included corrections for Z (atomic number), A (absorption), and F (secondary fluorescence) effects. Data obtained from spot analysis along line transects were then averaged and processed using Excel. Mean weight percentage (wt.%) values and standard error for each element were calculated for each of the five sampling spots. Weight percentage values indicate the concentration of an element in a mineral compound. In analyzes using bioapatites, wt.% values represented a wet weight, as water and organic components were not removed before analysis. Statistical analyzes were performed using BioStat 2009 (AnalystSoft, Alexandria VA, USA). Statistical significance was set at 5% probability level. 3.2. Energy-dispersive X-rays

Fig.1. Orientation of the longitudinal sectioning plane in dolphin teeth.

paper (Struers, Copenhagen, Denmark) for 4 minutes and ultrasonically cleaned for 3 minutes in water. Final polishing was performed with 9 lm and 1 lm diamond suspension (DP Suspension P, Struers, Copenhagen, Denmark) for 5 minutes each, and ultrasonically cleaned in between different suspensions. Final ultrasonic cleaning was done in ethanol for 3 minutes. Before analysis using EDX or WDX, specimens were surface-coated with 25 nm of carbon and mounted in microprobe holders. For Raman spectroscopy, uncoated natural tooth surfaces were used.

Energy-dispersive X-ray analyzes were carried out in a Zeiss Sigma VP FEG-SEM, housed at the Department of Anatomy and Structural Biology of the University of Otago. Analytical settings used were 20 kV accelerating voltage, 30 lm aperture size and a working distance of 9 mm. Elemental maps were produced to show the concentration and distribution of F, P, Cl, Ca, Sr, Mg, and Na in a selected region consisting of enamel, enamel–dentine junction and adjacent dentine. Scanning time was set to about 10 minutes for each sample. Data obtained from energy-dispersive X-ray analysis was processed using AZtec (Oxford Instruments NanoAnalysis, Oxfordshire, UK). 3.3. Raman spectroscopy Spectra were collected over a range of sampling positions from outer enamel to dentine of the sectioned teeth. Raman spectra were collected using the Senterra Raman microscope (Bruker Optics, Ettlingen, Germany) with 785 nm incident laser at 25 mW power. Measurements were taken over 5 s  60 co-additions using the 20 objective and 50 lm confocal aperture with 3 cm1

3. Methods 3.1. Wavelength dispersive X-rays The major elemental chemistry of dentine and enamel was determined by electron microprobe analysis on a JEOL JXA-8600 Superprobe, housed at the Department of Geology of the University of Otago. Wavelength dispersive X-ray spectroscopy analyzes (WDX) were carried out using 20 lm beam diameter, 15 kV accelerating voltage and 20 nA beam current. The microprobe has two spectrometers which count the number of X-rays of a specific wavelength diffracted by a crystal, thus providing a sensitive method for determining the chemical composition of a particular sample. Analyzes were calibrated using Smithsonian standards composed of natural mineral and oxides, particularly the standards for apatite (fluorapatite), volcanic glass-2 (VG-2), and strontium titanate (SrTiO3) (Jarosewich et al., 1980). Elements such as F, P, Cl and Ca were calibrated using apatite, while Na and Mg were calibrated using volcanic glass (VG-2) and Sr was calibrated with strontium titanate (SrTiO3). Instrument drift during the analyzes was corrected with repeated measures of the standards used. Selective spot analysis was used to measure the absolute concentration of elements at five different locations. Two spots were

Fig.2. SEM backscattered image identifying the location of sampling spots on enamel and dentine of Burmeister’s porpoise. Note the contrast between the mineral-rich enamel and the underlying dentine. The dark streaks in the dentine are the tubules. The section was made at the tooth midline and sampling was performed at the middle of the tooth crown.

C. Loch et al. / Journal of Structural Biology 185 (2014) 58–68

61

spectral resolution. Data intervals were 0.3 cm1 and the automated calibration was accurate to 0.1–0.3 cm1. This setup results in a confocal spot (measurement area) of about 2.4 lm in diameter. Five measurement regions were chosen with five spectra acquired from each measurement region to avoid subsampling of each region of interest. Peak ratios were calculated using the area under the peaks associated with phosphate, carbonate and amide I components of teeth (Yerramshetty et al., 2006). All spectra underwent rubber band correction (RBC) with 15 iterations before integrals were calculated to remove the baseline using OPUS 6.5 (Bruker Optics, Germany). The integral regions were defined as m1PO3 at 989– 4 925 cm1, m1CO2 at 1093–1057 cm1 and m(C@O) amide I at 3 1717–1608 cm1. In addition for the correlation of full-width half maximum (FWHM) with data obtained using WDX analysis, the region 909–999 cm1 was bandfitted using appropriate software (GRAMS AI 8.0, Salem NH).

EDX analyzes provided elemental maps for the distribution of P, Ca, Mg, and Na, allowing a qualitative assessment of mineral components across dolphin enamel and dentine (Fig. 4). Due to low concentrations of elements and constraints of the detection limits of EDX analyzes, distribution trends were not as clear for the other elements sampled (Cl, F, and Sr) and thus are not shown here. Analysis of image profiles along enamel and dentine revealed a decrease in Ca and P content from enamel to dentine, while Mg increased (Fig. 4b–d). There was a slight decrease in Na content from enamel to dentine (Fig. 4e).

