Journal of the North Atlantic T.D. Price 2015 Special Volume 7 71 Introduction A Scottish chemist, Frederick Soddy, coined the term “isotope” in 1912. He observed what now defines an isotope—that the number of protons in the atomic nucleus determines the chemical properties and identity of an element, but there can be different numbers of neutrons associated with the same number of protons, resulting in the same element having several variations with different weights (or masses). These different structural formations of an element are its isotopes. The beginnings of isotopic applications in archaeology probably dates to the mid-1960s with the analysis of lead isotopes in ancient metals and slags by Brill and Wampler (1965). Today the use of isotopes in archaeological chemistry is widespread and rapidly growing, with many kinds of applications. Various isotopes are analyzed in different kinds of materials to answer a variety of questions about the past. Isotopes of oxygen, carbon, nitrogen, lead, and strontium isotopes are commonly used in archaeological applications. For the study of human remains, these isotopes are important because they get into our bodies through the food we eat and the water we drink and are deposited in measurable amounts in our skeleton. Interest in the isotope chemistry of human skeletal remains began in the late 1970s as a spinoff from radiocarbon dating (e.g., Bender et al. 1981, Tauber 1981, Vogel and van der Merwe 1977). The isotopic analysis of human skeletal remains generally involves two types of questions: concerning either past diet or human provenience. Isotopic studies of ancient human remains began in almost 40 years ago with the recognition that different diets produced different carbon isotope ratios in the tissues of animals (e.g., DeNiro and Epstein 1978). Almost simultaneously there was a recognition that different pathways of photosynthesis produced different ratios of 13C/12C in plants (O’Leary 1981, Smith and Epstein 1971). This information was soon applied to the study of the spread of corn from its homeland in Mexico to North America (Bender et al. 1981, Vogel and van der Merwe 1977). Around the same time, the impact of marine foods on carbon isotope ratios in human tissue was reported in seminal papers by Tauber (1981) and Chisholm et al. (1982). The isotopic investigation of human provenience began in the mid-1980s with both practical and theoretic publications. Krueger (1985) measured strontium isotope ratios in a series of human and animal samples from the Maya region in Central America and reported significant geographic differences. That same year Ericson (1985) proposed the application of strontium isotopes to questions concerned with place of origin. The method slowly gained attention with an important dissertation published in South Africa (Sealy 1989) and papers by Price et al. (1994a, 1994b) concerned with place-oforigin studies in early Bronze Age Europe and the prehistoric southwestern US. Both questions—past diet and place of origin— are of interest in the investigation of the Norse colonization of the Atlantic. Such studies involve both the light isotopes of carbon, nitrogen, and oxygen, and the heavy isotopes of strontium and lead. Each of these isotopic methods will be introduced and described in some detail below. In addition, there are two primary types of tissues that have been used in such studies: bone and tooth enamel. These tissues are considered below prior to a discussion of the isotopic methods. A brief summary of sample preparation and measurement for these isotopic methods is provided in Appendix 1. An Introduction to the Isotopic Studies of Ancient Human Remains T. Douglas Price* Abstract - This paper provides an introduction to methods in archaeology that utilize light isotopes for helping to determine diet and heavier isotopes for information on mobility and provenience of specimens. The common isotopic systems of carbon, nitrogen, oxygen, strontium, and lead are described in terms of basic principles, specifically with references of human bone and tooth enamel. Isotopic analyses of carbon and nitrogen in collagen for past diet provides information on certain food types (e.g., C4 plants, marine resources, freshwater fish) and trophic level, while the analysis of oxygen, strontium, and lead can provide information on local vs. non-local origins, mobility, and place of origin. Analysis of tooth enamel provides information on childhood contexts, and the analysis of adult bone provides information on the later years of life. This paper is intended to provide the methodological background for the isotopic analysis of human and faunal materials from the Viking colonization of the North Atlantic. Viking Settlers of the North Atlantic: An Isotopic Approach Journal of the North Atlantic *Laboratory for Archaeological Chemistry, University of Wisconsin-Madison, Madison, WI, USA 53706; tdprice@wisc.edu. 2015 Special Volume 7:71–87 Journal of the North Atlantic T.D. Price 2015 Special Volume 7 72 Tooth Enamel and Bone Bone and tooth enamel incorporate chemical signals from different periods in an individual’s life. Bone is a relatively plastic material, containing both an organic (mostly collagen) and inorganic (mostly the mineral hydroxyapatite) phase. Collagen is the major structural protein in bone, and also forms the molecular cables that strengthen the tendons and vast, tough sheets of tissue that support the skin and internal organs. Because bone is a dynamic material, it is constantly changing and remodeling, adding new material and losing old. Thus, the chemical composition of bone reflects the chemistry of diet and place of residence of the later years of life. Enamel, on the other hand, forms during early childhood and is constructed from nutrients eaten by the mother and the young child (Hillson 2005). Calcification of the enamel crown of most of the permanent teeth (with the exception of M3) is normally completed by the age of six (AlQahtani et al. 2010, ElNesr and Avery 1994, Ten Cate 1998). Formation time for the permanent teeth (except M3) is shown in Figure 1. M3 begins forming between the ages of 8–10, and the crown of the tooth is complete between 12–16 years of age in most individuals. Because tooth enamel, composed primarily of the mineral hydroxyapatite, does not change during one’s lifetime, it retains the chemistry of the place of birth and childhood (Ericson 1985, Krueger 1985, Price et al. 1994b). A variety of studies have demonstrated that enamel is highly resistant to post-mortem contamination (e.g., Budd et al. 2000, Hoppe et al. 2003, Kohn et al. 1999, Lee-Thorp