Elemental classification of the tusks of dugong (Dugong dugong) by HH-XRF analysis and comparison with other species

The elemental composition was investigated and applied for identifying the sex and habitat of dugongs, in addition to distinguishing dugong tusks and teeth from other animal wildlife materials such as Asian elephant (Elephas maximus) tusks and tiger (Panthera tigris tigris) canine teeth. A total of 43 dugong tusks, 60 dugong teeth, 40 dolphin teeth, 1 whale tooth, 40 Asian elephant tusks and 20 tiger canine teeth were included in the study. Elemental analyses were conducted using a handheld X-ray fluorescence analyzer (HH-XRF). There was no significant difference in the elemental composition of male and female dugong tusks, whereas the overall accuracy for identifying habitat (the Andaman Sea and the Gulf of Thailand) was high (88.1%). Dolphin teeth were able to be correctly predicted 100% of the time. Furthermore, we demonstrated a discrepancy in elemental composition among dugong tusks, Asian elephant tusks and tiger canine teeth, and provided a high correct prediction rate among these species of 98.2%. Here, we demonstrate the feasible use of HH-XRF for preliminary species classification and habitat determination prior to using more advanced techniques such as molecular biology.

The dugong (Dugong dugon, Müller), or sea cow, is one of four herbivorous marine mammal species surviving in the family Dugongidae, order Sirenia. The dugong grazes exclusively on seagrass and is thus limited to coastal habitats where there is an abundance of seagrass meadows 1 . Currently, dugong populations are persistently declining due to anthropogenic causes, including illegal hunting, habitat degradation and fishing-related fatalities 2 . The dugong is a vulnerable species and has been placed in Appendix I of the Convention on International Trade in Endangered Species (CITES).
Unlike other mammalian species, the study of dugong teeth is not well-established. The dugong possesses a pair of tusks, formed by the first upper incisor teeth in both males and females, that are used as cutting instruments for foraging 3 . Sexual dimorphism is exhibited in dugongs by tusk eruption in males after puberty due to increased testosterone levels [4][5][6] , and also rarely in females older than 40 years 5 . Dugong tusks have been used for age estimation based on dentinal growth layer groups (GLGs) and cheek-tooth development using a multiple logarithmic regression model 4,[7][8][9] , as well as puberty prediction based on erupted teeth 4,5 . A relationship was also demonstrated between behavioral characteristics and testosterone content in tusks 6 . Furthermore, the results of an osteological study and skull measurements of dugongs from India suggested an osteological similarity between dugongs from India and the Red Sea, whereas a discrepancy in skull measurements was observed in the different regions 10 . From a literature review, we found only two publications reporting on elements in dugong tusks. A study in 1979 was able to identify real dugong tusks using strontium (Sr) content and microstructure 11 , while a second study in 1997 demonstrated the difference in elemental distribution in dentinal growth layer groups 1 Animal Bone and Joint Research Laboratory, Department of Veterinary Biosciences and Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand. 2 Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand. 3 Phuket Marine Biological Center, Phuket 83000, Thailand. 4 Center of Excellence in Elephant Research and Education, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand. Correspondence and requests for materials should be addressed to K.N. (email: korakot.n@cmu.ac.th) Handheld X-ray fluorescence. Teeth elemental analyses were conducted using a handheld X-ray fluorescence analyzer (HH-XRF) (DELTA Premium; Olympus, Tokyo, Japan), which uses a silicon drift detector, detecting from magnesium (Mg 12 ) through bismuth (Bi 83 ) on the periodic table. The collimator size was set at 0.3 mm for the analysis area diameter, using the standard mining plus mode. Calibrations were performed each day before the first use of the HH-XRF for sample analysis. Light elements (LE) were those with an atomic number lower than Mg (H 1 -Na 11 ), which could not be differentiated as single elements. For each scan (2 min each), the XRF unit was secured in a stand and the sample was placed directly adjacent to the puncture-resistant window of the machine to limit the distance between the detector and sample. Each element was expressed as a percentage. The XRF method was noninvasive, and samples were not manipulated or destroyed in the process of scanning.