4. Results

4.3. Ca/P ratios of enamel and dentine

4.1. Wavelength dispersive X-ray analysis

Ca and P are the major components in tooth enamel and dentine and their relative concentrations reflect the mineral content in different regions of the tooth. When comparing the Ca and P content of enamel and dentine in dolphins with values reported for other mammals (e.g. hippopotamids and humans), it was observed that dolphin values were comparatively lower than those reported for other mammals, especially for the enamel region (Fig. 5). While dentine Ca/P wt.% values were similar for human and dolphin samples, the Ca/P ratio of dolphin enamel was considerably lower than both human and hippo samples.

Results for WDX spot analysis for ten species of dolphins are summarized in Table 2. To consider general mineral composition, for most species, the dentine close to the EDJ (Spot 2) was the least mineralized sampled spot, followed by crown dentine (Spot 1). Higher values of weight percentage (wt.%) were registered for the three spots sampled on enamel in comparison to dentine. For most of the species sampled, there was a trend of increase in mineral content from inner enamel towards outer enamel (Spots 3–5). Statistical analyzes showed, however, that differences in mean mineral concentration values among sampling locations in enamel were not statistically significant (F = 1.778, p = 0.1882). When considering the variation in concentration of inorganic compounds among the different sampling locations, a similar general trend was observed in all the species studied (Fig. 3). The major components found were Ca and P, both for enamel and dentine. Ca was the main component, representing on average about 43% of mineral compounds in dentine and 49% in enamel. P, the second main constituent, contributed approximately 32% of mineral content in the dentine and 37% in enamel. The transition from dentine to enamel, as for teeth of all species, is thus marked by an increase in concentration of Ca and P. A consistent trend among species was also seen for the minor inorganic components (F, Cl, Na, Sr, and Mg). Na and Mg were the most important of the minor components, and showed an opposite trend in the transition from dentine to enamel. The concentration of Mg considerably decreased at the enamel–dentine junction and then remained constant from inner to outer enamel. Mg represented on average about 1.4% of mineral components in dentine and 0.5% for enamel. The concentration of Na on the other hand, showed a marked increase from dentine to the inner enamel, followed by a constant decrease towards the outer enamel. Na contributed on average 0.75% of minerals in dentine and 0.9% for enamel. For most species, the concentration of Cl showed a gradual increase from dentine to outer enamel, while the concentration of F remained relatively low within dentine as well as for inner and mid enamel, peaking at the outer enamel region (Fig. 3a and b). For some other species however, concentration of F remained low and did not show the previous trend observed. On average, F represented 0.6% of the mineral composition for dentine and 0.8% of enamel. Cl contributed with 0.9wt.% of the mineral composition of dentine on average, while in enamel it represented 0.2% of the

composition. For most of the species and locations sampled, the concentration of Sr was below detection limits. When detectable, Sr represented around 0.01% of the inorganic compounds of dolphin enamel. 4.2. Energy-dispersive X-rays

4.4. Raman spectroscopy‘ To provide a broader characterization of different chemical species in the tooth matrix (particularly carbonates and proteins), Raman spectra were taken for the Guiana dolphin, pilot whale and Amazon River dolphin at different sampling positions in enamel and dentine. These species were selected to provide representative samples of tooth size, enamel organization and feeding strategies across clades of living Delphinida. The peak positions and associated assignments of the bands from Raman microscope analysis of dentine and enamel are summarized in Table 3. Peak positions were calculated for crown dentine (Spot 1) and mid enamel (Spot 4), to avoid interference of interface regions and/or collecting spectra from the embedding medium. A graphic representation of the spectra for the Amazon River dolphin is shown in Fig. 6. Integral ratios provided an indication of how the respective amounts of the components change over the different sample regions (Fig. 7). The phosphate/carbonate peak ratio indicates the degree of change in substituted carbonate in the hydroxyapatite matrix across the different tooth regions. Thus, lower values in the graph indicate higher carbonate content in a particular region. All three tooth samples follow the trend of increased carbonate levels at crown dentine, progressively decreasing towards mid enamel (Fig. 7a). Outer enamel showed a relatively higher carbonate content than mid enamel. The phosphate/amide I peak followed the trend of being lowest for crown dentine, highest at inner enamel and dropping again towards outer enamel (Fig. 7b). This would imply a higher protein content in crown dentine and low protein content at inner enamel. Interestingly, the outer enamel region showed lower ratios of phosphate vs. carbonates and amides, indicating a higher proportion of the latter components in the outer layer compared to mid and inner enamel.

62

C. Loch et al. / Journal of Structural Biology 185 (2014) 58–68

Table 2 Summary of electron microprobe results (in wt.% ± SE). Total values are less than 100% as other components like carbonates, organic matrix, and water were not probed. Spots

