Forensic Characterization of Nacre (Mother of Pearl) by pXRF and DRIFTS Analysis

Iridescence in Nacre

Nacre’s iridescent, color-changing appearance is a form of structural coloration caused by the interaction of light with a mollusks unique layered structure of protein and calcium carbonate; this is an example of diffraction and thin- film interference, similar to soap bubbles or oil slicks [1, 3, 4]. The iridescent, rainbow-like colors of nacre also appear to separate into their constituent spectral components on close visual evaluation, a phenomenon that could be attributed to dispersion [5]. Sun J, et al. [6] reviewed the development

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of the carbonate crystal layers and Madhav D, et. al. [1] discussed how incident light interacts with the structure of nacre to display the color shifts generated by the surface. Individual calcium carbonate crystals act as “bricks” that are held together by an organic matrix “mortar” that accounts for the remaining 5 wt.% of the shell and includes components such as polysaccharides and glycoproteins [7].

The hierarchical architecture of nacre is complex with varying spacing, crystal thickness, and orientation of the grooves and indentations of the crystal carbonate structure creating grating; this grating interacts with light resulting in the multi-color luster typical of nacre [1, 3, 4, 6]. We observed this intricate crystalline structure that is characteristic of nacre in a Pteria sp. (winged oyster) shell using a Phenom XL Desktop Scanning Electron Microscope (Nanoscience Instruments, Inc., Phoenix, AZ, USA) (Figure 1).

Visually the nacre layer disperses the light causing an iridescent glow or luminosity. Tan TL, et. al. [3] and Liu Y, et. al. [4] have described this phenomenon in abalone and pearl oyster shells where a crystal grating, produced by the calcium carbonate, creates structural rainbow-like colors from the interaction with incident light. Synthetically manufactured Mother of Pearl look-alike materials often reflect the incident light to produce a shiny effect (e.g., glitter), but this phenomenon is based on refraction and reflection of light [8]. Other manufactured materials use polymers which display iridescence by employing thinly grated surface features that successfully create structural color, mimicking MOP iridescence. However, these polymers do not contain calcium carbonate [8, 9, 10, 11]. Although some iridescent imitations can be transparent and film-like, pigments, shimmery flakes, and stains have frequently been observed to also be incorporated in the synthetic material [8, 10, 11]. Some manufacturers indicated that nacre, or MOP, products could be synthesized by taking small flakes of natural nacre and mixing them with either acrylic or glue to produce thin sheets or scales [12, 13], this approach produces a substance we refer to herein as “crafted nacre”.

Nacre in Trade

Limited research conducted by Chand S, et. al. [2] between 2010 and 2013, to study tourist purchasing habits in Fiji, estimated that MOP shell products contributed approximately 2.73 million USD to the local community [14]. Commercial online marketplaces such as Amazon® and Etsy© that sell natural MOP products and inlay materials do not always list the species name [15, 16]. This could affect the ability to successfully monitor trade and can lead importers and exporters to circumvent applicable tariffs.

Government agencies monitoring artisanal fishing activities, such as the harvesting of nacre products, are nearly non-existent. There is also evidence of under-declaring export volumes, as well as unreported and illegal trade [17, 18]. Accurate monitoring of species in trade ensures population stability for ecosystem viability, recreational use, and commercial fishing demands. An example of overfishing of pinto abalone occurred in Washington state from 1980 to 1992, and although commercial fishing was never permitted, local authorities authorized recreational harvesting through “daily take” and size limits [19]. An estimated 40,000 abalone were harvested yearly from combined recreational and illegal fishing; however, a single poacher admitted to taking upwards of 40,000 abalones to sell to restaurants, locally and overseas, mainly from pinto abalone herds during spawning season [19, 20]. This kind of behavior drastically reduced the population to near extinction [19, 20].

