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Bone Chemistry Analysis:

The Theoretical Basis

Introduction

Although many tissues of the human body can be used to determine diet and nutritional status (Underwood

1977), bone is often times the only tissue recovered from an archaeological context. Due to its availability

the majority of archaeological dietary studies have used bone as the material for analysis (for an overview

see Price 1989; Price et al. 1985b; Schoeninger and Moore 1992; Schwarcz and Schoeninger 1991). Bone is

a complex cellular tissue that is composed of three major components: an organic matrix (mostly collagen),

an inorganic mineral fraction, and water. Both the organic and inorganic components are used in conducting

bone chemistry analysis; the inorganic mineral fraction is used for elemental analysis, and the organic

portion (collagen) is necessary for isotopic research.

The following chapter will outline the utility of isotopic and elemental analyses in reconstructing diet, and

examine how these analytical procedures can be used effectively in archaeological research. Stable carbon

and nitrogen isotopes have been used to identify differences in the consumption of meat, specific terrestrial

plant foods (such as maize), marine plants and mammals. Trace element analysis can distinguish relative

differences in the consumption of meat, plant foods, and marine resources. The post-mortum alteration of

bone (known as diagenesis) will also be discussed in light of its ability to alter the biological quantities of

isotopes and trace elements, thereby inhibiting dietary reconstruction. While diagenesis is pervasive in all

archaeological bone, it can be controlled through several new techniques that are discussed at the end of this

chapter.

Stable Isotope Analysis

Carbon Isotopes

The organic matrix of bone is composed mainly of collagen (approximately 90%), followed in abundance

by noncollagenous proteins, lipids and carbohydrates (Boskey and Posner 1984). Present within the bone

collagen are stable isotopes of carbon and nitrogen, that are frequently used to reconstruct ancient diet (for

reviews see Schoeninger and Moore 1992; Sihlen and Kavanaugh 1982). While the amount of carbon and

nitrogen in the body is under strict homeostatic control, the ratios of their stable isotopes (13C/12C and 15

in bone collagen vary according to the ratios found in food items and in the surrounding environment.

The differences in the isotopic ratios of carbon and nitrogen are due primarily to environmental differences

and biochemical or physiological reactions of organisms (Schwarcz and Schoenmger 1991). During these

reactions, one isotope is discriminated against in favor of the other, resulting in a change in the ratio of the

carbon and nitrogen isotopes involved. These ratios are expressed relative to a standard and in par per

million (%o) using the following formulas:

The standard used for carbon is PeeDee Belemnite Carbonate (PBD) a marine fossil limestone from South

Carolina (Craig 1957). The standard used for nitrogen is at.mospheric N (AIR), since its isotopic ratio is

constant worldwide (Mariotti 1980).

All terrestrial plants rely on atmospheric CO2 as their major carbon source but differentially fix CO2

according to one of three photosynthetic pathways: C3 C4 and CAM (crassulacean acid metabolism). The C3

and C4 pathways are so named because during the first stage of photosynthesis C3 plants produce a molecule

containing three carbon atoms, while C4 plants produce a molecule with four carbon atoms (OLeary 1981). C3 plants include wheat and rice, all root crops, vegetables, legumes, nuts, most fruits, and a rnajority of

temperate zone grasses. C4 plants are represented by the tropical grasses. C4 plants are represented by the

tropical grasses, such as millet, sorghm maize, some amaranths, and some chenopods. Tropical succulents,

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such as pineapple and various consumers of the next trophic leveis, such as carnivores or omnivores will

show an additional isotopic fractionation of approximately 1 %o from their chosen diets (Bender et al. 1981;

DeNiro and Epstein 1978b; Schoeninger 1985). Taking into consideration the above mentioned collagen enrichment factor and additional 1 %o trophic level fractionation, Chisholm (1989) provided a table of expected values for several different dietary regimes (Table 3.1). From Chisholms expectations, it would be difficult to distinguish individuals consuming large amounts of maize (-7.5% from those heavily dependent

on meat from C4-eating herbivores (-6.5%o).

Table 3.1. Anticipated a13C Values for Consumers and their Specific Diets (from Chisholm 1989:Table 2.1)

Ave. Dietary C (%) Consumer a13C (%)

C3 plants only -26.5 -21.5

Meat from herbivores on C diets -25.5 -20.5

C plants only -12.5 -7.5

Meat from herbivores on C diets -11.5 -6.5

Marine plankton only -19.5 -14.5

Meat from marine herbivores -18.5 -13.5

Meat from marine carnivores -17.5 -12.5

It appears then, that differential discrimination of carbon isotopes by living organisms allows for the

reconstruction of their dietary components. One of the first scholars to realize the potential of differential

fractionation by plant species as a method of reconstructing cacti are members of the CAM plants, and fix

atmospheric CO2 by either a C3 or C4 pathway depending on their environmental conditions. Some CAM

plants, such as Opuntia, rely almost entirely on the C4 pathway.

The biochemical differences in these pathways result in isotopic fractionation and 13C values that are

distinctive according to plant type. For example, C3 plants have 13C values ranging between -20 and -34%o,

with an average of -26.5%o. Modern C4 species range between -9%o and -16%o, and average near 12%o.

CAM plants that thrive m sunny, arid microhabitats fix CO2 in the same manner as C4 plants, and thus, have

a values similar to C4 species (OLeary 1481,1988; Smith and Epstein 1971). Although sorne environmental factors such as water availability, temperature, light intensity and available soil nutrients can effect the a13C

values of C3 plants, the degree of fractionation is so distinct from C4 plants that their is no overlap in their

a13C values (Farquhar et al. 1982; Tieszen 1991).

When an animal consumes plant foods, the carbon isotopes in the plant are further fractionated during

metabolism, causing a change in the animals a13C value (Bender et al. 1971; DeNiro and Epstein 1978a, b; van der Merwe and Vogel 1978). This change, known as the collagen enrichment factor, produces a 13C bone collagen value approximately 5%o more positive than the plant food originally consumed (Chisholm

1989; Chisholm et al. 1982,1983; Krueger and Sullivan 1984). For example, if an herbivore consumed only

C4 grasses, its bone collagen 13C value would be near -7.5% (-12.5 +5%o). It has also been reported that

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ancient diet was Robert Hall (1967a, 1967b, 1967c). In an attempt to explain anomalous C dates taken from

grass and com samples, Hall found that different plant species had characteristic 13C/12C ratios, that could be

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used to distinguish browsing animals (i.e. bison) from grazers (i.e. deer) (1967a: 5). Hall concluded his

paper with a great deal of insight, when he suggested that;

Presumably the diet of humans would be affected in sorne similar way. This opens up an obvious lime of investigation because of the reliance of sorne people on corn, others on bison flesh, others on acrns others

on marine sources and others on various combinations of these... (Hall 1967a: 5-6).

Seventeen years after Halls initial work, carbon isotope ratios have become so valuable to the reconstruction of ancient diets, that they are used in the majority of bone chernistry research. In addition to

carbon, the isotopes of nitrogenalso found in bone collagenhave been used with varying degrees of success to reconstruct past diet. Foliowing is a discussion of nitrogens value as a paleodietary indicator.

Nitrogen Isotopes

Nitrogens two stable isotopes, 15N and 14N are found almost exclusively either dissolved in the oceans or bound as N2 in the atmosphere (Marjotfi 1983). The ratio of these nitrogen isotopes can be used to

distinguish organisms that are N2-fixing from those that utilize

other forms of nitrogen, such as ammonia, soil nitrates, or animal urea (Ambrose 1993; Schoeninger &

Moore 1992). The biochemical reaction that take place in N2-fixing organisms produce 15N values very

similar to atmospheric N2 which approximates zero. Due to the greater amount of 15N relative to 14N soil

nitrates, ammonia and animal urea, plants that rely on these nitrogen sources have more positive 15N values

than N2 fixing plants. Thus, N2-fixing plants such as legumes, have lower 15N values than other terrestrial

plants, such as grasses (Delwiche & Steyn 1970; Wada et al. 1975). Many factors effect the degree of

nitrogen isotope fractionation in plants, including c1imate (Heaton 1987; Shearer & Kohl 1986), fertilizer

use (Aufderheide et al. 1988), soil type (Shearer et aL 19& and even altitude (Ambrose 1993). For this

reason, terrestrial plants Show (a wide range of 15N values, although many species have isotopic values

close to zero (Wada et al. 1975).