and Sponheimer 2003). Because of these differences in tissue formation and the inert nature of enamel, we have available in human skeletal remains the means to examine some of the conditions in the life history of a single individual (e.g., Sealy et al. 1995). Studies of paleodiet have focused primarily on bone, and specifically bone collagen, while studies of human provenience have concentrated on dental enamel. Collagen is usually extracted from cortical bone for the analysis of carbon and nitrogen isotopes. Procedures for this extraction have been described in detail in a number of publications (e.g., Ambrose 1990, Chisholm et al. 1983, Liden et al. 1995, Longin 1971). The principles for human proveniencing rely on isotopes that exhibit geographic variation and are deposited in the human skeleton in early childhood. Strontium, and sometimes lead and oxygen, in tooth enamel meet those criteria. Comparison of the isotope ratios in tooth enamel with the bone of the same individual or with measured local isotope ratios can provide an indication of local or non-local origin. In our analyses, we normally sample the M1 for enamel; other teeth are taken when the M1 is not available. Enamel is largely composed of apatite, so little preparation is necessary. The outer surface of the enamel is scoured, and 10–20 milligrams removed for analysis Larger samples are needed for lead. More information on method and procedure will be provided below. Carbon Isotopes in Collagen The measurement of carbon isotope ratios in bone collagen is well known in the study of marine resources or certain types of plants in human diets (e.g., Chisholm et al. 1982, Schoeninger and DeNiro 1984, Tauber 1981, van der Merwe and Vogel 1977). The ratio of carbon isotopes in collagen reflects the ratio Figure 1. Age and duration of enamel and dentin formation in teeth (Knipp er 2011). Journal of the North Atlantic T.D. Price 2015 Special Volume 7 73 of the diet. The method has been in use for a number of years and is well established. Carbon and nitrogen isotope analyses today are a routine part of the study of human skeletal remains. These ratios are usually calculated along with radiocarbon measurements at dating laboratories. There are other isotopes that may also be of interest in the study of diet such as hydrogen and sulfur, but these are less well understood and still experimental (e.g., Lee-Thorp 2008, Richards et al. 2001). Carbon isotopes in tissues like hair or nail collagen can provide information on the diet of the last days or weeks of an individual (e.g., Macko et al. 1999, O’Connell et al. 2001). There are a number of published summaries of carbon isotope analysis in archaeology available (e.g., Ambrose 1993, Ambrose and Krigbaum 2003, Hedges et al. 2006, Katzenberg and Harrison 1997, Lee-Thorp and Sponheimer 2003, Schoeninger 1995, Sealy 2001, van der Merwe 1992). Lee-Thorp (2008) provides a useful review of the development of the method and some suggestions for future research. Carbon is an element of biological interest with 3 stable isotopes: 12C, 13C, and 14C. Nitrogen has 2 natural stable isotopes: 14N and 15N. In each case, the lightest isotope is present in much greater abundance in nature than the others. For example, of the total carbon on earth, 12C = 98.89%, 13C = 1.1%, and 14C = 0.000001%. Carbon isotopes in biological materials are reported as a ratio of 13C to 12C. Ratios are used in order to standardize the value that is reported, regardless of the amount of material measured or the concentration of the element present. These ratios are then compared to the ratio of a standard, and reported as a “delta” value (δ), which is the ratio of the difference between the sample ratio and the standard ratio compared to that of the standard, given in parts per thousand or per mil (“‰”): δ13C‰ = 13C / 12Csample - 13C / 12Cstandard x 1000 13C / 12Cstandard Delta values are not absolute isotope abundances, but express the relative difference between a sample reading and a standard reference material. The standard reference material for carbon is the calcite from a mineral deposit known as PeeDee Belemnite (PDB). Carbon isotope ratios are reported as δ13C‰, and values for this ratio are negative in value and range from roughly 0.0 to -25‰. The numbers are negative because observed ratios are lower than the standard. A sample with δ13C = -5.0 means that the sample is 5 per mil (or 0.5%) lighter than PeeDee belemnite. Values for δ13C in human bone collagen range between approximately -5‰ and -25‰. This variation in human bone collagen is due in large part to different sources of 13C in the plants and animals we eat. There are two sources of heavier 13C isotopes: the sea and some species of plants, mainly tropical grasses, known as C4 plants. Two distinct photosynthetic pathways produce different carbon isotope ratios among terrestrial plant species (O’Leary 1981, Smith and Epstein 1971). Maize, sorghum, millet and other tropical plants (designated as C4 species) utilize a photosynthetic pathway that efficiently metabolizes carbon dioxide by initial conversion to a 4-carbon compound that incorporates more available 13C (van der Merwe 1982). C4 is a more efficient pathway for photosynthesis and is found in plants, grasses, sedges, and grains in drier, warmer regions. The C3 plants, more common in temperate areas, produce a 3-carbon compound. Woody, round-leafed species are 95% of all plants and utilize C3 photosynthesis. The carbon isotope ratios in the bone collagen of animals feeding on 3- or 4-carbon plants reflect these isotopic differences. Humans who consume C4 plants have more 13C and correspondingly heavier CO2 in their bone collagen and dental enamel. 