Crown dentine

EDJ dentine

Spot 1

Inner enamel

Spot 2

Mid enamel

Spot 3

Outer enamel

Spot 4

Spot 5

Bottlenose dolphin F Cl Na Ca P Sr Mg Total

wt.% 0.06 0.03 0.60 42.44 34.29 0.01 1.44 78.87

±SE 0.07 0.01 0.14 0.94 0.69 0.02 0.04 0.39

wt.% 0.07 0.04 0.76 43.39 33.67 0.01 1.36 79.29

±SE 0.08 0.01 0.10 0.60 0.68 0.01 0.01 0.09

wt.% 0.05 0.10 1.10 48.85 36.56 0.04 0.37 87.08

±SE 0.05 0.02 0.08 0.51 2.60 0.04 0.03 3.03

wt.% 0.02 0.17 0.98 49.91 38.01 0.00 0.41 89.50

±SE 0.03 0.01 0.07 0.22 1.19 0.00 0.04 1.35

wt.% 0.40 0.24 0.76 50.11 38.58 0.01 0.42 90.52

±SE 0.08 0.01 0.02 0.32 0.99 0.02 0.05 0.77

Striped dolphin F Cl Na Ca P Sr Mg Total

0.18 0.03 0.77 45.58 32.30 0.01 1.17 80.04

0.11 0.02 0.03 0.34 0.61 0.02 0.01 0.46

0.11 0.09 0.72 43.01 32.11 0.00 0.87 76.92

0.05 0.01 0.04 0.48 1.52 0.00 0.05 1.78

0.08 0.15 1.03 49.59 37.38 0.02 0.45 88.70

0.03 0.03 0.07 0.59 2.79 0.03 0.02 2.36

0.21 0.24 0.91 50.17 39.53 0.01 0.38 91.44

0.02 0.01 0.03 0.22 0.45 0.01 0.05 0.65

0.16 0.34 0.76 50.25 38.79 0.00 0.40 90.70

0.07 0.01 0.05 0.30 0.89 0.00 0.03 1.14

Hector’s dolphin F Cl Na Ca P Sr Mg Total

0.06 0.12 0.79 43.79 32.99 0.00 1.45 79.21

0.06 0.02 0.07 1.66 1.22 0.00 0.05 1.18

0.04 0.17 0.89 41.90 31.97 0.00 1.11 76.07

0.04 0.03 0.02 0.77 0.64 0.00 0.02 1.08

0.05 0.08 1.18 47.41 32.78 0.00 0.69 82.20

0.05 0.01 0.05 0.95 2.40 0.00 0.02 1.61

0.06 0.24 0.97 48.64 34.98 0.00 0.61 85.50

0.06 0.02 0.05 0.56 3.20 0.00 0.04 2.63

0.29 0.32 0.80 48.65 33.50 0.03 0.63 84.22

0.12 0.01 0.02 0.78 3.87 0.05 0.04 3.71

Pilot whale F Cl Na Ca P Sr Mg Total

0.03 0.02 0.87 43.60 34.16 0.00 1.10 79.78

0.03 0.01 0.13 0.95 0.78 0.00 0.09 1.20

0.09 0.04 0.87 43.33 30.67 0.00 0.87 75.87

0.03 0.01 0.06 0.29 2.53 0.00 0.08 2.87

0.00 0.13 1.02 49.44 33.95 0.00 0.40 84.95

0.00 0.02 0.04 1.19 1.28 0.00 0.03 2.46

0.01 0.27 0.83 50.18 38.13 0.00 0.38 89.80

0.01 0.01 0.03 0.37 1.60 0.00 0.06 1.42

0.04 0.43 0.66 50.41 36.19 0.01 0.41 88.16

0.04 0.02 0.02 0.65 1.46 0.01 0.02 2.11

Rough-toothed dolphin F Cl Na Ca P Sr Mg Total

0.09 0.02 0.99 43.35 34.68 0.00 1.66 80.79

0.06 0.00 0.07 0.93 1.53 0.00 0.06 2.50

0.00 0.07 0.94 41.97 31.61 0.01 1.27 75.87

0.00 0.01 0.05 0.70 3.21 0.01 0.06 3.69

0.04 0.11 1.16 49.40 38.45 0.01 0.41 89.58

0.04 0.03 0.02 0.30 0.51 0.01 0.02 0.79

0.03 0.19 1.05 49.72 38.18 0.00 0.41 89.58

0.04 0.03 0.12 0.26 0.60 0.00 0.02 0.53

0.02 0.22 1.04 49.92 38.81 0.00 0.42 90.43

0.03 0.06 0.09 0.47 0.22 0.00 0.03 0.68

Guiana dolphin F Cl Na Ca P Sr Mg Total

0.01 0.04 0.70 44.40 30.05 0.00 1.21 76.41

0.01 0.02 0.07 0.73 1.75 0.00 0.11 1.48

0.01 0.08 0.69 42.89 30.45 0.03 1.08 75.23

0.02 0.05 0.08 1.07 1.35 0.03 0.16 2.57

0.03 0.19 0.84 49.74 34.97 0.00 0.47 86.24

0.03 0.03 0.08 0.36 2.19 0.00 0.03 1.87

0.03 0.28 0.74 50.10 35.10 0.00 0.47 86.72

0.05 0.02 0.03 0.44 2.00 0.00 0.03 2.03

0.14 0.40 0.66 50.57 35.93 0.00 0.44 88.14

0.05 0.01 0.04 0.23 2.93 0.00 0.03 2.85

Franciscana F Cl Na Ca P Sr Mg Total

0.09 0.01 0.37 44.76 35.21 0.00 1.37 81.81

0.05 0.01 0.28 0.23 0.69 0.00 0.03 0.47

0.01 0.02 0.66 43.44 32.95 0.00 1.17 78.25

0.01 0.01 0.03 0.13 1.81 0.00 0.04 1.87

0.01 0.05 1.00 48.84 36.83 0.00 0.43 87.16

0.01 0.01 0.03 0.40 1.37 0.00 0.02 1.67

0.01 0.10 0.91 49.25 37.50 0.00 0.53 88.30