Study design and statistical analysis.
To more fully understand the elemental composition in dugong tusks and teeth, this research was categorized into seven studies. The samples used in each study were different, depending on the recorded history of samples. The results of each element were presented as a percentage. Moreover, the ratio between Ca and P was calculated in all studies because they are the major inorganic components of calcium phosphate [Ca 3 (PO 4 ) 2 ]; this interacts with calcium hydroxide [Ca(OH) 2 ] to form hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ], which can indicate teeth strength 16,17,38,39 . Study 1: Elemental analysis comparing the crown and root of dugong tusks. This study compared the elemental composition between the crown and root of dugong tusks. In this study, 10 tusks were used as subjects. We scanned the crown (2 or 3 different locations) and root (3 or 4 different locations) of each tusk (Fig. 1A). Elements were presented as a percentage and were compared between the two groups using Mann-Whitney U test for nonparametric data analysis. Study 2: Elemental analysis comparing different tusk layers. Dugong tusks are large, elongated upper incisor teeth. To study the difference of elemental accumulation between different tusk layers, longitudinal sections of 10 permanent tusks were used as subjects. We scanned the superficial, intermediate and medial layers of each tusk (Fig. 1B). Elements were presented as a percentage and were compared between the three groups by one-way ANOVA, followed by Mann-Whitney U test. Study 3: Elemental analysis comparing small and large permanent tusks. In this study a total of 39 dugong tusk samples, 15 small tusks (length < 7 cm) and 24 large tusks (length > 7 cm), were used as subjects (Fig. 1C,D). To study the difference of elemental accumulation by tusk size, the surface of each tusk was randomly scanned in 3-5 different locations. Elements were presented as a percentage and were compared between the two groups using Mann-Whitney U test for nonparametric data analysis. Study 4: Elemental analysis comparing dugong sexes. As is well-established, there is a discrepancy in the metabolism of males and females 17,40,41 . To study the accumulation of elements between sexes, a total of 42 tusk samples were examined, consisting of 17 tusks from males and 25 tusks from females. The surface of each tusk was randomly scanned in at least 3-5 different locations. Elements were presented as a percentage and were compared between the two groups using Mann-Whitney U test for nonparametric data analysis. To establish an equation for sex determination, we compared the elemental composition of male and female dugong tusks through a stepwise discriminant analysis with leave-one-out classification.  7 tusks taken from the Gulf of Thailand. The surface of each tusk was randomly scanned in at least 3-5 different locations. Elements were presented as a percentage and were compared between the two groups using Mann-Whitney U test for nonparametric data analysis. To establish an equation for habitat estimation, we compared the elemental composition of dugong tusks from the Andaman Sea and the Gulf of Thailand through a stepwise discriminant analysis with leave-one-out classification. Study 6: Elemental analysis comparing dugong tusks, dugong, dolphin and whale teeth. The aim of this study was to analyze the difference in elemental composition of dugong tusks and teeth compared with the teeth of two other marine mammals, dolphin and whale. A total of 43 dugong tusks (n of animals = 43), 60 dugong teeth (n = 30), 40 dolphin teeth (n = 10) and 1 whale tooth (n = 1) were subjected to HH-XRF. The surface of each dugong tusk was randomly scanned in 3-5 different locations, dugong and dolphin teeth in two different locations, and the whale tooth in six different locations. Elements were presented as a percentage and were compared between the four groups using one-way ANOVA, followed by Mann-Whitney U test. To establish an equation for species differentiation, we compared the elemental composition of tusks and teeth among dugong, dolphin and whale through a stepwise discriminant analysis with leave-one-out classification. Study 7: Elemental analysis comparing dugong tusks, Asian elephant tusks and tiger canine teeth. Elephant tusks were selected as representative of land mammals for purposes of comparison with marine mammals. In addition, tiger (Panthera tigris tigris) canine teeth were also included in the study due to their high value in the illegal market, similar to dugong and elephant tusks. A total of 43 dugong tusks (n of animals = 43), 40 male Asian elephant tusks (n = 40) and 20 tiger canine teeth (n = 5) were subjected to HH-XRF. The surface of each sample was randomly scanned in at least 3-5 different locations. To establish an equation for tusk species determination, we compared the elemental composition of dugong tusks, Asian elephant tusks and tiger canine teeth through a stepwise discriminant analysis with leave-one-out classification.