Employing a non-destructive analytical technique such as portable x-ray fluorescence spectrometry (pXRF) is useful in determining natural nacre. The portable XRF offers the ability for elemental analysis to be conducted in situ as well as the capability to analyze larger items that cannot fit into the chamber of typical micro-XRF systems like the EDAX, Orbis (AMETEK, Inc., Pleasanton, CA, USA) [21]. The objective of this study was to assess the ability of a portable XRF system to differentiate natural nacre from synthetic or imitation Mother of Pearl products employing elemental analysis. Additionally, we examined MOP and synthetic materials using diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) which allows for the identification of synthetic polymers used in look-alike MOP items.

Reference Materials and Samples

Natural mollusk shells (n=50), including samples of Haliotis spp. (abalone), Pteria spp. (winged oyster), and Pinctada spp. (oyster), that displayed natural nacre were analyzed. The shells were from multiple geographic locations including Mexico, Australia, the Philippines, and the United States of America (Table 1).

Elephant ivory specimens (n=11), Loxodonta spp. & Elephas maximus, and walrus ivory specimens (n=12), Odobenus rosmarus, were chosen to be used as controls for determining the daily accuracy of the pXRF because of their high calcium content [22, 23] and the ease with which the pXRF can detect this element. All shells and ivory are housed in the U.S. National Fish & Wildlife Forensic Laboratory (Ashland, OR) morphology collection. Synthetic MOP samples (n=18) and crafted nacre samples (n=2) were obtained from commercial sources and are summarized in Table 2 and Figure 2. Other artificial specimens were claimed by the manufacturer to be made of materials ranging from acrylic (polymethyl methacrylate or PMMA) to paint or adhesive (Table 2 and Figure 2).

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Visual Inspection for Iridescence

The presence of iridescence was determined by visual inspection via Video Spectral Comparator (VSC) 8000/ HS (Foster + Freeman Ltd., Evesham, Worcestershire, UK), utilizing a flood light and with a polarizing filter.

Portable XRF

Analysis of all samples listed in Table 1 and Table 2 were performed using a SciAps X-50 portable pXRF (SciAps Inc., Andover, MA, USA) featuring a Silicon Diode detector (SDD detector). Data was collected using Mining mode for quantitative determination of elemental composition using the following acquisition settings: exposure time of 30 seconds, the x-ray voltage set to 40 KeV and the current set to 32.4 µA. The diameter of the pXRF aperture is approximately 7mm. A set of five measurements per sample were collected from different locations, the flattest area of the sample surface was utilized for each measurement. No mechanical or solvent cleaning was performed on any of the samples prior to analysis.

Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) Analysis

Natural nacre shell specimens (n=5) and all the commercial synthetic specimens listed in Table 2 were analyzed using DRIFTS following the procedure described by Espinoza EO, et. al. [35] employing the Nicolet Nexus Smart Collector™ sample holder of the Nicolet iS50 FTIR (OMNIC v.9 software, Thermo Fisher Scientific Inc., Madison, WI, USA). Each DRIFTS spectrum collected consisted of 80 scans at a resolution of 4 nm that resulted in data spacing of 0.482 cm-1 with autogain, the 80 scans from one location were averaged to create each spectrum. Each item was sampled by sanding with 320 grit silicon carbide collection discs. The DRIFTS spectra of the commercial synthetic specimens were identified using a spectral match against a commercial polymer library and an in-house wildlife library containing appropriate refence spectra using OMNIC version 9 software (Thermo Fisher Scientific Inc., Madison, WI, USA).

Presence of Iridescence

Visual examination revealed the presence or absence of iridescence (Table 2). Iridescence was observed as a rainbow- like gradient that had a lustrous, luminosity (Figure 3A). In contrast, especially under polarized light, items that did not have iridescence were perceived as appearing dull and were nearly colorless (Figure 3B). Some items contained reflective or shimmery flakes in an attempt to mimic an iridescence from a distance, but they lacked the structural coloration in the form of rainbow-like colors that occur in natural nacre.