0.01 0.02 0.04 0.30 0.99 0.00 0.04 1.25

0.33 0.18 0.67 49.11 37.89 0.00 0.44 88.62

0.01 0.04 0.05 0.28 0.65 0.00 0.02 0.38

Burmeister’s porpoise F Cl Na Ca P Sr

0.06 0.46 0.68 43.90 30.01 0.00

0.06 0.06 0.07 0.21 1.31 0.00

0.04 0.47 0.73 43.75 30.67 0.00

0.04 0.06 0.05 1.31 3.04 0.00

0.04 0.10 0.80 46.24 35.23 0.00

0.03 0.01 0.08 0.37 1.18 0.00

0.04 0.06 1.09 47.42 36.28 0.00

0.05 0.01 0.04 0.49 1.55 0.00

0.13 0.05 1.10 47.06 35.63 0.00

0.07 0.01 0.02 1.11 1.89 0.00

63

C. Loch et al. / Journal of Structural Biology 185 (2014) 58–68 Table 2 (continued) Spots

Crown dentine

EDJ dentine

Inner enamel

Mid enamel

Outer enamel

Spot 1

Spot 2

Spot 3

Spot 4

Spot 5

Mg Total

1.53 76.64

0.08 1.07

1.60 77.25

0.12 1.83

1.20 83.61

0.13 0.93

1.03 85.92

0.08 1.21

1.05 85.01

0.05 1.67

Killer whale F Cl Na Ca P Sr Mg Total

0.04 0.04 0.88 38.91 31.01 0.00 1.82 72.71

0.04 0.01 0.06 1.99 0.41 0.00 0.06 1.96

0.04 0.04 1.05 42.40 34.86 0.00 1.38 79.78

0.03 0.01 0.19 1.72 1.29 0.00 0.44 2.90

0.01 0.15 1.13 46.35 37.20 0.00 0.54 85.38

0.02 0.02 0.05 1.56 1.45 0.00 0.03 2.93

0.00 0.25 0.87 46.53 36.05 0.00 0.54 84.25

0.01 0.01 0.11 1.42 1.46 0.00 0.03 0.33

0.10 0.36 0.75 46.68 38.22 0.01 0.50 86.62

0.01 0.02 0.09 1.47 0.52 0.02 0.03 1.88

Amazon River dolphin F Cl Na Ca P Sr Mg Total

0.08 0.01 0.62 42.47 34.31 0.00 2.66 80.16

0.01 0.01 0.05 1.38 0.82 0.00 0.08 2.09

0.04 0.03 0.77 42.90 33.73 0.00 2.46 79.92

0.05 0.01 0.04 0.34 0.39 0.00 0.09 0.23

0.01 0.10 0.99 49.85 37.87 0.00 0.32 89.14

0.02 0.01 0.02 0.23 0.22 0.00 0.03 0.33

0.05 0.16 0.88 49.83 38.31 0.00 0.38 89.62

0.03 0.01 0.03 0.30 0.08 0.00 0.02 0.29

0.00 0.31 0.69 49.42 38.36 0.00 0.34 89.13

0.00 0.08 0.06 0.22 0.18 0.00 0.02 0.36

Fig.3. Average results of WDX spot analysis in wt.% for the five locations sampled. The vertical axis shows the relative percentage, while the horizontal axis shows the five sampling regions. Major elements (Ca and P) are shown in the left chart, while minor elements (F, Cl Na, Sr, and Mg) are shown in the right chart. (a) Bottlenose dolphin. (b) Hector’s dolphin. (c) Amazon River dolphin.

An examination of the relationship between bandwidth (FWHM) of the m1 phosphate band and WDX results (Table 2) for different elements shows a qualitative trend for the wt.% of Ca and Mg. Increasing levels of Mg in the samples resulted in band broadening of the m1 phosphate band from 16 cm1 to 19 cm1 (Fig. 8a). Similarly, increased calcium content resulted in a narrower bandwidth from 19 cm1 to 16 cm1 as seen in Fig. 8b.

5. Discussion The general trends observed in regards to the distribution and concentration of mineral components within dolphin enamel and dentine are consistent with findings reported for other mammals, especially humans (Bodart et al., 1981; Cuy et al., 2002; Dorozhkin and Epple, 2002; Gutiérrez-Salazar and Reyes-Gasga, 2003),

64

C. Loch et al. / Journal of Structural Biology 185 (2014) 58–68

Fig.4. Elemental map of enamel and dentine of the Amazon River dolphin. (a) SEM image of sampled area. Black line shows the location where image profiles were calculated. (b) Map and image profile (Gray value/distance in lm) of Ca. (c) Map and image profile of P. (d) Map and image profile of Mg. (e) Map and image profile of Na.