Just as there is a collagen enrichment factor for carbon isotopes, the same appears to hoid true for nitrogen isotopes. The amount of enrichment between diet and bone, however, is somewhat unclear. Studies done on

pigs and mice in a laboratory setting (DeNiro & Epstem 1981; Hare et al. 1991) and on plants and animais

in terrestrial and marine ecosystems (Minagawa & Wada 1984; Vogel et al. 1990; Wada 1980), suggest that

bone collagen is approximately 3-4% more enriched in 15 than diet Research done on free-ranging

herbivores, however, illustrate that bone collagen enrichment may vary considerably on account of

environmental and physiological factors (Ambrose 1986; 1991; Ambrose and DeNiro 1986; Heaton et al.

1986; Schoeninger 1989; Sealy et al. 1987).

There also seems to be a step-wise enrichment of 15 withm a single trophic system (McConnaughey &

McRoy 1979). The 5 values increase by approximately 3%o as one moves up the food cham. This step-wise

enrichment has been demonstrated for both marine and terrestrial vertebrates and invertebrates (Schoeninger

1985, 1989; Schoenmger & DeNiro 1984; Wada 1980).

If marine food items have 15 values that are distinct from terrestrial food items, and terrestrial N2-fixing

plants can be distinguished from those that do not fix atmospheric nitrogen, then it should be possible to

determine the presence of marine foods and legumes (N2-fixing) in the human diet. Many studies have

identifled populations dependent primarily on marine foods from those dependent primarily on terrestrial

foods (Schoeninger et al. 1983, 1990; Medaglia et aL 1990; Walker and DeNiro 1986).

Studies attempting to demonstrate legume consumption, however, have been less successful (Minagawa and

Akazawa 1991; Schwarcz et al. 1985; White and Schwarcz 1989; Spielinan et al. 1990). Since legumes

usually have lower values, it is assumed that a heavy reliance on legumes produces lower than normal 3 bone collagen values. Unfortunately, to date there are no laboratory experiments that substantjate this claim.

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In addition, Spielman and co-workers (1990) analyzed archaeological beans and found that the 15 values

were much more positive than expected.

These elevated 15 legume values may be a consequence of the re-working of agricultural fields. When a

fleid is prepared for cultivation soil nitrates become more readily available and the 15N value of the soil

increases. With each successive use of the agricultural field, the soil 15N value continues to rise (Mariotti et

al. 1983). As these soil nitrates become available, legumes stop fixing atmospheric nitrogen and begin to

utilze this new nitrogen source, producing 15N values that are more postive than atmospheric nitrogen

(15Na near O %o) (Shearer et al. 1983). Thus, the elevated 15N values present in prehistoric bean samples

may represent plants grown in cultivated fields and utilizing soil nitrates rather than fixing atmospheric

nitrogen.

The 3%o enrichment of 15 by trophic level raised hopes among scholars that nitrogen isotopes could be

used to identify the consumption of animal protein by ancient human populations (Schoeninger 1985).

Unfortunately, since humans are neither exclusively carnivorous nor herbivorous, the 3%o 15N enrichment

factor is not detectable. Instead, most humans groups consume a percentage of animal protein in their diet,

ranging from approximately 10% to 25% in human societies world wide (Schoeninger and Moore 1992). As

Schoeninger and Moore (1992) illustrate, there is only a O.5% difference in the 15N values of those

consuming 10% animal protein and those consuming 25%. Consequently, the very small difference in 15

values limits nitrogens use in determining meat consumption between human populations.

It appears then, that nitrogen isotopes are of rather limited utility. While nitrogen can be used to distinguish

between marine and terrestrial components, it has been less effective as an indicator of meat and/or legume

consumption. Carbon isotopes, on the other hand, have been used more successfully to reconstruct ancient

dietary components. A new approach suggests that carbon ratios can even be more reliable in reconstructing

diet when taken from both bone collagen and bone apatite. The foliowing section outlines this new

technique.

Collagen versus Apatite as a Paleodietary Indicator

While many of the studies discussed aboye rely on a predictable relationship between collagen and the

isotopic composition of diet to differentiate between social groups, collagen may not accurately reflect the

sum importance of the lipid, carbohydrate and protein fraction in the diet (Ambrose 1993; Chisholm 1989;

Chisholm et aL 1982; Krueger and Sullivan 1984). Carbon atoms used to synthesize collagen may come

from different dietary fractions according to the amount of protein consumed. According to a model

proposed by Krueger and Sullivan (1984), when protem intake is low, protein carbon will be deposited in

bone coilagen, since it is used primarily for growth and maintenance of bodily tissues (Krueger and Sullivan

1984). Lipids and carbohydrates, on the other hand, are used primarily for energy metabolism (DeNiro and

Epstein 1978a). When lipids and carbohydrates undergo cellular metabolism, the associatecj carbon atoms

are synthesized into blood bicarbonates, after which they are incorporated into the apatite fraction of bone

(DeNiro & Epstein 1978a). Consequently, since the protein fraction appears to contribute carbon atoms to

collagen, while carbohydrate and lipid carbon settles in bone mineral, it has been suggested that only the

protein fraction of the diet will be discernible in collagen carbon isotope ratios (Chisholm et al. 1982).

Krueger and Sullivan (1984) further hypothesized that if different dietary fractions were responsible for

collagen and apatite synthesis, than the ratio between apatite and collagen a 13C values (13CA-C would be

smaller in carnivores than in herbivores, due to a differential emphasis on lipids, carbohydrates and protein

in the diet. Herbivores acquire protein primarily from plant protein, and energy from plant carbohydrates,

while carnivores utilize animal flesh for protein, and lipids and animal protein for energy metabolism. Since

lipids are low in 13C relative to carbohydrates and protein (DeNiro & Epstein 1978a; Jacobson et al 1972;

Winlder & Schmidt 1980), and carnivores have a greater reliance on 13C depleted lipids for energy

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metabolism than herbivores, then carnivores will have lower 13CA-C values. Thus, it was hypothesized that

while carnivores had the lowest 13CA-C L values, and herbivores the highest, human populations should fall

between this range depending on their meat intake. Throughout their research, Krueger and Sullivan (1984)

implied that collagen 13C values reflect primarily protein intake, and apatite 13C values reflect the energy

portion of the diet.

New data generated by Ambrose and Norr (in prep., as cited in Ambrose 1993), suggest that this division

between protein and collagen, and lipids carbohydrates and apatite, may be more complex than reported. In

a recent controlled, laboratory study, Ambrose and Norr found that collagen carbon reflect protein intake

only when adequate protein is available in the diet for collagen synthesis. When the diet is very low in

protein, however, carbon atoms from both lipids and carbohydrates form the major component of bone

collagen. In other words, the origm of collagen carbon will depend directly on the quantity of protem

consumed by an individual. Consequently, while collagen 13C values have been used to reconstruct levels of

C3 and C4 (maize) plants in the diet (for a review see Schoeninger and Moore 1992), this is only an accurate

predictor when low-protein diets are consumed. When enough meat is included in the diet to supply protein

for collagen formation, the collagen 13C will reflect the animal flesh consumednot the plant material. As Ambrose explains, ... in diets with levels of animal protein sufficient for collagen synthesis, carbohydrate and lipid carbon may be severely underrepresented (Ambrose 1993:105).