13C/12C ratios in seawater bicarbonate are higher than in atmospheric carbon dioxide. These differences are observed between the plants from the land and sea, as well as in the bone collagen of the animals that feed on these species. Carbon isotope ratios are reported in δ13C values that generally become more positive (less negative) along the continuum from plants to herbivores to carnivores in marine and terrestrial regimes. Terrestrial C3 diets appear as δ13C‰ values in collagen around -20‰. Marine or largely C4 diets exhibit δ13C‰ values in human bone collagen around -10‰. Thus, less negative values for collagen in bone mean either marine foods or C4 plants in the diet, or both. Thus, human diets that include marine foods, C4 plants, or the animals that ingest C4 plants can be distinguished using stable carbon isotope ratios. Numerous published studies of δ13C have documented various aspects of past human diets in antiquity. Lee-Thorp et al. (2003), for example, reported measurements of carbon isotope ratios in the bone collagen of early African hominids. Richards et al. (2000) and Bocherens et al. (2001) examined Neanderthal diets using carbon isotope ratios. Sealy and van der Merwe (1988) documented Holocene marine diets from prehistoric South Africa. Ambrose et al. (2003) documented status differences in diet ca. AD 1000 in the midwestern US. The list goes on and on, and the range of applications continues to expand. Journal of the North Atlantic T.D. Price 2015 Special Volume 7 74 be reflected in carbon isotopes in carbonate when consumed in small amounts, whereas they will be reflected in collagen only when consumed in sizeable proportions (Harrison and Katzenberg 2003). These experiments showed that when the protein and bulk diet have the same δ13C values, collagen and apatite are enriched by 5.0‰ and ~9.4‰, respectively, relative to the total diet. Thus, the apatite-collagen offset (Δ13Cap–co) is 4.4‰. The results of these experiments permit more detailed reconstruction of the isotopic composition of prehistoric human diets. A spacing of less than 4.4‰ indicates that dietary protein is isotopically heavier (more positive) relative to the whole diet. If the spacing is greater than 4.4‰, then dietary protein is isotopically lighter (more negative) than the whole diet (Ambrose et al. 1997, 2003; Jin et al. 2004). Harrison and Katzenberg (2003), on the other hand, found that a δ13C apatite–diet value of +12.0‰ and a δ13C collagen–diet value of +5.1‰, with a resulting Δ13Cap–co offset of 6.9 to be more accurate for human samples. Δ 13Cap-co has also been used to improve estimates of marine foods in the diet (Ambrose et al. 2003) and to discover C4 foods in largely C3 diets in Neolithic China (Hu et al. 2006). Marine foods, rich in protein, will contribute disproportionately to the amino acids in collagen compared to terrestrial plants. Moreover, being enriched in 13C, marine proteins will disproportionately increase the collagen δ13C values relative to the bulk diet, and relative to apatite δ13C (Ambrose and Norr 1993). In marine contexts with no C4 plants, protein comes mainly from 13C-enriched marine animal resources, while carbohydrates and some proteins come from 13C-depleted C3 plants and C3-feeding animals. Because the marine protein source is more enriched in the heavy carbon isotope, the diet-to-collagen spacing (Δ13Cdiet–coll) should be greater than 5‰, and collagen-to-carbonate spacing (Δ13Ccoll–carb) should be less than 4.4‰. Because the marine protein source is more enriched in 15N, collagen δ15N values should also be high. In a coastal environment lacking C4 plants, a positive correlation should exist between collagen δ13C and δ15N, and a negative correlation should occur between δ15N and Δ13Cdiet-coll. In terrestrial high-latitude diets, the entire foodweb is based on 13C-depleted C3 plants, so the bulk diet and dietary protein should have very similar δ13C values. The diet-collagen spacing and the apatite-collagen spacing should be 5‰ and at least 4.4‰, respectively . In an effort to simplify these relationships, Kellner and Schoeninger (2007) and Froehle et al. (2012) have published models of the relationships between apatite and collagen carbon and nitrogen. Variation is summarized in a scatterplot of discriminant functions Carbon Isotopes in Apatite Most of the paleodiet work using carbon isotopes has focused on the organic collagen in bone. Carbon is present in the mineral, or carbonate, portion of bone and tooth enamel as well and also contains information on diet (Cerling and Harris 1999, Froehle et al 2012, Lee-Thorp et al. 1989, Sullivan and Krueger 1981, Tieszen and Fagre 1993). Tykot (2004) reports that δ13C values in apatite are approximately 7‰ more positive than in collagen. This offset is variable depending on diet and is discussed in more detail below. Although there are potential problems with contamination in apatite (e.g., Hedges 2002, Krueger 1991, Schoeninger and DeNiro 1982), this carbon isotope ratio can provide substantial insight on questions regarding diet and place of origin of the individual from whom the sample was taken. This tissue provides different information on diet than bone collage. Tooth enamel—and the carbonate and phosphate minerals where carbon is bound—forms during childhood. Whereas bone collagen provides a record of adult diet, tooth enamel provides information about the diet of early childhood. Differences in stable carbon isotope ratios between the apatite and collagen compartments of bone in the same individual were first reported by Krueger and Sullivan (1984). They proposed that consumer collagen carbon was derived from dietary protein and apatite from dietary energy sources. They used this model to explain systematic differences in the isotopic composition of collagen and apatite of non-human herbivores versus carnivores and omnivores, and marine versus terrestrial human diets. In protein-deficient diets, some of the carbon for collagen may be taken from carbohydrates. Loftus and Sealy (2012), in a comparison of collagen and apatite carbon in archaeological samples from South Africa, found a strong correlation between δ13Cenamel and δ13Ccollagen and indicated that both ratios were good proxies for past human diet. The also examined intertooth (intraindividual) isotopic variation, measuring each tooth from one side of 3 different mandibles. The range of values within each mouth varied from 1.1 to 1.6%, substantially less than reported intrapopulation variation. Loftus and Sealy concluded that any tooth could be used for dietary reconstruction. Experimental studies with rats and mice have shown that collagen carbon comes largely from dietary protein, whereas apatite carbon more accurately reflects the isotopic composition of the whole diet (Ambrose and Norr 1993, Jin et al. 2004, Tieszen and Fagre 1993). Protein-poor foods will Journal of the North Atlantic T.D. Price 2015 Special Volume 7 75 that summarizes the relationship among these 3 variables (or a cross plot of δ13C for collagen and apatite) and identifies the major components of diet in terms of C3, C4, marine protein, and non-protein input. Thus, carbon isotope ratios in bone and enamel apatite can provide information on childhood diet and on whole diet when compared with collagen. A number of case studies have been published. Ambrose et al. (2003) examined bone collagen and apatite carbon in human remains from the western Pacific and suggested the presence of previously unknown foods in the diet. Price et al. (2012) compared collagen and