artiodactyls (Barnicoat, 1959; MacKenzie et al., 2011; Brügmann et al., 2012) and other non-cetacean marine mammals (Cruwys et al., 1997; Edmonds et al., 1997; Labrada-Martagón et al., 2007). The regular distribution pattern of the major and minor components suggests that the general pattern of tooth chemistry is consistent and conserved among the Mammalia in spite of widely varying diets and orodental function. The main components of hydroxyapatite, Ca and P, are reported to increase from dentine towards the outer enamel surface (Barnicoat, 1959; Cuy et al., 2002; Brügmann et al., 2012), corroborating the findings reported here for dolphin teeth. The concentration patterns of Ca and P across enamel were stable and varied by about 1%, but variations for dentine were more prominent, particularly the transition from dentine to enamel, as reported by Brügmann et al. (2012) in hippopotamid teeth. The enamel–dentine junction is marked by sharp

concentration changes not only in Ca and P content, but also in the concentration of minor components such as Mg, Cl, Na, F, and Sr (Cuy et al., 2002; Brügmann et al., 2012). The small variation in concentration patterns of Ca and P occurs because these elements are stoichiometrically controlled in order to avoid the precipitation of different apatite phases (Brügmann et al., 2012). For the minor constituents incorporated in the hydroxyapatite structure of dolphin teeth, only Mg and Cl showed the same trends as reported for human (LeGeros et al., 1995; Cuy et al., 2002; Gutiérrez-Salazar and Reyes-Gasga, 2003) and hippopotamid dental samples (Brügmann et al., 2012). While Mg progressively decreased from dentine to outer enamel, Cl slightly increased. For Na, Cuy et al. (2002) and Brügmann et al. (2012) detected a progressive decrease from dentine towards the outer enamel. However, the results of the present study show a progressive increase

65

C. Loch et al. / Journal of Structural Biology 185 (2014) 58–68

Fig.5. Comparative Ca and P content in teeth from cetaceans (this study), hippos (data from Brügmann et al., 2012) and humans (data from Cuy et al., 2002). Note that the Ca and P content increases from lower left to upper right in the graph.

Table 3 Major peak positions and associated assignments for dentine and enamel of the Guiana dolphin, pilot whale and Amazon River dolphin. Vibrational assignments followed Kirchner et al. (1997) and Penel et al. (1998). Guiana dolphin

Pilot whale

Amazon River dolphin

Peak position

Peak position

Peak position

Dentine

Enamel

Dentine

Enamel

Dentine

Enamel

429 w

431 w

430 w

430 w

433 w

431 w

586–610

581–610

586–608

588–607

591

582–609a

961 s

960 vs

960 s

960 vs

961 s

960 vs

1025 vw

1028 vw

1045 vw br

1049 vw

1036 vw

1044 w

1037 vw

1045 vw

1071 w

1070 w

1071 w

1071 w

1072 w

1070 w

1453 w 1666 vw br

1450 w 1674 w br

2943 s

2952 vs

1452 w 1649 w br 2905 m 2945 s

Assignment

m2PO3 4 m4PO3 4 m1PO3 4 m3PO3 4 m3PO3 4 m1CO2 3

b-type carbonate substituted apatite apatite b-type carbonate substituted apatite b-type carbonated apatite

b-type substituted d(CH2) scissors m(C@O) amide I – m(CH2) symmetric

Descriptors on the relative intensity and shape of the peaks are w = weak, vw = very weak, vs= very strong, sh = shoulder, br = broad. a = Broad feature with a series of bands.

in Na concentration from dentine to inner enamel, followed by a decrease in concentration from inner to outer enamel. This ‘‘umbrella-shaped’’ trend was also observed by Gutiérrez-Salazar and Reyes-Gasga (2003). Pinheiro et al. (1999) observed a higher concentration of Sr in human dental samples from fishermen compared to miners, and attributed this finding to a diet richer in fish. The detection of Sr in some of the samples used here, even though by a small amount, may also be related to the piscivorous feeding habit of the dolphin species sampled. However, it could equally reflect the Sr content of waters draining from river systems or sourced by deep-sea sediment pore waters (Peek and Clementz, 2012). In a survey of Sr/ Ca and Ba/Ca variations in marine foodwebs, Peek and Clementz (2012) reported that odontocetes have the lowest Sr/Ca in marine systems, due to biopurification of Sr obtained through their diet. In relation to F, Cuy et al. (2002) did not find a consistent trend in human dental samples, but mentioned a slight increase in the concentration of this anion in the outer layer of enamel. Dorozhkin and Epple (2002) also mentioned the presence of fluoride at the enamel surface, although the overall content was quite small. These findings corroborate our results with dolphin dental samples. For some species there was no significant trend (e.g. Amazon River

dolphin, rough-toothed dolphin), but other species showed a moderate increase in concentration of F in the outer enamel layer (e.g. bottlenose dolphin, Hector’s dolphin). In humans and other vertebrates, the incorporation of fluoride into the hydroxyapatite lattice has been implicated in protection against demineralization by acids (Enax et al., 2012), a pathological condition documented for dolphins in previous studies (Loch et al., 2013c). However, it is not known whether the F present in the outer layer of dolphin enamel has the same effects or if it comes from dietary sources or from superficial bonding of F ions present in the aquatic environment. The similar distribution pattern and small variation in average concentrations of major and minor elements in dentine and enamel of dolphins suggest that there is a strong physiological control of the concentration of these elements. The same general trends have been observed in the enamel of many other mammals, implying that during amelogenesis the fundamental chemical processes of mineralization follow common trends in different mammal groups (Brügmann et al., 2012). The final composition of the hydroxyapatite which forms enamel is controlled by fluids generated during the secretion and maturation phases of amelogenesis. Hydroxyapatite crystallites both precipitating during secretion and undergoing

66

C. Loch et al. / Journal of Structural Biology 185 (2014) 58–68

Fig.6. Raman spectra of enamel and dentine of the Amazon River dolphin (1 = crown dentine, 2 = dentine near EDJ, 3 = inner enamel, 4 = mid enamel, 5 = outer enamel).