In addition, contrary to the view that bone apatite 13C values represent only the lipid and carbohydrate

fraction of the diet (DeNiro & Sullivan 1978a; Krueger and Sullivan 1984), Ambrose and Norr found that,

collagen reflects mostly the protein 13C value, while the apatite reflects the whole diet 13C value, not simply the energy component (Ambrose 1993:109). Thus, when the collagen 13C value is compared to the whole diet, apatite 13C value, both trophic levels and dietary fractions can be distinguished (Ericson et al.

1989; Ezzo 1993; Lee Thorp et al. 1989).

A comparison of collagen and apatite 13C values may further aid in identifying status-based dietary

differences of ancient populations. While collagen carbon has been the primary source of a values in most

research, it reflects only specific fractions of the diet. Further, these fractions (lipids, carbohydrates,

proteins) will be differentially reflected in collagen depending on the level of protein intake. For example, if

two sub-populations both consume 70% of their daily calones from maize (a C4 plant), yet only one group

has su dietary intake of a C3 protem source, the 13CA-C values will not be equivalent between the groups. It is

likely that only the protein-deficient group will have collagen with a C4 signal, since the collagen carbon

was derived primarily from the maize carbohydrates and lipids, thereby reflecting a C signal. In the collagen

of the C3 protein eaters, carbon from maize will be underrepresented, since their collagen carbon comes

from the C3 animal source. If the apatite values reflect the entire diet, not just the protein source like

collagen, thai a comparison of the collagen and apatite 13C values would allow for a more thorough

comparison of the differential reliance of dietary fractions throughout the population.

One of the potential problems with bone apatite is its susceptibility to post-mortem alterations (diagenesis)

(Schoerunger & DeNiro 1982; Land e al. 1980; for reviews see Lee Thorp 1989; Klepinger 1984).

Biological apatite contains carbon in the form of carbonate ions (Lowenstam & Weiner 1989), which can be

found in two primarv locations: (1) within the crystal lattice, as a substitute for Sr, Ba, Pb, hydroxide or

phosphate ions (Baud & Very 1975; Le Geros et al. 1967), and (2) absorbed in the hydration shell or on the

crystal surface (Baud & Very 1975; Eanes & Posner 1970). Due to the propensity of ionic exchange at the

crystal surface, carbonate jons exposed to the surface are readily exchanged with other ions in bodily fluids

(Newesly 1989; Piepenbrink 1989). In a post-mortem enviromnent, the surface (absorbed) carbonate in

archaeological bone has a tendency to be replaced with ions from the surrounding soil or groundwater.

A number of scholars have argued that a chemical pretreatment of bone apatite with an acid, such as acetic

acid, will remove most diagenetic effects, leaving only the original 13c/12c ratio (Lee Thorp 1989; Lee

Thorp et a!. 1989; Ericson et al. 1981; Haynes 1968; Krueger and Sullivan 1981). It appears that an acid

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pretreatment should substantially reduce diagenetic effects, since researchers who analyze for trace

elements, and thereby rely exclusively on bone mineral have been using similar techniques with great

success (Price et al. 1991; Sillen & Kavanaugh 1982).

While the potential for using apatite for carbon isotope ratios appears promising, questions still remain

concerning dietary fractions and their influence in the formation of both bone collagen and apatite. Until

more controlled, laboratory feeding experiments are conducted, carbon isotope ratios of bone apatite should

be interpreted with caution. The above discussion, however, assumes that the bone collagen used for

analysis is clean and well-preserved. What happens when archaeological bone is buried for thousands of

years? How does post-mortem alteration (diagenesis) affect bone collagen, and in turn, carbon and nitrogen

isotc ratios? The following section will explore some of the problems associated with diagenesis and

isotopic analysis.

Diagenesis iii Isotopic Analysis

Scholars eager to apply isotopic analysis to the reconstruction of ancient diets have only very recently

considered the role of diagenesis. White & Hannus (1983) were among the first scholars to recognize

diagenetic processes in the organc fraction They suggested that the chemical weathering of collagen

(initiated by micro-organisms) was the first stage in the decomposition of bone tissue. It was unclear,

however, the minerals released during collagen diagenesis existed via groundwater or remained and

recrystallized within the mineral fraction. Thus, the movement of minerais m the organic fraction could

render the minera! fraction vulnerable to diagenesis.

Several scholars have examined diagenetic effects on both the organi and inorganic bone fractions

(Schoeninger & DeNiro 1982; Nelson et al. 1986). Nelson and co-workers analyzed a series of terrestrial

and marine mammals for elemental strontium, strontium isotopes, carbon ratios from collagen, and carbon

ratios from apatite. They found that diagenesis was more pronounced m the mineral than in the orgamc

fraction; marir and terrestrial samples could not be differentiated using the carbon isotol ratios in apatites,

however, carbon ratios from collagen were successful in separating the animal types in both modern and

archaeological assemblages.

In an attempt to identify the purity of collagen, and hence, the extent of diagenesis, several scholars have

analyzed the amino acid composition and the C/N ratio of ollagen (DeNiro 1985; DeNiro & Weiner 1988;

Hare 1980; Hare & Estep 1983; Hare et al. 1991; Kennedy 1988; Schoeninger et al.1989; Tuross et al.

1988). Unlike other animal tissues, collagen is unique in possessing the amino acid hydroxyproline and

glycerin in excess of 30% (Hare 1980). Since each amino acid has a specific stable isotope ratio, the loss of

one amino acid can greatly affect the isotopiv ratio of collagen as a whole (Hare & Estep 1983). According

to Hare et al. (1991), isotopic analysis should be conducted on a single collagen amino acid rather than

collagen as a whole. They argued that single amino acid could be easily tracked through the food chain,

since the isotopic composition of a molecule records its biochemical history in the food chain (1991:286). Results from their analyses show that carbon isotope rafias taken from individual amino acids were almost

identical to those fornid in the diet.

Most collagen samples used for isotopic analysis have an atomic C/N ratio that normally ranges between 2.9

to 3.6 (DeNiro 1985). Although some of this variafion may be due to experimental error in measuring

calcium and nitrogen concentrations, the higher values may also indicate collagen contamjnation. According

to Kennedy (1988), C/N ratios higher than 3.4 may be the result of contamination by humic acid, lipids,

carbonates or other carbon-rich rnaterjals. While these contaminants may cause a shift in 13C values away

from the original biological signal, Ambrose (1993) argues that samples with C/N ratios aboye 3.4, shoul not be rejected unless the isotopic composition of the contaminant differs greatly from that of the tissue

analyzed (p.75)

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Although the mineral fraction of bone is more susceptible to diagenetic forces than the organic fraction,

collagen preservation can vary greatly within a particular site. Tests assessing the integrity and purity of

colla should be conducted before the resulting isotopic ratios are used to reconstruct paleodiets. Amino acid

assays and C/N ratio of collagen have been successful in identifying diagenesis in the organic bone fraction.

all methods must be used on every sample; Schoeninger (1989) suggest that bone samples with less than

25% organic content by weight should use atomic C/N ratio to assess collagen integrity, while those with

less than 10% organic by weight should utilize an amino acid assay.

Trace Elements

Introduction

Unlike stable isotope analysis, trace element analysis makes use of the inorganic portion of bone. This

inorganic, mineral fraction is compose primarily of calcium and phosphate, but also includes a number of

minor and trace elements that are incorporated into the lattice structure of the crystals during bone

manufacture (Lowenstam and Werner 1989). WhiJ the amount of calcium and phosphorus are tightly

regulated in the body quantities of many trace elements vary according to physiological and biochemical

processes. Interest in the movement of trace elements through the human body and biosphere was largely a

consequence of research conducted on the by-products of nuclear testing during the 1950s. The radioactive isotope Strontium 90 was considered harmful to human health, and as such, its concentrations were

carefully recorded throughout the environment, foodwebs, and human and animal tissues (Alexander &

Nusbaum 1959; Alexander et al. 1956, Bowen & Dymond 1955; Comar 1963; Comar et al. 1955; Elias et al.