apatite carbon from the bones and teeth of the early colonial inhabitants of the colonial Mexican town of Campeche. Harrison and Katzenberg (2003) reported on bone collagen and apatite δ13C and used the offset to better estimate the contribution of maize and marine foods in prehistoric populations in Ontario and California, respectively. Nitrogen Isotopes in Collagen Nitrogen isotopes in bone collagen are also informative with regard to past human diets and are reported as δ15N‰. There are 2 isotopes used in this ratio, 14N (99.63% in nature) and 15N (0.37% in nature). The ratio is calculated in a similar fashion as δ13C in parts per thousand or per mil (‰), as the difference between the sample and a standard, divided by the standard: δ15N‰ = 15N / 14Nsample - 15N / 14Nstandard x 1000 15N / 14Nstandard As noted earlier, these delta values are not absolute isotope abundances, but express the relative difference between a sample reading and a standard reference material. The standard reference material for nitrogen is air. Natural δ15N levels in biological materials typically range from -5‰ to +10‰ (Ambrose 1991, Bocherens and Drucker 2003, Sealy et al. 1987). Variations in nitrogen isotope ratios are largely due to the role of leguminous plants in diet and the trophic level (position in the food chain) of the organism (e.g., Minagawa and Wada 1984, Post 2002, Schoeninger 1985). The nitrogen isotope ratios for plants depend primarily on how they obtain their nitrogen—by symbiotic bacterial fixation or directly from soil nitrates. Atmospheric nitrogen (δ15N = 0‰) is isotopically lighter than plant tissues; values in soil tend to be even higher. Non-nitrogen-fixing plants, which derive all of their nitrogen from soil nitrates, can thus be expected to be isotopically heavier than nitrogen-fixing plants, which derive some of their nitrogen directly from the atmosphere. Nitrogen isotope ratios can vary widely among different plant species in the same area (e.g., Dawson et al. 2002, Dijkstra et al. 2003). Drier environments tend to have higher food-web δ15N values (Ambrose 1991). Grazing animals show δ15N enrichment relative to the plants they consume; predators show further δ15N enrichment relative to their prey species. Thus, stable nitrogen isotopes tend to mirror trophic level in the food chain. In one sense, nitrogen isotopes reflect the proportion of animal protein in the diet. δ 15N values in the food chain are passed along accompanied by an approximately 2–3‰ positive shift for each trophic level, including between mother and nursing infant (Tykot 2004). Human consumers of terrestrial plants and animals typically have δ15N values in bone collagen of about 6–10‰, whereas consumers of freshwater or marine fish, seals, and sea lions may have δ15N values of 15–20‰ (Schoeninger and DeNiro 1984, Tykot 2004). A more positive nitrogen isotope ratio generally reflects a higher trophic position (e.g., DeNiro and Epstein 1981, Hedges and Reynard 2007, Pate 1994). Fogel et al. (1989), for example, report on the use of nitrogen isotope ratios in identifying the age of weaning in archaeological skeletal populations containing infant burials. Oxygen Isotopes in Apatite Oxygen isotopes have been widely used as a proxy for temperature in many climate and environmental studies and vary geographically in surface water and rainfall (Dansgaard 1964). Oxygen isotopes in archaeology are used primarily to study ancient environments and to examine past human mobility. Oxygen isotopes in ancient human skeletal remains are found in both tooth enamel and bone. Oxygen is incorporated into dental enamel during the early life of an individual and it remains unchanged through adulthood. Samples for the analysis of human skeletal remains are normally taken from dental enamel due to better preservation and resistance to diagenesis. Oxygen isotopes are also present in bone apatite and are exchanged through the life of the individual during bone turnover, thus reflecting place of residence in the later years of life. Thus, oxygen isotopes have the potential to be used to investigate human mobility and provenience (Bowen et al. 2005). The use of both hydrogen isotopes in bone collagen has also been suggested (O’Brien and Wooller 2007), but this application remains largely experimental. Journal of the North Atlantic T.D. Price 2015 Special Volume 7 76 rected: δ18OC(PDB) = (0.97 x δ18OC(SMOW)) - 29.98. Thus, a drinking water δ18OC(SMOW) value of -6.0‰ to -6.5‰(SMOW) yields an enamel carbonate δ18OC(PDB) value of approximately -4.0‰. The oxygen isotope ratio in the skeleton reflects that of body water, and ultimately of drinking water (Kohn 1996, Luz and Kolodny 1985, Luz et al. 1984), which in turn predominantly reflects local rainfall. Water from food and atmospheric oxygen are minor, secondary sources. Isotopes in rainfall are greatly affected by enrichment or depletion of the heavy 18O isotope relative to 16O in water due to evaporation and precipitation (e.g., Dansgaard 1964). Major factors affecting rainfall δ18O values are latitude, elevation, amount of precipitation, and distance from the evaporation source (e.g., an ocean), i.e., geographic factors (Fig. 2). Rainwater, can contain either isotope, and H2 18O has a greater mass than H2 16O, and requires more energy to evaporate and more energy to stay in the atmosphere. When water evaporates over the ocean, it becomes relatively lighter isotopically (more 16O) than the water left behind. As this moisture moves over land, the first precipitation contains more of the heavy isotope and as the clouds move inland (and to higher elevations) the rain becomes even more depleted in the heavier isotope. Thus, oxygen isotope ratios have some potential Oxygen has three isotopes—16O (99.762%), 17O (0.038%), and 18O (0.2%)—all of which are stable and non-radiogenic. As with carbon and nitrogen, isotope measurements of oxygen are always reported as a ratio of one isotope to another, lighter and more common cousin. Oxygen isotopes are commonly reported as the per mil difference (‰ or parts per thousand) in the ratio of 18O to 16O between a sample and a standard. This value is designated as δ18O. This value can be measured in either carbonate (CO3)-2 or phosphate (PO4)-3 ions of apatite in tooth and bone. Phosphate and carbonate produce comparable results for oxygen isotope ratios (Sponheimer and Lee- Thorp 1999). Less sample is needed for carbonate, preparation is less demanding, and results between laboratories are more comparable (e.g., Bryant et al. 1996, Chenery et al. 2012, Sponheimer and Lee- Thorp 1999). The standard used is commonly Vienna Standard