Fig.8. The bandwidth (FWHM) of the m1PO3 4 band for tooth spectra across different sampling positions against WDX results for (a) Mg and (b) Ca wt.%. Deviations from best fit values appear to be sensitive to FWHM to about 0.5 cm1. GD = Guiana dolphin, PW = Pilot whale, ARD = Amazon River dolphin.

Fig.7. Ratios of peak integrals across sampling positions. (a) Phosphate to carbonate ratio. Lower values in the graph indicate higher carbonate content in a particular region, while higher values indicate low phosphate content. (b) Phosphate to amide I. Lower values in the graph indicate higher amide I content in a particular region, while higher values indicate low amide I content. (1 = crown dentine, 2 = dentine near EDJ, 3 = inner enamel, 4 = mid enamel, 5 = outer enamel).

crystal growth during maturation remain in equilibrium with this evolving fluid, finally resulting in crystalized bioapatite which replaces the original enamel fluids and the scaffolding primordial organic matrices. The enamel fluid becomes continuously depleted in Mg and Na but enriched with Cl during maturation, which results in the formation of an enamel layer rich in Mg and Na but poor in Cl near the EDJ, and conversely poor in Mg and Na but rich in Cl near the outer surface (Brügmann et al., 2012). Previous studies have reported trace elements such as Pb, Fe, Cu, and Zn present in mammalian teeth (Bodart et al., 1981; Cruwys et al., 1994; Pinheiro et al., 1999), relating their occurrence to environmental influences and contamination. None of these elements was detected in our analysis. The previous studies reported that most of these elements occurred in low concentrations, lower than the detection limits of our analytical instruments (around 0.01%), which means that they could have been present in the samples analyzed, but not detected. Changes in concentration of F, Cl, Na, Sr, and Mg have some limited effects on the mechanical properties of dental tissues, but the Ca/P content is considered the most likely source of variation in hardness and elasticity of enamel and dentine (Cuy et al., 2002; Gutiérrez-Salazar and Reyes-Gasga, 2003). Loch et al. (2013b) evaluated the mechanical properties of enamel and dentine in dolphins using nanoindentation and reported mean values of hardness and elastic modulus lower than other vertebrates. This finding suggested that dolphins have a less biomechanically demanding dental function than other vertebrates that masticate and process their food. The considerably lower Ca/P content in dolphin enamel and dentine reported in this study when compared to values for human

C. Loch et al. / Journal of Structural Biology 185 (2014) 58–68

and hippopotamid dental samples (Cuy et al., 2002; Brügmann et al., 2012) also corroborates a less critical mineralization process in dolphin dental tissues, implying reduced biomechanical functional demand. Due to analytical constraints, two important components of hydroxyapatite could not be quantitatively measured in this study: the anions hydroxyl (OH) and carbonate ðCO2 3 Þ. While hydroxyl is part of the original composition of hydroxyapatite, carbonate is further incorporated into enamel and dentine during formation and maturation (Sydney-Zax et al., 1991; LeGeros et al., 1995; Wentrup-Byrne et al., 1997). Carbonates have profound effects on both physical structure and chemical stability of enamel, and have been implicated in susceptibility to dental caries and acid erosion (Nelson, 1981; Sydney-Zax et al., 1991; LeGeros et al., 1995). Sydney-Zax et al. (1991) reported a carbonate content of about 5% in human enamel at time of deposition, decreasing to about 3.5% in mature enamel. While the quantification of carbonate content on dolphin enamel and dentine still remains unknown, the changes in carbonate content across tooth regions were qualitatively shown using Raman spectroscopy. Quantification of carbonate content in enamel and dentine would require Raman analyzes of carbonate-apatite standards with known content in order to relate the peak area with the relative carbonate concentrations. Raman spectroscopy investigation of dolphin enamel and dentine showed characteristics similar to carbonated hydroxyapatite, with the strongest peak for the phosphate PO3 stretching mode 4 at 960–961 cm1. Other important features include phosphate peaks at the 400–600 cm1 range and the main carbonate CO2 3 stretching mode at around 1070–1072 cm1 (Kirchner et al., 1997; Wentrup-Byrne et al., 1997; Schulze et al., 2004). For all three species analyzed (Amazon River dolphin, pilot whale and Guiana dolphin), dentine samples revealed a higher diversity of peaks representative of organic components and proteins than enamel samples, mainly in the 1200–1700 cm1 range. Although the intensity of these peaks was not very high, this finding is consistent with a higher proportion of organic matter in dentine compared to enamel (Kirchner et al., 1997; Wentrup-Byrne et al., 1997). Integral ratios between the main phosphate peak and main carbonate and amide peaks suggested a change in the concentration of organic and inorganic components across enamel and dentine. The phosphate/carbonate ratio progressively increased from dentine towards mid enamel, suggesting a progressive decrease in carbonate content. According to the analysis reported here, the phosphate/carbonate ratio in enamel was about twice that of dentine, similar to the findings of Schulze et al. (2004). A higher phosphate/carbonate ratio was seen in the mid enamel region, followed by outer and inner enamel, suggesting a higher proportion of carbonate in the extremities of enamel compared to its mid region. Conversely, Wentrup-Byrne et al. (1997) reported a higher concentration of carbonates in the enamel area close to the EDJ in human dental samples. Both carbonate/amide and phosphate/amide ratios followed the trend of progressively increasing from dentine towards mid enamel, then slightly decreasing at outer enamel. This observation suggests a higher proportion of organic components in dentine, as expected (Kirchner et al., 1997; Wentrup-Byrne et al., 1997; Schulze et al., 2004). As in the trend observed for carbonates, the outer enamel layer seems to have a higher organic content compared to mid and inner enamel. This finding seems to correlate with the observed lower elastic modulus in the outer enamel of dolphins compared to inner and mid enamel (Loch et al., 2013b). The qualitative trend perceived for the association between bandwidth of the m1 phosphate band and wt.% of Ca and Mg showed that a higher concentration of Mg in dentine samples compared to enamel resulted in band broadening of the m1 phosphate peak. On the other hand, increased calcium content in enamel compared to