1982; Harrison et aL 1955; Odum 1951, 1957; Steadman et al. 1958; Taylor et al. 1962; Thurber et aL 1958;

Turkian & Kulp 1956; Wasserman & Comar 1956).

These initial studies demonstrated that strontium concentrations varied according to physiological and

biochemical processes such that plant species were identifiable by strontium content. Toots and Voorhies

(1965) were the first to apply these data to distinguish grazmg fossil herbivores from browsers by measuring

bone strontium leveis. However, it wasnt until the 1970s that anthropologist began to recognize the potential of trace element analysis to reconstruct paleodiets (Boaz & Hampel 1978; Browns 1973; Gilbert

1975; Kavanaugh 1979; Lambert et al. 1979; Schoeninger 1979a, b; Szpunar 1977; Szpunar et al. 1978).

Trace element analysis heid great promise, because rather than rely on artifacts or floral and faunal remains

to indirectly estimate diet, this analysis was capable of measurrng the skeletal material directly.

As interest in trace element analysis continued to grow, it gained wide spread acceptance for reconstructing

ancient subsistence strategies (Schoeninger 1981; Sillen 1981a, b; Katzenburg 1984; Edward et al. 1984;

Bisel 1980; Blakely & Beck 1981; Brown & Blakely 1985; Connor & Slaughter 1984; Geidel 1981, 1982;

Gilbert 1985; Harch & Geidel 1983; Katzenburg & Schwarcz 1984; Lambert et al. 1982, 1983; Price 1985,

1986; Frice & Kavanaugh 1982; Price et al. 1985a, b, Schoeninger & Peebles 198 While many trace

elements were used to reconstruct diet, most of these studies focused on the unportance of strontium, due to

its theoretical ability to distinguish between meat and vegetable components in the diet This theoretical

foundation is presented below.

Theoretical Basis

Trace element analysis rests on an understanding of bone microstructure and physiology. Mature bone

mineral is composed primarily of a crystalline form of calcium phosphate, known as hydroxyapatite. With

an unit celi formula of Caio (PO4)6(OH)2 calcium plays a major role in the formation of hydroxyapatite

(Neuman & Neuman 1958; Sillen 1989; Baron 1990). Bone hydroxyapatite can fluctuate from this predicted

structural formula, however, during an ionic exchange between body fluids and the bone minera! (Neuman

& Neuma 1958; Pate & Hutton 1988; Sillen 1989; Pate et al. 1991).

Due to the large, highly-charged surface area of hydroxyapatite crystals, various ions in body fluids (such as

strontium, barium and lead) are attracted to the crystal and easily incorporated by displacing the calcium

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component (Neuman and Neuman 1980; Johnsson 1986; Molleson 1990; Pate et al. 1991; McLean & Urist

1955; Ortner & Putschar 1985; Underwood 1977). As a result, rather than being a pure, insoluble crystalline

form, trace amounts of strontium, barium and lead are commoniy found within the lattice structure of the

hydroxyapatite crystal.

During mammalian metabolism, a proportionateiy greater amount of calcium is absorbed relative to the non-

nutrient alkaiine earth elements, such as strontium (Elias et al. 1982), which resuits in a fractionation of

strontium in favor of calcium. Since calcium is one of the principie elements used in bone and cell wall

construction, more calcium than strontium is absorbed and deposited in bone mineral (Avioli 1988; Comar

1963; Silverberg 1990; Wasserman & Comar 1956). Although only approximately 20% of ingested

strontium is actually absorbed into the bloodstream, 99% of the strontium absorbed is deposited in mineral

fraction of bone (Elias et al. 1982; Hogue et al. 1961; Kshurager et al. 1966; Lough et al. 1963; Rosenthal et

al. 1972; Samachson 1967; Schachter 1963; Spencer et al. 1973; Schroeder et al. 1972; Taylor et al. 1962;

Wasserman 1963).

This compositional variability of bone mineral is the basic tenet of trace element analysis, since

environmenta! exposure and dietary intake are assumed to be responsible for such chemical alterations

(Klepinger 1984; Parker & Toots 1980). Theoretically then, the quantity of trace elements consumed in the

diet should correlate with the quantity retained in the bone mineral.

Since strontium intake is reflected in bone mineral, and all mamn discriminate against strontium in favor of

calciurn during metabolisn there should be a decreased concentration of bone strontium as one advances up

the food chain. Research indicates that strontium levels are highest in bedrock, followed by soil, plants,

herbivores and carnivores in a given micro-habitat (Comar et al. 1957; Elias et al. 1982; Menzel & Heid

1959; Wallace & Romney 1971). Plants absorb between 50-100% of the available strontium from the soil.

An herbivore consuming leafy matter will absorb only 20% of the strontium it ingests relative to calcium. A

carnivore who thrives on herbivore flesh will not only ingest less strontium than the herbivore, but will only

absorb 33% of the ingested strontium. Therefore, in examining a particular food chain, herbivore have the

highest strontium values, carnivores the lowest, and omnivores such as huinans, have intermediate values.

Scholars wary of this principie argue that its simplistic nature overlooks many of the variables that make

regional, trophic-level comparisons difficult (Klepinger 1984; Pate et al. 1991; Radosevich 1989a, 1993;

Sanford 1993; Schoeninger 1981; Silien & Kavanaugh 1982; Sillen Smith 1984). At the base of the food

chain, soil chemistry can cause a great deal of variability in the Sr/Ca ratios available to organisms (Pate et

al. 1991; Radosevich 1989a). For example, in many arid regions, calcium carbonate nodules are a common

occurrence (Ezzo 1991). The calcium build-up caused by these nodules could significantly alter soil Sr/Ca

rati and ,in turn, effect the Sr/Ca ratios throughout the foodchain, making regional comparisons difficult.

It should not be assumed, however, that ah soil elements are chemically mobile. Interaction among elements

depends on their availabilitY within the soil. Many factors effect the availability of jons, including

temperature, precipitation, porosity of the soil, solubility and weathering rates of a given minera! and soil

pH (Buol et al. 1981; Mitchehl 1957). Soil pH can have a dramatic effect on ionic mobility such that a drop

in pH levels greathy increases the potential for ionic exchange (Buole et al. 1981; Isermann 1981). Ionic

availability is slowly gaining in importance as a factor that can affect both trophic level Sr/Ca ratios and the

geochemistry of archaeological bone ( Pate & Brown 1985; Pate & Hutton 1988; Pate et al. 1989, 1991;

Radosevich 1989a,).

Plant uptake and physiology can also alter the Sr/Ca ratios of a food chain. As Radosevich (1993) suggests,

root placement in either strontium rich or calcium-rich soil horizons can greatly influence Sr/Ca plant ratios.

For example, in soils with a high caliche content only the roots of trees and shrubs are massive enough to

permeate the calcareous subsurface. The Sr/Ca ratios of these plants will be much lower than the ratios of

grassy vegetation, whose root system is not expansive enough to penetrate the calcium-rich subsoil.

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Therefore, browsing animals who rely on leaves from trees and shrubs will have lower bone mineral Sr/Ca

ratios than grazing animals who consume various grasses. Differences in strontium content are not solely the

result of plant species. As Isermann (1981) suggests, different parts of the same plant (stem, leaf, frult) can

also differ in strontium content by an order of magnitude.

As discussed aboye, many variables can affect strontium concentrations in the food chain. Variations in soil

chernistry and the chemical characterization of its parent material, fluctuations in plant physiology and

uptake, trophic level and even animal behavior can a Sr/Ca ratios. The role of strontium in the biosphere is a

cornplex issue and all variables must be explored before assumptions regarding strontium distribution can

be made.