Mean Ocean Water (VSMOW or SMOW) for phosphate oxygen, and PeeDeeBee dolomite (PDB) for carbonate oxygen. These 18O values for carbonate and phosphate oxygen using different standards are comparable though calculation. Chenery et al. (2012) defined the relationship between the δ18O value of drinking water and δ18O in enamel carbonate as δ18OC = (δ18ODW + 48.634) / 1.59 relative to SMOW. Measurements made using a PDB standard must be further cor- Figure 2. Oxygen isotopes in hydrological cycles and geographic variation. Journal of the North Atlantic T.D. Price 2015 Special Volume 7 77 seen at a given site. There are also reservoir effects. Water in lakes, ponds, and storage vessels can have higher δ18O values due to evaporation of the lighter isotope. Through-flowing rivers can have δ18O that differs from local rainfall values. Even beveragepreparation techniques can affect mean dietary δ18O (Knudson 2008). In addition, considerable variability may be expected among the teeth of a single individual or even within a single tooth (Fricke and O’Neil 1996, Weidemann et al. 1999, Wright and Schwarcz 1998). Because oxygen isotopes are much lighter and have a much greater relative mass difference than strontium, the ratio of 18O to 16O is highly sensitive to environmental and biological processes, and fractionation is not uncommon. Since most permanent teeth form over the span of 2–4 years, seasonal fluctuations may well be visible in dental δ18O values, if measurements are sufficiently precise. Cultural practices, such as long-term water storage, cooking, diet, and breastfeeding can influence the δ18O of human skeletal tissues (White et al. 2007, Wright and Schwarcz 1998). Because of the high variation present in oxygen isotope ratios, the application of this method for to vary geographically and provide information on past human movement. At the same time, there are significant difficulties in the application of oxygen isotope ratios to human proveniencing (e.g., White et al. 2004). There are potential difficulties with diagenesis (e.g., Sharp et al. 2000). As noted, oxygen isotope ratios vary with latitude with pronounced variation in the arctic region. Many parts of the temperate and tropical regions of the world, however, have similar δ18O values, ranging broadly from approximately -2.0‰ to -8.0‰ (Fig. 3), so that finding distinctive differences in these regions is difficult (Bowen et al. 2005). We have also observed unexplained variation on the order of ±2‰ in δ18O values among individuals from the same location in our investigations (see also Huertas et al. 1995). Moreover, the δ18O of human tissues may differ from that of rain falling in the same landscape. Several different factors appear to affect the final values measured in the human skeleton. Rainfall δ18O levels vary from year to year and over time in the same area (e.g., Rozanski et al. 1993). This annual variability is undoubtedly a major contributor to the broad range of δ18O values Figure 3. Isoscape map of global d 18O values (Bowen 2012, Bowen and Revenaugh 2003). Journal of the North Atlantic T.D. Price 2015 Special Volume 7 78 provenience studies must be done with caution and remains experimental in many regions. Strontium Isotopes in Apatite Strontium isotope analysis provides a robust means for identifying human mobility in the past. There are several published summaries of the method (Bentley 2006, Montgomery 2010, Price 2000, Slovak and Paytan 2011). Analytical methods are described in detail in a number of publications (e.g., Frei and Price 2012, Price et al. 1994b, Slovak and Paytan 2011, Sjögren et al. 2009). Numerous examples of the application of strontium isotope ratios to archaeological questions have been published (e.g., Benson et al. 2006, Hedman et al. 2009, Knudson 2008, Montgomery et al. 2003, Price et al. 2011, Wright 2005). The principle is straightforward. The strontium isotope ratio of 87Sr/86Sr varies among different kinds of rocks and sediments. Because the 87Sr forms through a radiogenic process as a product of decay from rubidium-87 over time, older rocks with more rubidium have a higher 87Sr/86Sr ratio, while younger rocks with less rubidium are at the opposite end of the range with low ratios (e.g., Montgomery et al. 2006). The half-life for this process is on the order of billions of years, so the abundance of 87Sr does not change on the time scales of human prehistory, but can accumulate appreciably on geologic time scales such that regions with old rocks have more 87Sr than regions with relatively younger rocks. Strontium moves into humans from rocks and sediment through the food chain (Price 1989, 2000; Sillen and Kavanagh 1982). Strontium is chemically similar to calcium and substitutes for calcium in enamel and bone. Because some rocks and sediments contain higher concentrations of strontium than others, a correction is made by using a ratio of the radiogenic 87Sr to a non-radiogenic 86Sr. The amount of 87Sr in nature varies but is ~7% of total strontium and 86Sr is 10%. This ratio normally varies from about 0.700 in young rocks with low Rb to >0.730 in high-Rb rocks that are billions of years old. Most measurements fall in the range of 0.703 to 0.723. This ratio generally varies with geology and is not significantly altered by biological processes, so that the 87Sr/86Sr ratio in enamel reflects that of the underlying geology where one was born and raised when the enamel formed. If one moves to a new location with a different geologic setting, or is buried in a new place, the enamel isotopes will not match those of the new location, allowing us to identify the individual as an immigrant. An essential question regarding strontium isotope analysis concerns the local strontium isotope signal for the area in which the burial is located. In actual fact, levels of strontium isotopes in human tissue may vary from local geology for various reasons (Price et al. 2002) and, for this reason, it is necessary to measure bioavailable levels of 87Sr/86Sr to determine local strontium isotope ratios. The local bioavailable isotopic signal of the place of burial can be determined in several ways: in human bone from the individuals whose teeth are analyzed, from the bones of other humans or archaeological fauna at the site, or from modern fauna in the vicinity. This baseline information on isotope values across an area needs to be obtained in order to make useful and reliable statements about the origins of the human remains under study (Frei and Price 2012, Price et al. 2002). Lead Isotopes in Apatite Lead (Pb) behaves remarkably like strontium in terms of provenience