Study 1: Elemental analysis comparing the crown and root of dugong tusks.
A total of 20 elements plus LE were detected in the crown and root (Table 1). There were seven elements that were significantly higher in the crown compared with the root: titanium (Ti), vanadium (V), manganese (Mn), Fe, silver (Ag), tin (Sn) and antimony (Sb). The Ca/P ratio was higher in the root than in the crown (p > 0.05).

Study 2: Elemental analysis comparing different tusk layers. A total of 22 elements and LE were
detected in three different layers of the tusks ( Table 2). Only 18 elements were found in all three layers, including aluminum (Al), silicon (Si), P, sulfur (S), Ca, chromium (Cr), nickel (Ni), copper (Cu), Zn, zirconium (Zr), cadmium (Cd), Ti, V, Mn, Fe, Ag, Sn and Sb. Four elements were found only in some layers: chlorine (Cl) and potassium (K) were not detected in the superficial layer, while tungsten (W) and lead (Pb) were not detected in the intermediate and medial layers of tusks. Three elements (Ni, Cu and Zr) showed a similar content (p > 0.05) in all three layers. The Ca/P ratio in the superficial layer was significantly lower compared with the other two layers.

Study 3: Elemental analysis comparing small and large permanent tusks.
A total of 20 elements plus LE were detected in small and large dugong tusks (Table 3). Three elements were significantly higher in small tusks, including P, Ca and Zr, while Fe, Pb, LE and Ca/P ratio was significantly higher in large tusks.

Study 4: Elemental analysis comparing dugong sexes.
A total of 20 elements plus LE were detected in tusks from both sexes (Table 4). Only one element (Ag) showed a significant difference between sexes (higher in males than females). LE in females were significantly higher than in males. The elemental composition of dugong tusks was used for creating an equation for assigning sex, as follows: An overlapping area of discriminant values obtained from the Andaman Sea and the Gulf of Thailand was observed (Fig. 3). We found that this equation could correctly identify dugongs from the Andaman Sea with high accuracy, 93.7%, but with a low level of correct prediction for dugongs from the Gulf of Thailand (Table 7). The overall accuracy for estimating location was 88.1%.     Table 8). We found that for two elements, nickel (Ni) and lead (Pb), there was not a significant difference between the four tooth types. The Ca/P ratio was significantly higher in dugong tusks, and significantly lowest in dolphin teeth. We created an equation for differentiating dugong tusks and dugong, dolphin and whale teeth based on their elemental composition, as shown in Fig. 4. Dolphin teeth were able to be correctly predicted 100% of the time, followed by dugong tusks, dugong and whale teeth, with correct prediction rates of 79.3%, 75.3% and 75%, respectively ( Table 9). The whale tooth was misidentified as a dugong tusk 25% of the time ( Table 9). The overall accuracy of differentiating species by this equation was 79.3% (Table 9).        Study 7: Elemental analysis comparing dugong tusks, Asian elephant tusks and tiger canine teeth. To differentiate among dugong and Asian elephant tusks and tiger canine teeth, their elemental composition was used to establish an effective equation using stepwise discriminant analysis. As shown in Fig. 5, a discrepancy of discriminant values between dugong tusks and teeth, Asian elephant tusks and tiger canine teeth was noted, indicating a high correct prediction rate among species of 98.2% (Table 10). However, there was a small misclassification within tiger canines, which were predicted as Asian elephant and dugong tusks 26.3% and 2.6% of the time, respectively.