Multi-Element Research

While sorne studies focus on one or two elements, other scholars use a multielemental approach to

reconstruct paleodiets (see reviews in Buikstra et al. 1989; Sandford 1992; Ezzo 1992). The trace elements

are generally chosen because of their ability to distinguish between plant a animal resources in the human

diet. For example, magnesium, manganese, strontium and vanadium are used to infer plant consumption,

while selenium, zinc, copper and molybdenum are considered to be indicators of animal protein (Arrhemus

1990; Beck 19 Edward et al. 1984; Edward & Benfer 1993; Francalacci 1988, 1989; Francalacci & Tan

1988; Geidel 1981, 1982; Gilbert 1975. 1977; Hatch & Geidel 1983, 1985; Lambert et al. 1979; Liden 1990;

Morgan & Schoening 1989). Most multi-elemental studies have focused on the shift from hunting gathering

to maize horticulturalists (Beck 1985; Buikstra et al. 1989; Gilbert 1975; Lambert et al. 1979; Szpunar 1977;

Szpunar et al. 1978 or status based dietary differences ( Blakely & Beck 1981, Blitz 1993, 1994; Brown &

Blakely 1985; Lambert et al. 1979, 1982). The basic tenet behind trace element research is that elemental

concentrations in bone mineral vary as a consequence of environmental exposure and dietary intake. It is

therefore imperative to understand the association between dietary intake and skeletal concentrations before

an element can be used to reconstruct consumption patterns. While most scholars argue that multi-element

dietary reconstructions are more accurate than those based on a single element, there is almost no

information concerning the distribution and function of magnesium, manganese, zinc, seIenium copper, or

molybdenum in both pre- and post-mortum contexts (Klepinger 1984; Ezzo 1991).

Pate and associates (1991:59) include magnesium in their, list of dietary indicators, however, swine feeding

experiments demonstrate otherwise. (Klepinger 1990). In Klepingers research, animals fed magnesium enriched diets had bone magnesium levels only slightly higher than those of the control group. She argued

that if a greatly inflated magnesium intake had such a limited affect on bone magnesium concentration, than

dietary variations found among most human groups would not be detected by differences in the bone magnesium concentration (1990:516).

In a thorough review of trace elements used as possible dietary indicators, Ezzo (1991) suggests that only

barium and strontium should be used for paleodietary reconstructions. As he rightly notes, although it is possible other elements may be found to have potential as dietary

indicators, models based on physiological and biochemical data have yet developed (1991:49). Considering the available data and uncertainty surrounding many of the elements, only barium and strontium will b used

in this research for dietary reconstruction.

While the aboye noted studies emphasize elemental distribution in living bone mineral, an increasing

number of scholars are turning the attention towards elemental distribution in post-mortem bone tissue

(Byrne & Paris 1987; Edward 1987; Klepinger et al. 1986; Kyle 1986; Pate Hutton 1988; Pate et al. 1991).

Not only can diagenesis affect bone colla as discussed previously, but it can also alter the mineral fraction

thereb obscuring the biological trace element signal.

Diagenesis in Elemental Analysis

10

The structural and chemical alteration of post-mortem bone tissue is known as diagenesis. Diagenesis

primarily results m either the recrystallization of hydroxyapatite crystals, or isoionic and heteroionic

exchange of its chemical components. The earliest anthropological studies used strontium to reconstruct past

diet with little consideration for diagenetc processes (Brown 1973: Schoeninger 1979a, b). Over the Iast

decade, diagenesis has surged to the forefront as a process that must be understood before elemental

analysis can be used with any degree of confidence (Price et al. 1991).

Once a bone enters a post-mortem environment, its deterioration is mfluenced by a number of biochemical,

geochemical and organic processes. The results of these processes are cumulative; the longer a bone remains

in a postmortem matrix, the more physical and chemical alteration will take place (Deotare et al. 1988; Price

1989). Diagenetic changes in bone tissue generaily occur in one of three ways (Parker & Toots 1980; Pate &

Hutton 1988; Pate et al. 1991; Radosevich 1993; Sandford 1992; Sillen 1989): (1) Chemical components

from biological hydroxyapatite can be exchanged with soluble jons from the surrounding soil (Ezzo 1991;

Lambert et al. 1979, 1982, 1983, 1984a, 1985b), (2) Mineral components such as calcite (CaCO3) and barite

(BaSO4) can be precipitated to fill the tiny cracks and pores in bone tissue (Parker & Toots 1980), and (3)

During crystallization, hydroxyapatite crystals can be altered from biological apatite to a variety of

geochemical forms (Sillen 1989).

These processes have been grouped according to their role as either intrinsic or extrinsic forces in bone

tissue alteration (Von Endt & Ortner 1984). Intrinsic factors, such as porosity, density, size, and chemical

composition of bone greatly affect the degree of diagenesis. In general, bones that are less dense, more

porous or contain a high percentage of amorphous mineral crystais are more susceptible to diagenesis

(Grupe 1988; Newsley 1988, 1989b).

Due to their distinct degrees of calcification and density, cortical (compact) and trabecular (cancellous) bone

should respond differently to diagenetic processes. Cortical and trabecular bone differ markedly in their

degree of calcification; cortical bone is 80-90% (by volume) calcified, while only 15-20% (by volume) of

trabecular bone is calcified. Consequently, the solid, cortical bone is much denser and less susceptible to

diagenesis than trabecular bone. Trabecular bone, on the other hand, is much more porous due to its

principie function as the mineral reservoir of the body (Baron 1990; Underwood 1977), and thus has a

greater tendency to be physically or chemically altered (Currey 1984; Price 1985; Sillen 1990)

Several studies have emphasized the differential effects of diagenesis on bone tissue. Lambert and his

associates (Lambert et al. 1979; Szpun 1977; Szpunar et al. 1978) analyzed human remains from Woodland

sites in West Central Illinois to elucidate dietary and biological patterning. When it became apparent that

diag was effecting elemental biological signais in the bone tissue, their research shifted to an examination of

post-depositional processes and diagenesis (Lambert et 1982, 1983, 1984a, b, c, 1985a, b; Vlasak 1983).

In their latter research, they compared the elemental differences between femurs and ribs from Woodland

sites in Illinois. They argued that unless diagenesis had occurred, the dense, cortical bone of the femur

should have the same strontium levels as the trabecular rib bone. An elemental analysis of both ribs and

femurs demonstrated that the ribs contained elevated leveis of elements usually associated with soil

contaminants, such as potassium, iron, magnesium and aluminum. Sodium and calcium were lower in ribs

than in femurs, while the elements assumed to be dietary indicators (strontium, zinc and magnesium) were

found in almost equivalent levels. These results led them to conclude that the problem with diagenesis lies

in the incorporation of extraneous elements rather than diagenetic loss of material (Lambert et al. 1982:291).

In 1989, Buikstra and her associates re-analyzed Lamberts research using matched rib-femur samples. While similar leveis of zinc and lead were found in both types of bone, the ribs had a higher concentration

of magnesium, copper, potassium, iron, manganese and aluminum than the femora. Only calcium, strontium

and sodium were higher in the femur than in the rib. The researchers suggest that elemental differences in

11

the bone pairs could be due to a greater permeability for ribs, differential cleaning according to bone type, or

difference in bone remodeling rates.

Grupe (1988) and Brtter et al. (1977) also reported elemental differences in cortical and trabecular

archaeological borie. Unlike Buikstra, both studies found that trabecular bone contained a significantly

higher concentration of strontium and barium than cortical bone. Upon examining the mass ratio of

spongiosa/compacta bone at specific locales throughout the human body, it was determined that trabecular

bone is much more susceptible to ionic additions due to its porous nature. In addition, trabecular bone

showed enormous variation between samples, even if they were taken only centimeters apart.

