Isotopic analysis is destructive and requires that a specimen retains its original (biogenic) chemical composition. A specimen's relative abundance of calcium and phosphorous (Ca/P) or carbonate and phosphate (CO3/PO4) is often used to assess preservation. If a specimen's Ca/P or CO3/PO4 is similar to modern specimens, a specimen's isotopic composition may be biogenic. However, most methods for measuring these proxies are destructive. Moreover the relationships between Ca/P, CO3/PO4 and isotopic preservation are poorly established. In this study, we assessed the ability of handheld X-ray fluorescence (hXRF) to non-destructively evaluate a specimen's preservation by characterizing the calcium to phosphorous ratio (Ca/P). We first established that surface Ca/P (Ca/Psurface) for modern specimens was consistent with expectations for unaltered bone (1.3–2.3). Several specimens had slightly larger ratios, suggesting the currently accepted range may need to be expanded. Second, we tested the ability of Ca/Psurface to detect alteration using twenty Quaternary mammal teeth from Big Bone Lick, Kentucky. Ten specimens had Ca/Psurface between 1.3 and 2.3 and ten had larger ratios, suggesting alteration. Because most methods measure Ca/P in powder (Ca/Ppowder), we compared Ca/Psurface, Ca/Ppowder, and the enamel subsurface (Ca/Psubsurface). With two exceptions, Ca/Psubsurface and Ca/Ppowder were below 2.3, regardless of Ca/Psurface, suggesting that Ca/Ppowder and Ca/Psubsurface underestimate alteration. We next compared Ca/Psurface, CO3/PO4, and carbon (δ13C) and oxygen (δ18O) isotope values for the fossil teeth. Fourteen specimens were identified as altered or unaltered by both proxies, but six specimens only had one altered proxy. Specimens with both proxies altered had lower, less variable δ13C values than specimens with both proxies unaltered. Median δ18O values were similar between these groups. Individuals with altered Ca/Psurface but unaltered CO3/PO4 isotopically resembled specimens with both proxies altered. Conversely, specimens with unaltered Ca/Psurface and altered CO3/PO4 were similar to specimens with both proxies unaltered. Notably, all individuals with both proxies altered had relatively low δ13C values, including a horse and mammoth, which are normally considered grazers (and therefore should have higher δ13C values). These and other altered specimens may be isotopically compromised. Overall, our results suggest that Ca/Psurface is effective at detecting alteration non-destructively, quickly, and affordably, making it an attractive approach for analyzing unique specimens.

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Most researchers assume minimal impact of pretreatment on strontium isotope ratios (87Sr/86Sr) for bones and teeth, and methods vary tremendously. We compared 14 pretreatment methods, including no prep other than powdering enamel, ashing, soaking in water, an oxidizing agent (bleach or hydrogen peroxide) or acetic acid (0.1 M, 1.0 M, and 1.0 M buffered with calcium acetate), and a combination of these steps. We prepared and analyzed aliquots of powdered molar enamel from three proboscideans (one modern captive Indian elephant, Elephas maximus indicus; one Pleistocene mastodon, Mammut americanum; and one Miocene gomphothere, Afrochoerodon kisumuensis). Each pretreatment was performed in triplicate and we measured 87Sr/86Sr, Sr concentration, and uranium (U) concentration, using the same lab space and instrumentation for all samples. Variability in 87Sr/86Sr and Sr and U concentrations was considerable across pretreatments. Mean 87Sr/86Sr across methods ranged from 0.70999 to 0.71029 for the modern tooth, 0.71458 to 0.71502 for the Pleistocene tooth, and 0.70804 to 0.70817 for the Miocene tooth. The modern tooth contained the least Sr and negligible U. The Pleistocene tooth contained slightly more Sr and measurable amounts of U, and the Miocene tooth had approximately 5x more Sr and U than the Pleistocene tooth. For all three teeth, variance in 87Sr/86Sr, Sr concentrations, and U concentrations among replicates was statistically indistinguishable across pretreatments, but there were apparent differences among pretreatments for the modern and Pleistocene teeth. Both contained relatively little Sr, and it is possible that small amounts of exogenous Sr from reagents, building materials or dust affected some replicates for some pretreatments. For the modern tooth, median 87Sr/86Sr varied considerably (but statistically insignificantly) across pretreatments. For the Pleistocene tooth, variability in median 87Sr/86Sr was also considerable; some pretreatments were statistically distinct but there were no obvious patterns among methods. For the Miocene tooth, variability in median 87Sr/86Sr was much smaller, but there were significant differences among pretreatments. Most pretreatments yielded 87Sr/86Sr and Sr concentrations comparable to, or lower than, untreated powder, suggesting selective removal of exogenous material with high 87Sr/86Sr. Further evaluation of the mechanisms driving isotopic variability both within and among pretreatment methods is warranted. Researchers should clearly report their methods and avoid combining data obtained using different methods. Small differences in 87Sr/86Sr could impact data interpretations, especially in areas where isotopic variability is low.