Discussion
When we compared the elements found in dugong teeth and tusks using a HH-XRF with either tusks or teeth of other species, the data obtained consisted of four important points. (i) Three major elements with quantity > 1%, i.e. Ca, P and Si, could be detected in dugong tusks and teeth, while Al was found only in dugongs (at a low level) but in no other species. (ii) Elemental analysis can likely be used for differentiating dugong tusks from different habitats, with a high accuracy rate (88.1%). (iii) It was feasible to distinguish dugong teeth from two other marine  Table 9. Classification result of species prediction using the equation obtained from elemental composition. a Cross-validation is done only for those cases in the analysis. In cross-validation, each case is classified by the functions derived from all cases other than that case.  Table 10. Classification result of tusk species prediction using the equation obtained from elemental composition. a Cross-validation is done only for those cases in the analysis. In cross-validation, each case is classified by the functions derived from all cases other than that case. mammal species (dolphin and whale), with a 79.3% accuracy rate. (iv) Moreover, dugong tusks could be distinguished from elephant tusks and tiger canine teeth with 100% accuracy by elemental analysis.
Elements in dugong tusks. From studies 1-3, it was shown that the distribution of elements was not even in tusk samples. Most of the elements accumulated in a greater amount at the surface of the crown as compared with the root. However, there was no difference in the distribution of the major elements (Ca, P, and Ca/P ratio). A comparison of the three different layers of dugong tusks revealed that most elements showed the highest accumulation in the surface layer, whereas the Ca/P ratio was lowest in the superficial layer. These results were in contrast with a study of the elements in human, bovine, porcine and ovine teeth, in which the Ca/P ratio was higher in the superficial (enamel) layer than in the dentine layer 26 . However, the surface area of dugong tusks is not covered with enamel, as in other species; the enamel covers only the tips of tusks when dugongs are young, but the enamel disappears in older animals 4 . It is possible that because of the continued growth of tusks from inside to outside throughout life 4 , the change of elemental accumulation in the superficial layer occurs over a longer period than in the medial layer. The Ca/P ratio is representative of calcium hydroxyapatite and is indicative of the strength of bones and teeth 16,17,38,39 . There was no difference in the Ca/P ratio between the crown and root of dugong tusks, but it was significantly higher in the intermediate and medial layers compared with the superficial layer. However, we found that large tusks had a significantly higher Ca/P ratio than small tusks. Moreover, dugong tusks had a higher Ca/P ratio than the other three marine tissues studied (dugong teeth, dolphin teeth and whale tooth).
Elements and the estimation of sex and habitat. In this study, we analyzed the elemental content in tusks for two main purposes: (i) sex and (ii) habitat estimation. We found that most of the elements were not significantly different between sexes, except Ag which was significantly higher in males. Silver is one of the heavy metals and can be toxic to aquatic animals 43 . However, we cannot explain why Ag was higher in male dugong tusks. According to some reports in the literature, it is believed that gender slightly influences the accumulation of heavy metals in the body 18,31,44 . However, it was previously demonstrated in striped dolphin (Stenella coeruleoalba) that gender exhibited little effect on the accumulation of some heavy metals (Fe, Mn, Zn, Cu, Pb, Ni, Cd and Hg) in body tissues 44 . Moreover, in a recent study of 47 canine compact bone types, little relation was found between gender and the accumulation of heavy metals 31 . The best equation for determining sex based on the data of elemental content in dugong tusks provided a moderate accuracy rate of 68.9%. This result indicated that elemental content may not be pertinent for assigning sex in dugongs. Also, the elemental content in male and female elephant tusks is not much different 25 . In humans, the elemental content in bone (cranium, humerus, os coxae and their combination) has been used for sex classification, but it only gave an accuracy rate ranging from 60 to 67% 21 . Taken together, we have concluded that elemental analysis might not be a worthy tool for sex identification in dugongs.