studies. Both elements substitute for calcium in hydroxyapatite in skeletal tissue, are transferred with negligible fractionation from soils into plants and through successive trophic levels, and are found as trace constituents in plants and animals. Virtually the entire body burden of both elements is deposited in the skeleton. Bone concentrations of both elements are proportionate to dietary (and hence environmental) abundances, and both elements undergo trophic-level biopurification. Most importantly, both elements have stable and radiogenic isotopes, such that the ratios of these isotopes depend upon the local geology and are thus geographically variable. In contrast to strontium, there is very little lead in seawater. Lead is the element with the heaviest stable isotope (208) in nature. Lead, in addition to nonradiogenic 204Pb, has not one, but three, radiogenic isotopes: 206Pb, 207Pb, and 208Pb. Measurable differences in stable lead isotopic compositions are caused by the differential radioactive decay of 238U (t1/2 = 4.5 x 109 years), 235U (t1/2 = 0.70 x 109 years), and 232Th (t1/2 = 1.4 x 1010 years) to form 206Pb, 207Pb, and 208Pb, respectively (Faure and Mensing 2005). The relative abundances of these isotopes in nature are 204Pb = 1.4%, 206Pb = 24.1%, 207Pb = 22.1%, and 208Pb = 52.4%. Normally the relative abundance of the different isotopes of an element varies little across the surface of the earth, but that is not the case with lead. Stable lead isotopes, and their ratios, locally vary according to both the geologic age of the terrain and the amounts of the parent isotopes of uranium and thorium. Importantly, lead isotope ratios, like strontium Journal of the North Atlantic T.D. Price 2015 Special Volume 7 79 A variety of isotopes are used in the study of human remains. Carbon and nitrogen isotopes have often been measured in bone collagen for information on diet and are especially useful for detecting certain types of plants or marine foods in the diet. At the same time, smaller differences in past human diets may also be recorded in these isotopes and thus they are potentially useful for further differentiation among populations. Carbon and oxygen can be measured in both bone and tooth enamel, in the mineral or apatite component of the tissue. Carbon isotope ratios in enamel apatite provide information on childhood diet. Carbon isotope ratios in bone apatite provide information on adult diet. Comparison of this ratio in apatite and collagen can provide some indication of the differences between protein intake and whole-diet composition. Oxygen isotopes vary geographically with changes in rainfall and provide information sometimes useful for human proveniencing. The oxygen isotope ratio in apatite reflects that of drinking water and ultimately rainfall or snow intake. However, oxygen isotopes are quite variable in human remains, and the approach remains experimental. Strontium isotopes in enamel apatite provide a fairly robust method for investigating past human mobility. Strontium isotopes reach the human skeleton from the food chain, and ultimately from the rocks and sediments on which plants are growing. Strontium isotope ratios in tooth enamel provide a signature of the place of birth and early childhood that can be compared to the value for the place of burial to determine if an individual is local or not. It is important to remember that while strontium isotopes are useful for identifying non-local individuals in a population, it is much more difficult to determine place of origin because of the widespread occurrence of certain strontium isotopic signatures. It is also essential in the investigation of past human mobility to have information on the local and regional isotope values in order to have a baseline for comparison. These baseline values cannot be directly predicted from local geology and need to be measured to determine the bioavailable strontium isotope ratios of the place of burial. Price et al. (2015 [this issue]) considers the bioavailable strontium isotope ratios in the potential homelands and destinations of the Viking settlers of the North Atlantic. This baseline information is important to have prior to an examination of values in the tooth enamel of these individuals. Lead isotopes follow principles of distribution and deposition similar to strontium. Lead is isotope ratios, are not changed by biological or other low-temperature chemical or physical processes. The ratios present in dental enamel reflect those of dietary intake and, in the New World prior to the modern industrial era, that of the local geology. The four stable isotopes of lead provide a large number of potentially useful ratios, but the values of 206Pb/204Pb, 207Pb /204Pb, 208Pb /204Pb, 207Pb /206Pb, and 208Pb /206Pb are commonly used in isotopic provenience studies. Lead isotope analysis was initially applied to lead-rich artifacts, such as those made of copper, bronze, and lead-bearing ores, to study provenience questions. This method was later used with other artifact classes such as ceramics. Lead isotopes can also be used as a tracer in past human-mobility studies (e.g., Augustine 2002; Budd 1999, 2000; Carlson 1996; Corruccini et al. 1987; Gulson et al. 1997; Molleson et al. 1986; Montgomery et al. 1999; Reinhard and Ghazi 1992). There are several problems with lead isotope analysis, including the low concentrations of this element in dental enamel, the poorly known geographic distribution of most lead isotope values, and the solubility of lead resulting in potential contamination of modern and ancient samples, particularly from the combustion products of leaded gasoline (Albarède et al. 2011). The ubiquity of these pollutants means that an ultraclean lab is often necessary to avoid contamination issues. These factors make interpretation of ratios for lead isotopes considerably more complex than for strontium isotopes. Although there is evidence for diagenesis of lead in buried bone (e.g., Kyle 1986), this contamination does not appear to be present in tooth enamel in most cases (Montgomery et al. 1999, Waldron 1983). Because of their variability, lead isotopes have had a successful history in archaeometric research for artifact provenience studies. More pertinent here, lead isotopes have been used in recent years to identify prehistoric human immigrants in a number of different contexts (e.g., Budd 1999, 2000; Carlson 1996; Gulson et al. 1997; Molleson et al. 1986; Montgomery et al. 1999; Reinhard and Ghazi 1992). Conclusions Major questions pursued in isotopic studies of human remains focus on past diet and human mobility. Two major types of