Sperm Whale, Elephant or Hippo? Using Raman Spectroscopy as a Non-destructive Method to Identify Unknown Ivory Samples

Abstract Body : The trade in ivory is a worldwide problem. Although many countries have banned the sale of modern elephant ivory, there are variations in regulations surrounding the trade in ivory from different animal species. Ivory is a highly mineralized material with the chemical structure Ca10(PO4)6(CO3)H2O. Current methods used for determining the species of origin of an ivory sample at customs are DNA and/or isotope analysis. These are destructive and time-consuming methods, and owners are reluctant to perform these techniques on potentially legal objects of high monetary or cultural value. Although it is possible to visually tell the difference between the ivory of different species in its natural state using Schreger lines, once samples have become worked or carved, this becomes much harder. Previous work has demonstrated that Raman spectroscopy, a vibrational spectroscopic technique, can identify biochemical differences in elephant and mammoth ivory.
The hypothesis of this project was that the biochemistry of ivory between different species is distinguishable using Raman spectroscopy. This hypothesis was tested using slices of ivory from Physeter macrocephalus (sperm whale), modern Elephantidae (elephant, species and genus unknown) and Hippopotamus amphibius (hippo). These samples were scanned with an inVia Raman micro spectrometer (Renishaw Ltd, Glostershire, UK) equipped with an Olympus 50×/0.5 long working distance objective lens and a 785nm laser, ~200mW at source. Each spectrum was collected for 60s (6 × 10 s accumulations). Spectroscopic analyses were taken at a minimum of twenty different sites for each sample to provide replicates and all spectra were acquired in the spectral range ~600-1700 cm-1. Line maps were used to analyse differences from the medulla to the cortex of each tusk. Data was analysed with Matlab (v2021a, The Mathworks, Inc., Natick, MA, USA) and baseline corrected using a sixth order polynomial subtraction and vector normalised using an in-house script. Principal component analysis (PCA) and PCA-linear discriminant analysis (PCA-LDA) was performed on this data. Data was grouped by species. Univariate analysis of the phosphate to amine III peak was performed using Microsoft Excel (Microsoft Corporation) to compare the mineral to collagen ratio of samples.
The results of this study demonstrate differences and separation in the spectral ‘fingerprint’ of ivory from different species of mammal. PCA and PCA-LDA analysis demonstrates distinct grouping of different species with some inter-sample variation. Line maps demonstrate differences in the hydroxyapatite peaks from cortex to medulla, suggesting an increase in mineralisation, with the sample sperm whale demonstrating the thickest outer layer of enamel. Calculating the peak intensity ratios between organic v(CH) collagen peaks and inorganic v(PO) hydroxyapatite peaks also demonstrates clear differences between species.
Raman spectroscopy demonstrates potential as a tool in the identification of ivory samples of unknown origin. Further work aims to compile a larger database of samples to be used as reference spectra. This would aid in the development of a quick and easy method of determining ivory species to prevent illegal trade. Increased surveillance and monitoring of samples passing through customs worldwide using Raman spectroscopy could act as a deterrent to those poaching endangered and critically endangered species of elephant.