In addition, we used the elemental content of dugong tusks for the estimation of the location of the dugongs' habitat. It was found that Al and W were present in dugong tusks from the Andaman Sea, and that there was a significant difference in S and Ti content between tusks from the Andaman Sea and the Gulf of Thailand. The differences in these four elements (Al, W, S and Ti) might be due to industrial contamination of the coastline, which is the habitat of dugongs, or because some elements are found in a particular area. For instance, W is a rare earth mineral which is found in some areas of Thailand 45 . It is possible that these elements accumulated in seagrass, which is the main food of dugongs [46][47][48] . An equation for discriminating dugong tusks from the Andaman Sea and the Gulf of Thailand showed an accuracy rate of 88.1%. Previously, our team utilized elemental content to estimate the origin of elephant tusks (Asia or Africa) with an accuracy rate of 94% 25 . This evidence demonstrates that elemental content could serve as an effective method for differentiating the origin or habitat of animals.
Elements and species identification. Aluminum was detected in dugongs only (both teeth and tusks), but not in dolphin and whale teeth. In a recent study of the elemental distribution in eight species (deer, dog, elephant, horse, human, monkey, dolphin and crocodile), Al was detected in elephant, human and monkey teeth 24 . This suggests that Al might be involved in some physiological pathway of dugongs, elephants, humans and monkeys. Dugongs and elephants belong to the same clade, Tethytheria, while humans and monkeys belong to the same order, Primatomorpha. To clarify our hypothesis, further work should be done.
In comparing dugong tusks, and dugong, dolphin and whale teeth, there was a correct prediction rate of 100% for dolphin teeth. Dugong tusks were correctly predicted 79.3% of the time; the misclassification rate was 20.7%, consisting of 15.7% predicted as dugong teeth and 5% predicted as whale tooth. A previous study showed an accuracy rate of 78.4% for identifying eight tooth species based on their elemental content 24 . Furthermore, the elemental content among dugong tusks, Asian elephant tusks and tiger canine teeth could be differentiated with an overall accuracy rate of 98.2%. We found that dugong tusks could be completely discriminated from Asian elephant tusks and tiger canine teeth, while some Asian elephant tusks were predicted as tiger teeth (0.7%) and some tiger teeth were predicted as dugong tusks (2.6%) and Asian elephant tusks (26.3%). It is plausible that dugongs and the other two animals have a significantly distinct habitat (land and marine), resulting in a clear difference in elemental composition in spite of the fact that they were scanned at the same region (upper incisor) in both species. Similarly, a previous study demonstrated that the identification of tusks from elephants living on different continents, Asia and Africa, also showed a high accuracy rate of 94% 25 . Limitations. There were some limitations of this study. HH-XRF equipment is convenient to transport and use in the field; however, it only scans elements at the surface of a sample. Thus, if the surface is treated with chemical agents or protectants that contain elements detected by XRF, the percentages could be altered and lead to inaccurate measurements and false identification. For this reason, all samples were untreated and cleaned of any dirt and debris before scanning. The HH-XRF machine does not provide probing volumes, so we could not calculate elemental concentrations; rather, these were presented as percentages. Elemental composition was compared through stepwise discriminant analysis. Light elements in samples that were not quantifiable by HH-XRF analysis had an impact on some elemental proportions, so the use of methodology to quantify these components might provide additional information that could lead to further improvements in discrimination functions. Finally, the number of samples from some species, whales in particular, was low because of the rarity of samples.

Conclusion
The elemental composition of biological materials is useful in a wide range of sciences. Our results have demonstrated the utilization of elements in tusks and teeth for differentiating animal species. In addition, we observed that elemental analysis of tusks and teeth could not be used to distinguish between male and female dugongs. In contrast, elemental analysis was shown to be an excellent candidate tool for estimation of animal habitat, as it could distinguish dugong tusks from the Andaman Sea and the Gulf of Thailand. These results may be beneficial for law enforcement authorities in resolving the difficulty of identification of tusks. Finally, we described the potential feasibility of utilizing handheld XRF to examine tusks and teeth for the preliminary determination of species, prior to using more advanced techniques such as molecular biology.