In addition to archaeological samples, modern bone tissue has also been analyzed for differential elemental

concentration by bone type. Tanaka et al. (1981) studied strontium concentrations in the human skeleton of

modern Japanese populations. In support of Buikstras analysis of archaeological remains, they found that the cancellous, rib bone contained significantly lower levels of strontium than did the cortical bone of the

femurs.

The available research demonstrates that differences exist in the elemental concentrations of trabecular and

cortical bone, however, the direction of this difference is unclear. With ah the uncertainly surrounding

elemental concentrations in clean, modern bone, archaeologists should not use elemental differences in rib

versus femur as a method of identifying diagenesis. More research on modern humai from the same

population consuming the same diet must be carried out before this method can be used with any degree of

confdence. In addition as will be discussed in the following section, there are other reliable mea for

assessing the presence of diagenesis.

While it is clear that intrrnsic factors have an important effect on diagenesis, extrinsic factors also play a

vital role (Von Endt & Ortner 1984

Extrinsic factors such as temperature (Hare 1980;Von Endt & Ortner 1984) groundwater (Hare 1980; White

& Hannus 1983), soil pH (Garlick 1969; Gordon & Buikstra 1981), and soil chernistry (Becker et al. 1968;

Benfer 1984; Katzenburg 1984; Keeley et al. 1977; Lambert et al. 1984a; Nelson & Sauer 1984) have all

been considered by various scholars.

A common thread running through these studies is that although sorne elements, such as iron, manganese,

aluminum, and potassium are more susceptible to postmortem alterations than others (strontium, lead and

zinc), no one element is immune from diagenetic processes. For example, strontium, once thought to be

generaily stable in post-depositional environments (Parker & Toots 1970, 1980; Pate & Brown 1985), can

be highly mobile under certain geologic, geographic and temporal conditions (Nelson et al. 1986; Pate et al.

1991; Radosevich 1989a, b, 1993).

Pate and his associates (Pate & Hutton 1988; Pate et al. 1989) attempted to develop a model of ionic

exchange between bone and the surrounding soil matrix. They focused on the solubility of ions m arid soils

of South Australia and their presence in archaeological bone. Analyzing for a suite of elements, they found

that archaeological bone was depleted in magnesium, yet enriched in aluminum, iron, barium, strontium,

potassium and manganese. Only zinc and sodium were found m near equal quantities in both bone and soil.

Their results suggest that as bone deteriorates, the dissolution of apatite crystals permits the incorporation of

carbonates and other ions into the crystailine structure. It was also demonstrated that there is an association

between collagen degradation and degree of diagenesis; as collagen decomposes the physical and chemical

alteration of the mineral fraction will also increase. When microbes decompose collagen, they form acidic

by-products that accelerate the rate of ionic exchange in the hydroxyapatite crystal (White & Hannus 1983).

Pate and his colleagues found that the enrichment of aluminum, iron and potassium was probably due in part

to soil contaminants that were incorporated into the mineral fraction, but not completely removed before

analysis.

12

Price (1989) studied the effects of diagenesis on bone samples from Skateholm, Sweden, Nelson Bay Cave

in South Africa, and the Price] site in Wisconsin. Utilizing multi-elemental analysis, he found an association

between degree of diagenesis and age of sites in the Skatel sample. The older samples from the Skateholm II

site, had lower bon calcium and collagen levels than did the those from the younger Skateholm 1 site. In

addition, sodium, strontium, copper, magnesiuni manganese and aluminum were much more abundant in the

older samples than the younger. The lower calcium and collagen leveis at t older site indicate a greater

degree of bone degradation and diagenesis. The reduced calcium levels also appear to make the other

elements loi more abundant in proportion, which account for the high leveis of the elements in the older

samples. The strong positive correlation betwee strontium and iron, and between magnesium, aluminum and

iron suggest that many of the samples contained soil components that were incorporated into the bone

mineral. During diagenesis, bone calcium was replaced by soil ions, thereby reducing calcium levels and

increasing th quantity of magnesium and alummum.

At Nelson Bay Cave, Price found calcium values fairly stable over ti with an average of 35.7% (suggesting

good preservation). There was a very low correlation between calcium and the other elements analyzed

(aluminum, copper, iron, magnesium, manganese, sodium, strontium, and zinc). The strongest positive

correlations occurred between iron an aluminum, and sodium and magnesium. The similar behavioral

pattern of rnagnesiurn, iron and aluminum probably suggest the presence of soil contaminants. The elevated

sodium levels in the older samples suggest incorporation of these ions during apatite recrystallization, which

increases with length of deposition.

The data from the Price III site show that calcium values decreased with the sample age. The only

significant positive correlation was between iron and aluminum, while strontium was not strongly correlated

with any other elernent. A strong negative correlation was fornid between calcium and manganese. The

decreasing calcium values with sample age suggests a diagenetic loss of calciurn over time. Since

manganese is h mobile in post-depositional environments (Lambert et al. 1984a), its negative correlation

with calcium supports the idea of diagenesis through ionic exchange. The positive correlation between iron

and aluminum suggests bone mineral enrichment by soil contaminants.

Prices research dernonstrates that while diagenesis is prevalent in archaeological bone, the forrn and degree of diagenetic alterations are dependent upon geologic, geographic and temporal conditions. Before dietary

patternmg can be inferred, multi correlations should be conducted to assess the degree of diagenesis.

Elemental correlations can indicate bone mineral enrichment by soil contammants, apatite recrystallization,

ionic exchange, or bone deterioration.

While the problem of diagenesis has been considered by many scholars in their research, only now are sorne

devising methodologies to control for its affects. Lambert and co-workers (1989, 1990, 1991), have experimented with sample cleaning procedures. Their methods for obtaining elemental biogenetic

concentrations include: (1) abrading and washing the sample in distilled water, (2) physically removing 1-3

mm the outer bone cortex, (3) treating the powdered bone with 1N acetic acid, and (4) repetitively washing

the powdered bone in an acetate buffer.

Since each method appears to be beneficial in obtaming the biogenetic signal of specific elements, the

authors endorse a combined approach in processing bone samples for analysis. They also contend that

element such as magnesium, potassium, sodium, and zinc can be used as dietaz indicators when bone is

processed correctly (Lambert et al. 1991). Washing powdered bone in an acetate buffer restores original dietary zinc levels, while the removal of the outer bone cortex removes most diagenetic effects of

potassium, magnesium, and sodium.

A number of other washing procedures have been developed to control for diagenesis (Burton & Price

1990a, 1990b; Ezzo 1991, 1992; Fn et al. 1991; Sillen 1986, 1989; Sillen and LeGeros 1991). Sillens procedure calls for washing powdered bone in an acetate buffer with a pH of 4.5. The solution is

13

centrifuged, the supernatent is decanted and saved for elemental analysis, and more buffered acid is added to

the residue. This sequence is then repeated another 20-25 times, so as to create a solubilil profile (Sillen 1989). Siliens methodology was ground-breaking in that it demonstrated that Ca/P ratios of archaeological bone could resemble those of modern bone by means of a sequential buffered acid wash. Although his

methodology holds great promise for diagenetic research, his procedure (which calis for 20-25 supernatants

per sample), is both labor intensive and time-consuming. In addition, Siliens methodology analyzes the liquid decanted from the sample rather than the digested bone residue.

Price and associates (Burton & Price 1990a,1990b: Price et al. 1991) improved upon Siliens methodology in several ways. They wash whole bone in 1N unbuffered acetic acid, ash (to remove all organics), grind to

a powder, and digest in nitric acid. Unlike Siliens method, in which the bone powder was centrifuged and removed before analysis, Price and Burtons method analyzes the bone in solution Using this procedure, they demonstrated that archaeological bone Ca/P ratios could be reduced to modern levels (approx. 2.1),

thereby eliminating most diagenetic affects and recovering biogenetic signals. Perhaps the most important

improvement is the speed with which the samples can be processed and analyzed. Since the powdered bone

ash is digested only once, as opposed to the 20 times as in the procedures of Sillen, elemental analysis can

be done with much less investment of time and labor.