skeletal tissue—enamel and bone—are used in such studies. Enamel retains the chemistry of early childhood, while bone contains the chemistry of the last decade or so of life. The two types of tissue can provide insight into part of the life history of an individual. Journal of the North Atlantic T.D. Price 2015 Special Volume 7 80 spectives on questions about past diet (e.g., Hedges et al. 2008, Katzenberg et al. 1995, Schwarcz and Schoeninger 2011). Carbon and oxygen in apatite are also measured simultaneously in a number of labs and routinely used together in studies of past diet and movement. Using more than 2 isotopes can provide even more information (e.g., Jay et al. 2007, Knudson and Price 2007). Wright and Schwarcz (1998) measured 3 isotopes—carbon, oxygen, and nitrogen—in the investigation of infant diets in the Maya region. Price et al. (2012) in a study of a colonial cemetery in Campeche, Mexico, employed all of the isotopes discussed in this essay—carbon and nitrogen in collagen, carbon and oxygen in enamel apatite, and strontium and lead in enamel—to assess the places of origin of the buried individuals. Larger samples of burial populations and multiple isotope investigations will provide significantly more information on past diet and mobility. Isotopic studies can also be used together with molecular approaches such as use of DNA to learn even more about past human populations in terms of diet, genetic relationships, and mobility. Haak et al. (2008) documented a variety of information on these questions for a group of burials from Neolithic Germany. The potential for isotopic studies is enormous. This work has been ongoing for about 30 years and is in its early childhood. The next 30 years are going to be very exciting. Literature Cited Albarède, F., A.-M. Desaulty, and J. Blichert-Toft. 2011. A geological perspective on the use of Pb isotopes in archaeometry. Archaeometry 54:853–857. AlQahtani, S.J., M.P. Hector, and H.M. Liversidge. 2010. Brief communication: The London atlas of human tooth development and eruption. American Journal of Physical Anthropology 142:481–490. Ambrose, S.H. 1990. Preparation and characterization of bone and tooth collagen for isotopic analysis. Journal of Archaeological Science 17:431–451. 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While strontium occurs in most rocks and sediments, lead is not present everywhere, and isotopic variation in lead is not completely understood. Lead isotopes are not commonly employed in human mobility studies because of these issues and others. Ultraclean labs are required because of the ubiquity of contamination, and the analysis is expensive. At the same time, the larger number of isotopes present in lead provides several different kinds of information for investigation. In general terms, use of lead isotopes remain an experimental method in the search for past human provenience. As with any method in archaeological research, there are problems and uncertainties in the isotopic investigation of past diet and mobility. Some of these issues have been addressed in various publications (e.g., Fuller et al. 2005, Montgomery et al. 2007, Pollard 2011, Sillen et al. 1989, Van Strydonck et al. 2005). The science is evolving as evidenced by the numerouse changes that radiocarbon dating has experienced over the last 60 years as adjustments and corrections are introduced to fine tune the method. These calibrations are an inevitable part of an inexact science. As Pollard (2011) has observed, the addition of people to the analysis complicates our questions and answers enormously. Nevertheless, the final criteria by which these studies must be judged is what has been learned and where we go from here. Problems and uncertainties are part of the process. Each of these different isotopic methods offers interesting information and potential. At the same time, there are often questions and uncertainties that make decisions about diet or past mobility difficult. Two directions point to some solution to these problems: larger samples and multi-isotope analyses. In many studies to date, the number of samples has been only a small proportion of the larger burial population under investigation. 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Residential histories of the human sacrifices at the Moon Pyramid, Teotihuacan: Evidence from oxygen and strontium isotopes. Ancient Mesoamerica 18:159–172. Wiedemann, F.B., H. Bocherens, A. Mariotti, A. von den Driesch, and G. Grupe. 1999. Methodological and archaeological implications of intra-tooth variations (δ13C, δ18O) in herbivores from Ain Ghazal (Jordan, Neolithic). Journal of Archaeological Science 26:697–704. Wright, L.E. 2005. Identifying immigrants to Tikal, Guatemala: Defining local variability in strontium isotope ratios of human tooth enamel. Journal of Archaeological Science 32:555–566. Journal of the North Atlantic T.D. Price 2015 Special Volume 7 86 Initial Preparation Tooth enamel The permanent first molar is the preferred tooth for isotopic analyses both for consistency and the fact that the enamel of this tooth forms during late gestation and very early childhood. Teeth are mechanically pre-cleaned by hand and subsequently repeatedly washed ultrasonically in ultrapure (MilliQ™) water until the water remains visually clear. After drying, part of the surface of the tooth is initially abraded using a dental drill fitted with a diamond burr to remove contamination. Small pieces of enamel are removed by means of a small diamond blade saw and/ or with chromium steel pliers. Extreme care is taken to separate dentin material from enamel. Sample sizes are about 20 mg of material, with approximately 10 mg for strontium isotopes, 2 mg of fine enamel powder for enamel apatite carbon and oxygen isotopes, and the remainder reserved for possible subsequent analysis. For lead isotope analysis, an additional part of the tooth is prepared, and another 30 mg of enamel are removed. Bone Most of the carbon and nitrogen isotope measurements from the North Atlantic human remains were carried out by other projects and data has been shared. For bone apatite samples for strontium and lead, a sample of the exterior and interior surfaces of a section of cortical bone, weighing from about half a gram to a few grams, is heavily abraded and then broken into several small pieces (less than 1 cm). These pieces are placed into a 20-ml glass vial filled with deionized water and are repeatedly sonicated in an ultrasonic water bath, replacing any cloudy water with clean deioinzed water until the water remains clear after sonication. The samples are sonicated with 5% acetic