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Portable XRF Analysis

Portable XRF (pXRF) elemental analysis of the samples is summarized in Table 3. The natural nacre shell samples, and the two composite crafted nacre samples revealed higher relative concentrations of calcium (Ca) and strontium (Sr), and the results are shown graphically in Figure 4 and Figure 5. Giliken DP, et al. [36] showed that Ca/Sr ratios are strong indicators of biological control in marine aragonitic shells, and we have estimated these ratios for the samples analyzed. The calculated ratio of all the samples in this set is shown in Figure 6.

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The pXRF software can estimate the cumulative percentage of light elements (LE), atomic number <16, present in a sample. The relative concentration of LE is estimated from the intensity associated with Bremsstrahlung background intensity [37]. This phenomenon occurs due to the low energy emitted from LE being reabsorbed into the sample matrix or detector itself and scattered into the background (Bremsstrahlung) radiation [37, 38]. The graphical ploy of the LE percentage associated with the samples tested is shown in Figure 7 and it is important to note that synthetic polymers, which are primarily composed of low-Z elements (e.g., C, H, N, O) exhibited the highest LE percentages (Figure 7).

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Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) Analysis

DRIFTS analysis was done by collecting spectra from reference nacre shell specimens (n = 5) (three specimens of Haliotis rufescens; one specimen of Haliotis kamtschatkana; and one specimen of Pteria sterna), commercial synthetic specimens (n = 18), and crafted nacre (n = 2). The DRIFTS spectra of the tested samples are shown in Figure 8 and includes the averaged spectrum (n = 5) collected from five nacre specimens as well as the spectrum from one synthetic acrylic (PMMA) sample and of one crafted nacre composite sample consisting of a combination of both nacre and synthetic polymer.

The spectrum associated with the natural nacre (Figure 8A) shows a broad peak at 1420 cm-1, a unique peak 1085 cm-

1, a band stretch at 870-848 cm-1, and doublet peaks between 699 and 716 cm-1 that may be characteristic of calcium carbonate, specifically in the form of aragonite [39, 42]. Other distinguishing bands include a peak at 2926 cm-1, a lower intensity peak at 2853 cm-1, and a group of peaks at 1790 cm-1 and 1645 cm-1 that may be attributed to bond stretches and vibrations of the organic matrix [39, 42]. In contrast, the acrylic (PMMA) spectrum is distinct from the spectrum of the natural nacre where the peaks associated with aragonite are absent (Figure 8B). The acrylic (PMMA) spectrum also showcased a set of peaks that may suggest an organic moiety between ~3066 cm-1 and 2800 cm-1; a C=O bond stretch or vibration peak at 1745 cm-1, and possible unique peaks suggesting C-C-O bond presence at ~1246 cm-1 and ~1200 – 1155 cm-1.

DRIFTS analysis of the crafted nacre showed the spectrum to be like that of a variety of polymers and inconsistent with the reference spectra of nacre-producing mollusks. As previously stated, the manufacturer of the crafted nacre claimed that this material was a mixture of synthetic polymer and natural nacre flakes, and the DRIFTS analysis confirms that the spectrum was consistent with reference spectra of epoxy resin (Table 4). When comparing the spectrum of the crafted nacre (Figure 8C) to the natural nacre and acrylic (PMMA) spectra (Figure 8A and 8B), characteristic peaks and band stretches of esters that were seen in the acrylic spectrum are also observed in the crafted nacre spectrum. A small set of peaks that likely are associated with organic moieties are seen at 3018 cm-1 – 2800 cm-1 in the crafted nacre spectrum similar to the acrylic (PMMA) sample. Additionally, an ester element is also seen in the crafted nacre sample with peaks at 1745 cm-1, 1254 cm-1, and ~1116 cm-1.

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Figure 8: Natural nacre averaged DRIFT spectra of nacre shell (n = 5) specimens including one Haliotis kamtschatkana (pinto abalone), three specimens of Haliotis rufescens (red abalone), and one Pteria sterna (Pacific winged oyster) (A). The DRIFT spectrum of acrylic (PMMA) (B) and crafted nacre (C) are inconsistent with the natural nacre averaged spectrum.