In addition to washing methods, a number of other methods can be used to assess the nature and degree of

diagenetic change m bone tissue. As mentioned aboye, the ratio of calcium to phosphorus m archaeological

bone is a good indicator of post-mortem contamination. Since both calcium and phosphorus function

primarily as the crucial components for bone mjneralizafion, their quantities are constant in bone tissue

(Underwood 1977).

Phosphorus accounts for approximately 17% of the mass of modern bone mineral, while calcium levels

approach 37% (Avioli 1990; Broac 1990; Woodard 1962). Therefore, as Katzenberg (1984) and

Sillen(1981b) suggest, archaeological bone, free of diagenesis, should have the same Ca/P ratio as that

found in modern bone (approximately 2.15). Ratios deviating from this value suggest either enrichment or

depletion of calcium and/or phosphorus. For example, when calcium is depleted from bone mineral, the

Ca/P ratio is lower than 2.15, and the other element may appear to be present in higher quantities. Because

spectrographic analysis measures the amount of one element in relation to all other elements in the sample,

if one element, such as calcium, is low, the other elements may appear high in proportion. A Ca/P ratio

higher than 2.15 can occur when bone mineral is enriched in Ca during diagenesis.

The percentage of ash content of archaeological bone also provides a measure of diagenetic change. Living

bone consists of approximately 70% inorganic mineral distributed throughout an organic matrx (Underwo

1977). When ashed, clean, modern bone will have about 70% (by weight) remaining as a mineral fraction.

To assess the integrity of archaeological bone, then, the bone ash % should be very near to 70%. If the bone

ash % is lower, it may suggest that diagenesis is removing mineral components from the bone. If the bone

ash % is higher than 70%, it can indicate one of two processes: (1) an enrichment of bone mineral by

anionic or cationic substitution from the surrounding matrix, or (2) the deterioration of the organic portion,

which would cause the minera! portion to appear greater in quantity.

Another method that has been used successfully to identify diagenesis is x-ray diffraction (Kyle 1986;

Piepenbrink 1986; Schoeninger 1981, 1982; Schoeninger et al. 1989; Sillen 1981a, b, 1989). This

methodology is based on the principie that normal, unaltered bone mineral has a diffraction pattern similar

to apatite. Thus, geological hydroxyapatite (with a different chemical composition) can be distinguished

from biological hydroxyapatite crystais on the basis of differing diffraction patterns. When archaeological

bone displays patterns that vary from expected biological apatite, such as the presence of large amounts of

calcium carbonate, diagenesis or recrystallization is assumed to have taken place.

14

It has also been suggested that archaeological fauna! remains be used as a guide or baseline for identifying

human elemental values that extend far beyond the expected range (Brown 1973; Katzenberg 1984; Price

1985; Price et al. 1985a; Schoeninger 1979a, b, 1981, 1982; Sillen 1981). While both carnivores and

herbivores are equally important in defining a range for human values, carnivores have been used much less

frequently (Radosevich 1993). The limitation of using carnivores stems from their variable diets, which can

be supplemented with bone and plant materials (Katzenberg 1984; Schoeninger 1981, 1982), and their

scarcity from archaeological sites. Sorne scholars are instead, advocating the use of herbivores since their

diets are assumed to be homogeneous and relatively stable over time, even in the face of human interaction

(Elias et al. 1982).

A number of other techniques have been used to identify and assess the effects of diagenesis. These include

the analysis of archaeological and their surrounding soil (Becker et al. 1968; Benfer 1984; Katzenber 1984;

Keeley et al. 1977; Lambert et al. 1984a; Nelson & Sauer 1984), the comparisons of trabecular to cortical

bone (Lambert et al. 1982), the comparison of bone tissue to dental tissue (Parker & Toots 1970, 1974)

comparison between prehistoric populations and modern equivalent (Edward et al. 1984; Hancock et al.

1987, 1989; Gilbert 1975; Lambert et 1985a; Parker & Toots 1970, 1974, 1980; Szpunar 1978), and

microprobe analysis (Gilbert 1975;Hassan & Or 1977; Hende et al. 1983; Lambert et al. 1983, 1984a,b,c,;

Schoeninger 1979a, b,; Waldron 1981; Vlasak 1983).

Diagenesis versus Diet at Monte Albn

In an attempt to elucidate inter-elemental relationships, a correlation matrix was constructed for the log-

normal values (Table 3.2). The values prmted in boid represent correlation coefficient that are significant at

p less than or equal to 0.05. These statistically significant correlations raise interesting questions about the

nature of bone and human diet, and will be addressed below.

TABLA QUE BORR 3.2 Table 3.2. Correlation Matrix for Multi-Elemental Values from Monte Albn

One of the most numerous inter-elemental correlations involves aluminum (Al). As one on the most

common metais in the earths crust (Hammond 1975), aluminum is often an indicator of a soil contammant when correlated with potassiuni (K), iron (Fe) and manganese (Mn) (Ezzo 1992; Lambert et al. 1979,

1985a). As Table 3.2 illustrates, the strongest correlatjons are between Al, Fe, K, Mn and Zn. The

correlation between Al and Ba is perhaps evidence of oxide contaxnination, which often results in a

correlation between these 2 elements (Parker and Toots 1972). I would also like to point out that a

correlation between Al and K does not necessarily signal contamination or diagenesis. Table 3.3 shows the

results of a correlation matrix of 20 samples from modern cow bone (elemental values usted in Appendix C)

Even in a clean, modern bone, free of contaminants, there is still a atatiscally signicant correlation between

Al and K

Table 3.3. Correlation Matrix for Modern Cow Samples

The strongest correlation in the Monte Albn matrix is between Ca and P. This is expected since these

elements are the major components of bone, constituting 37% and 18% of bone respectively. The linear

relationship between Ba and Sr is reflected in Table 3.2 as the strongest correlation that either Ba or Sr have

with any other element. The positive correlation suggests that because the 2 alkaline earth elements mirror

on another in behavior, the data set have not been substantially altered by diagenesis.

Diagenesis is a problem that cannot be avoided. It affects all buried bone to sorne degree, and in certain

circumstances, can completely alter the dietary signal of the bone. As discussed in Chapter 4, several

15

methods have been used in this research to test for and reduce the impact of diagenetic alteration of the

mineral fraction. Since bone ash (which is largely mineral in composition) is approximately 67% of whole

dry bone, the elevated ash% values usted for the Monte Albn samples (Appendix E) seem to indicate an

addition to the mineral fraction via diagenesis. As Table 3.2 illustrates, however, although there is a

correlation between several soil elements, the presence of a statisti significant correlation between Sr and Ba

demonstrates that the dietary signal is still retained. Perhaps the high ash% values do not indicate an

addition to the mineral fraction, but rather a depletion of the organic fraction over time. Adding strength to

this hypothesis is that during preparation for isotopic analysis, only 40 of the original 50 samples had

enough collagen to analyze. If 20% of the samp!e lost almost all their organic fraction, than it can be

assumed that the other 80% lost sorne of their organic fraction as well. When ah unes of evidence are

examined, although the Monte Albn samples may have been affected by diagenetic processes, the mineral

portion is reflectjve of diet and can be used in reconstructing past behavior.

While alrnost all of the eleven trace elements usted have been used in the reconstruction of ancient diet

(Buikstra et al. 1989; Klepinger 1990), only Sr, and to a lesser extent Ba, have been shown to be reliable

indicators of paleodiet (Burton and Price 1990a, b; Elias et al. 1982; Ezzo 1991; Weydert 1990). Therefore,

due to the uncertainty surrounding the utility of the trace elements, 1 will emphasize only Ba and Sr in this

research purposes of dietary reconstruction.