acid, and rinsed again with ultrapure deionized water. The bone chips are then ashed in a muffle furnace at 750–800 °C for 8 hours, and the resulting ash is used for strontium and lead isotopes. Carbon and Oxygen in Enamel Apatite References: Loftus and Sealy 2012, Stephan 2000, Schwarcz et al. 1991. Enamel apatite carbonate isotopic analysis (sample weight = ~700 μg) was performed by reacting the prepared finely powdered enamel with 100% phosphoric acid in the presence of silver foil at 70 °C in a Kiel III automated carbonate reaction device coupled to a MAT 252. Carbon and oxygen isotope ratios were simultaneously determined on the CO2 generated by this reaction. The isotope ratio measurement was calibrated based on repeated measurements of NBS-19 and NBS-18 and precision is ± 0.1 ‰ for δ13C and ±0.06‰ for δ18O (1s). The carbonate–CO2 fractionation for the acid extraction is assumed to be identical to that of calcite. Replicate analyses of apatite were not performed. Analytical error on this instrument is ±0.05‰ for δ13C and ±1.0‰ for δ18O (Balasse 2002). Carbon in Bone Apatite References: Lee-Thorp et al. 1989. Bone samples were treated as follows. Carbonate from apatite samples was extracted using established techniques, specifically the removal of organic components using bleach (24 hrs for enamel, 72 hrs for apatite) and non-biogenic carbonates using buffered 1 M acetic acid (24 hrs). Carbonate samples were analyzed using a similar Finnegan MAT Delta Plus XL mass-spectrometer, coupled with a Kiel III device that produces CO2 gas using 100% phosphoric acid injected into individual sample containers. Carbon and Nitrogen in Bone Collagen References: Ambrose 1990, Chisholm et al. 1983, Grupe and Piepenbrink 1987, van Klinken 1999. Sample preparation for the simultaneous measurement of carbon and nitrogen isotopes in bone collagen generally follows the procedure described here with minor differences from lab to lab. Whole and fragmented bone (about 1 g) was first cleaned using ultra-sonic vibration and distilled water. From the cleaned bone, 10 mg of bone powder were extracted for apatite analysis. Following initial treatment with 0.1 M NaOH to remove humic acids, bone collagen was extracted using 2% HCl for 72 hrs. Following a second 24-hr treatment with NaOH, residual lipids were separated with a mixture of methanol, chloroform, and water. After drying, the collagen was weighed to determine percent yield, with 1% or more considered reliable for isotope analyses. Duplicate 1-mg samples were then weighed into tin cups, and analyzed using a CHN analyzer coupled with a Finnigan MAT Delta Plus XL stable isotope ratio mass spectrometer set up with a continuous flow. The reliability of isotope measurements was also validated by C and N gas yields and C:N ratios during processing on the mass spectrometer, with values between 2.9 and 3.6 considered equivalent to those found in collagen. The precision of the analyses is about ±0.1‰ for carbon and ±0.2‰ for nitrogen. Results are reported relative to the PDB and AIR standards, respectively. Strontium Isotopes in Enamel Apatite References: Bentley 2006, Montgomery 2010, Price et al. 1994a, Slovak and Paytan 2011. Appendix 1. A brief summary of sample preparation and measurement for these isotopic methods is provided. This information is widely available in a number of publications as referenced in each section. Samples of tooth enamel are taken for oxygen, carbon, strontium, and lead. Samples of bone are taken for carbon and nitrogen in collagen. This appendix first describes the preparation of enamel and bone for analysis and then summarizes the procedures used for isotope ratio measurement for carbon and oxygen in enamel apatite, carbon and oxygen in bone apatite, carbon and nitrogen in bone collagen, strontium in enamel and bone apatite, and lead in ena mel apatite. Journal of the North Atlantic T.D. Price 2015 Special Volume 7 87 For 87Sr/86Sr, samples weighing approximately 3 mg were dissolved in 5-molar nitric acid. The strontium fraction was purified using EiChrom Sr-Spec resin and eluted with nitric acid followed by water. The eluent was dried, and the Sr residue was placed on a tantalum filament. Isotopic compositions were obtained on this strontium fraction using a VG (Micromass) Sector 54 thermal ionization mass spectrometer (TIMS) in the Department of Geological Sciences, University of North Carolina at Chapel Hill or a VG Sector 54 IT mass spectrometer at the Isotope Laboratory in the Institute of Geography and Geology, University of Copenhagen. Regular and repeated 87Sr/86Sr analyses of the NIST 987 (National Institute of Standards, Standard Reference Material) strontium carbonate yielded a value of 0.710259 ± 0.0003 (2 SE). Internal precision (standard error) for the samples is typically 0.000006–0.000010, based on 100 dynamic cycles of data collection. Total procedural blanks for strontium typically were below 100 picograms, which is insignificant relative to the amounts of strontium in the samples. Lead Isotopes in Apatite References: Budd et al. 1999, 2000; Carlson 1996; Gulson et al.1997. Samples weighing 15–30 mg are placed into 7-ml Teflon beakers (Savillex™), and are then dissolved in a 1:1 mixture of 0.5 ml 6 N HCl (Seastar) and 0.5 ml 30% H2O2 (Seastar). The samples are typically decomposed within 5–10 minutes, after which the solutions are dried down on a hotplate at 80 °C. Enamel samples were taken up in a few drops of a 3:1 mixture of 1.5N HBr and 2N HCl and then loaded on disposable mini extraction columns (1-ml pipette tips with a fitted frit at the bottom) charged with 0.1 ml intensively pre-cleaned mesh 100 anion resin (BioRad AG 1x8). The elution recipe essentially followed that described by Frei and Kamber (1995), tailored to the small size of the herein used extraction columns. Lead samples were dissolved in 2.5 ml of a 1 M H3PO4 and loaded together with a silica gel activator onto previously outgassed 99.98% single rhenium filaments. Samples were measured at 1100–1200 °C in static multi-collection mode on a VG Sector 54 IT mass spectrometer equipped with 8 faraday detectors (Institute of Geography and Geology, University of Copenhagen, Denmark). Repeated analyses of 10-ng loads of the NBS 981 Pb standard were used to control the mass bias of the sample analyses. Mass fractionation amounted to 0.103% / AMU (atomic mass unit) relative to the values for this standard reported by Todt et al. (1996). Total lead procedure blanks were ~50 picograms. These amounts are insignificant relative to the total amounts of lead in a sample, and therefore blank corrections were not undertaken.