67

dentine resulted in a narrower bandwidth. As the bandwidth of the m1 phosphate relates to inhomogeneities in the mineral structure, the greater bandwidth indicates an increase in disorder within the unit cell also consistent with substitution of Ca with a different cation, in this case Mg (Thomas et al., 2007; 2011). Understanding the spatial distribution of mineral and organic contents in enamel and dentine are fundamental steps to elucidate the interaction between structural organization and biomechanical properties of dental tissues (Wentrup-Byrne et al., 1997). Together with its chemical composition, the morphology and anisotropic nature of dolphin enamel ultrastructure should also have a clear influence in its mechanical properties (Loch et al., 2013a,b). To date, this is the first effort to characterize the elemental and chemical composition of cetacean teeth. The results reported here are provisional due to the limited number of species and specimens analyzed, but a clear trend of the elemental variations can be perceived for all the dolphin teeth sampled. Uncertainties could arise because calculated elemental values derive from varied analytical techniques. Future studies should also explore other analytical techniques such as thermogravimetry and X-ray powder diffraction. Nevertheless, this study showed the value of a broad and multidisciplinary characterization of the chemistry of cetacean dental tissues, setting the foundations for future contributions with a wider range of species and specimens. Acknowledgments Thanks are extended to Andreas Auer and Kat Lilly (Department of Geology, University of Otago) for their technical assistance with WDX and EDX analyzes. We are very grateful to Paulo C. SimõesLopes (UFSC, Brazil), Danielle dos Santos Lima (IEPA, Brazil), Karen Stockin and Wendi Roe (Massey University, New Zealand) for providing specimens for this study. Thanks also to Robert Boessenecker (Department of Geology, University of Otago) for designing Fig. 1. Mark T. Clementz provided insightful comments in early drafts of this manuscript. Two anonymous reviewers also contributed with valuable suggestions to the final version of this text. C. Loch acknowledges the University of Otago for a PhD scholarship and Publishing Bursary. References Barnicoat, C.R., 1959. Wear in sheep’s teeth. N. Z. J. Agric. Res. 2 (5), 1025–1040. Bodart, F., Deconninck, G., Martin, M.T., 1981. Large scale study of tooth enamel. IEEE Trans. Nucl. Sci. 28 (2), 1401–1403. Brügmann, G., Krause, J., Brachert, T.C., Kullmer, O., Schrenk, F., Ssemmanda, I., Mertz, D.F., 2012. Chemical composition of modern and fossil hippopotamid teeth and implications for paleoenvironmental reconstructions and enamel formation – part 1: major and minor element variation. Biogeosciences 9, 119– 139. Committee on Taxonomy, 2012. List of marine mammal species and subspecies. Soc. Mar. Mammal. , consulted on 15 January, 2013. Cruwys, E., Robinson, K., Davis, N.R., 1994. Microprobe analysis of trace metals in seal teeth from Svalbard, Greenland, and South Georgia. Polar Rec. 30 (172), 49– 52. Cruwys, E., Robinson, K., Boyd, I.L., 1997. Measurements of calcium and phosphorus concentrations in the neonatal dentine of Weddell and crabeater seals using energy-dispersive X-ray analysis. Polar Rec. 33 (184), 21–28. Cuy, J.L., Mann, A.B., Livi, K.J., Teaford, M.F., Weihs, T.P., 2002. Nanoindentation mapping of the mechanical properties of human molar tooth enamel. Arch. Oral Biol. 47, 281–291. Dorozhkin, S.V., Epple, M., 2002. Biological and medical significance of calcium phosphates. Angew. Chem. Int. Ed. Engl. 41 (17), 3130–3146. Edmonds, J., Shibata, Y., Prince, R.I.T., Preen, A.R., Morita, M., 1997. Elemental composition of a tusk of a dugong, Dugong dugon, from Exmouth. W. Austr. Mar. Biol. 129 (2), 203–214. Enax, J., Prymak, O., Raabe, D., Epple, M., 2012. Structure, composition and mechanical properties of shark teeth. J. Struct. Biol. 178, 290–299. Flower, W.H., 1885. An Introduction to the Osteology of the Mammalia. Macmillan, London. Gutiérrez-Salazar, M.P., Reyes-Gasga, J., 2003. Microhardness and chemical composition of human tooth. Mater. Res. 6 (3), 367–373.

68

C. Loch et al. / Journal of Structural Biology 185 (2014) 58–68

He, L.H., Swain, M.V., 2008. Understanding the mechanical behaviour of human enamel from its structural and compositional characteristics. J. Mech. Behav. Biomed. Mater. 1 (1), 18–29. Hillson, S., 2005. Teeth. Cambridge Manuals in Archaeology, Cambridge. Jarosewich, E., Nelen, J.A., Norberg, J.A., 1980. Reference samples for electron microprobe analysis. Geostand. Newslett. 4, 43–47. Kirchner, M., Edwards, H., Lucy, D., Pollard, A.M., 1997. Ancient and modern specimens of human teeth: a fourier transform Raman spectroscopic study. J. Raman Spectrosc. 28 (2–3), 171–178. Labrada-Martagón, V., Aurioles-Gamboa, D., Castro-González, M.I., 2007. Relation of dental wear to the concentrations of essential minerals in teeth of the California sea lion Zalophus californianus californianus. Biol. Trace Elem. Res. 115 (2), 107– 126. LeGeros, R.Z., Kijkowska, R., Bautista, C., LeGeros, J.P., 1995. Synergistic effects of magnesium and carbonate on properties of biological and synthetic apatites. Connect. Tissue Res. 33 (1–3), 203–209. Loch, C., Duncan, W., Simões-Lopes, P.C., Kieser, J.A., Fordyce, R.E., 2013a. Ultrastructure of enamel and dentine in extant dolphins (Cetacea: Delphinoidea and Inioidea). Zoomorphology 132 (2), 215–225. Loch, C., Swain, M.V., Jansen van Vuuren, L., Kieser, J.A., Fordyce, R.E., 2013b. Mechanical properties of dental tissues in dolphins (Cetacea: Delphinoidea and Inioidea). Arch. Oral Biol. 58 (7), 773–779. Loch, C., Grando, L.J., Schwass, D.R., Kieser, J.A., Fordyce, R.E., Simões-Lopes, P.C., 2013c. Dental erosion in South Atlantic dolphins (Cetacea: Delphinidae): a macro and microscopic approach. Mar. Mamm. Sci. 29 (2), 338–347. MacKenzie, C.S.K., Clough, M.J., Broders, H.G., Tubrett, M., 2011. Chemical and structural composition of Atlantic Canadian moose (Alces alces) incisors with patterns of high breakage. Sci. Total Environ. 409 (24), 5483–5492. McGoverin, C.M., Rades, T., Gordon, K.C., 2008. Recent pharmaceutical applications of Raman and terahertz spectroscopies. J. Pharmaceut. Sci. 97 (11), 4598–4621. Myrick Jr., A.C., 1991. Some new and potential uses of dental layers in studying delphinid populations. In: Pryor, K., Norris, K.S. (Eds.), Dolphin Societies: discoveries and puzzles. University of California Press, Los Angeles, pp. 251– 279. Nelson, D., 1981. The influence of carbonate on the atomic structure and reactivity of hydroxyapatite. J. Dent. Res. 60 (3), 1621–1629.

Peek, S., Clementz, M.T., 2012. Sr/Ca and Ba/Ca variations in environmental and biological sources: a survey of marine and terrestrial systems. Geochim. Cosmochim. Acta 95, 36–52. Penel, G., Leroy, G., Rey, C., Bres, E., 1998. Micro-Raman spectral study of the PO4 and CO3 vibrational modes in synthetic and biological apatites. Calcif. Tissue Int. 63 (6), 475–481. Pinheiro, T., Carvalho, M., Casaca, C., Barreiros, M.A., Cunha, A.S., Chevallier, P., 1999. Microprobe analysis of teeth by synchrotron radiation: environmental contamination. Nucl. Instrum. Methods Phys. Res. B 158 (1), 393–398. Schulze, K., Balooch, M., Balooch, G., Marshall, G.W., Marshall, S.J., 2004. MicroRaman spectroscopic investigation of dental calcified tissues. J. Biomed. Mater. Res. A 69 (2), 286–293. Simpson, G.G., 1945. The principles of classification and a classification of mammals. Bull. Am. Mus. Nat. Hist. 85, 1–350. Sydney-Zax, M., Mayer, I., Deutsch, D., 1991. Carbonate content in developing human and bovine enamel. J. Dent. Res. 70 (5), 913–916. Thomas, D.B., Fordyce, R.E., Frew, R.D., Gordon, K.C., 2007. A rapid, non-destructive method of detecting diagenetic alteration in fossil bone using Raman spectroscopy. J. Raman Spectrosc. 38, 1533–1537. Thomas, D.B., McGoverin, C., Fordyce, R.E., Frew, R.D., Gordon, K.C., 2011. Raman spectroscopy of fossil bioapatite – a proxy for diagenetic alteration of the oxygen isotope composition. Palaeogeogr. Palaeoclimatol. Palaeoecol. 310 (1– 2), 62–70. Ungar, P., 2010. Mammal teeth: origin, evolution and diversity. The Johns Hopkins University Press, Baltimore. Veis, A., 1984. Bones and teeth. In: Piez, K., Reddi, A.H. (Eds.), Extracellular Matrix Biochemistry. Elsevier, New York, p. 329. Wentrup-Byrne, E., Armstrong, C.A., Armstrong, R.S., Collins, B.M., 1997. Fourier transform Raman microscopic mapping of the molecular components in a human tooth. J. Raman Spectrosc. 28 (2–3), 151–158. Werth, A.J., 2000. Feeding in marine mammals. In: Schwenk, K. (Ed.), Feeding: form, function and evolution in tetrapod vertebrates. Academic Press, San Diego, pp. 487–526. Yerramshetty, J.S., Lind, C., Akkus, O., 2006. The compositional and physicochemical homogeneity of male femoral cortex increases after the sixth decade. Bone 39 (6), 1236–1243.