Summary

Reconstructing diet on the basis of isotopic ratios and quantities of trace elements began almost 20 years

ago. During that time, carbon isotopes strontium were used alrnost exclusively to identify dietary

components in human diet. Advances in technology and an increase in laboratory research suggested that

nitrogen isotopes and a number of other trace elements could be used as dietary indicators. While mtrogen

can be used to determine a marine component in the diet, its ability to detect meat and legume consumption

is still unclear. Another innovation in isotopic analysis involves the comparison of bone collagen and bone

apatite ratios to determine specific dietary components. While the role of lipids, carbohydrates and protein

in the synthesis of collagen and apatite carbon is still unclear, new research by Ambrose and Norr (in

preparation, as cited in Ambrose 1993) suggest that the 13C value of apatite reflects the diet as a whole,

while the 13C collagen reflects only the protein cornponent. Consequently, a comparison of collagen and

apatite 13C values may provide some of the data needed to distinguish different dietary fractions among

ancient populations. Innovations in trace element analysis stressed the utility of nurnerous elements in

reconstructing the importance of specific food items to the diet (i.e. magnesium for nut consumption).

Unfortunately, of all the assumptions surrounding various trace elements, only barium and strontium have

been found useful as indicators of past diet.

Along with research on new dietary indicators carne an interest in the post-mortum contamination of

archaeological bone (diagenesis). An increased awareness of diagenesis spurred numerous advances in the

analytical techniques used for its detection, and the sample preparation methods used to lessen its

destructive forces. Although all archaeological bone is chemically or physically altered over time (Badone

& Farquhar 1980; Sandford 1993), the presence of diagenesis does not impede the ability to obtain

paleodietary information from the sample. The use of multiple methodological and analytical strateg such as

acid washing faunal and soil analysis, Ca/P ratios, and multi-elemental analysis, have been used to

determine diagenetic processes and recover biogenetic signals. In order to successfully test for diagenesis

and conduct meaningful isotopic and elemental analyses of the Monte Albn skeletal population, a set of

procedures for the selection and preparation of the samples was devised. The methodology and reasoning

behind these procedures are presented in the foliowing chapter.

Notes:

16

1. Prior to taking the naperian logarithm of the entire data set, all negative Fe and Mg values had to be

eliminated (see Appendix D). Using Buikstra et al.s (1989) method, all elemental values were increased by 100 and then multiplied by 10. Once these mathematical procedures were completed, the values were

logarithmically transformed and a correlation matrix constructed

Sample Selection and Preparation

Introduction

For isotopic and elemental analysis to yield meaningful results, the role of diagenesis and the original

research question must be considered when selecting and procuring samples for analysis. To this end, 1

applied four separate criteria when selecting the Monte Albn sample. To limit the affects of diagenesis,

samples were selected so that variability in bone preservation and skeletal part were kept to a minimum.

Moreover, sinsce the primary focus of th.is research rests on the question of social inequality and dietary

patterning, samples were selected on the basis of interment type and provenience. By selecting samples

from a continuum of burial contexts (tomb to shallow grave), and locations (site core to periphery), it was

assumed that a more representative sample of social status levels would be collected. Once the samples were

chosen, they were prepared the laboratory for both isotopic and elemental analysis. The following chapter

describes the criteria used for sample selection and the procedure followed for sample preparation.

The Monte Albn Sample

Background

The Monte Albn burial population was sampled during June and July 1990, and June 1991, at its two

curated locations; the Laboratory of Physical Anthropology in the National Museum of Anthropology and

History, Mexico City, and the store rooms of the historic ex-monastery in Cuilapan, Oaxaca. The samples

from Mexico City were collected under the auspices of Sergio Alonso Lopez, then Director of the

Department of Fhysical Anthropology. The samples from Cuilapan were collected under the direction of

Ernesto Gonzalez Licn, then Director of Archaeology at Instituto Nacional de Arqueologia e Historia

(INAH), Oaxaca City, and Lourdes Marquz Mrfin, then director and curator of Physical Anthropology

also at INAH, Oaxaca City. Before sampling was conducted, a list of many of the burials at Monte Albn by

age and sex was provided to me by Dr. Richard Wilkinson of the State University of New York at Buffalo.

A complete list of the burials recovered from Winters (1972) excavation of Monte Albn also was provided by Dr. Marc Winter of INAH, Oaxaca City, the principal investigator, and Dr. Richard Wilkinson, the

physical anthropologist associated with the project.

Skeletal Part and Preservation

Bone samples were selected for analysis on the basis of body part and age of individual. As discussed m

Chapter 3, elemental concentrations vary widely throughout the human skeleton. Research demonstrates that

intra-skeletal concenfratio have a greater amount of variability than do same bone comparisons between

individuals with similar diets (Brtter et al. 1977). The tremendous amount of variability within one

individual is due to the different physical characteristics of trabecular and cortical bone.

Cortical bone, which is dense and compact, interacts much differently with trace elements than does porous,

trabecular bone. One means to use. to limit the natural variability found throughout the skeleton is to select

the same skeletal part throughout the analysis. Research conducted by Grupe (1988) suggests that elemental

concentrations from the shafts of long bones represent the average elemental concentration of the entire

skeleton. In this research, therefore, I collected only long bone shafts for analysis. Initially only mid-shaft

sections of the femora were collected. However, in some instances the femur was either in a complete state

and could not be sampled, or was missing, and so the tibia was instead sampled. If both the femur and tibia

were unavailable, the humerus midshaft was collected. In several instances, the skeleton was so incomplete

17

that other bones were sampled. Table 4.1 provides a summary of the skeletal elements sampled from the

Monte Albn burial population. Por a more complete listing, see Appendix A.

Approximately one gram of bone was removed from each long bone shaft by means of a small, notched-

tooth saw. Each bone was placed in plastic bag and labeled by burial number. A paper tag was placed

around the remaining cut bone shaft, indicating that it had been artificially altered for the purposes of

chemical analysis. A list was made of all samples collected and placed on file with the Departments of

Physical Anthropology at the National Museum of Anthropology and History, Mexico City, and at INAH,

Oaxaca City.

Table 4.1. Skeletal Elements Sampled

Element n

Femur 152

Tibia 39

Humerus 24

Fibula 10

Ulna (cubito) 1

Radius 1

TOTAL 227

Ah sample collection was conducted under the supervision of Jos Jimenz Lpez in Mexico City, and

Lourdes Marquz Mrfin in Oaxaca City. These physical anthropologists also supplied information

regarding the sex and age of specific individuals when these data were missing from the master lists. For the

purpose of this research, only adults (over 15 years of age) were selected for analysis, since elemental

values taken from children fluctuate widely and inconsistently (Price 1989). Table 4.2 ihlustrates the

breakdown of the Monte Albn sample by sex. For a more detailed account, see Appendices A and B.

Table 4.2. Monte Albn Sample by Sex TOTAL 227

Interment Type

The individuals collected for a were recovered from several different burial contexts. Those sampled from

Caso and Acostas excavations at Monte Albn were labeled with a specific nomenclature according to interment type (Romero 1942). The term tomb referr an interment consistmg of one or more underground chambers.

According to Romero, the individual occupying the primary chamb tomb was considered the principal

occupant, and labeled T.l through T.172 according to tomb number.

The term entierro (interment) had a very generalized usage, and referred to any individual who was not

buried as the principal occupant a tomb. Thus, entierros were found in simple, shallow graves on the slopes

of the site, in stone-lined graves on the Main Plaza, or as secodary burials of a tomb. Usually, the labels for

entierros were more specific than tombs, indicating both entierro number and fleid season. For example in

the label Ent. IX-3, the roman numeral stands for the ninth field season, and the 3 represents the third entierro found during that field season.

Sorne confusion arises, however, when several individuals were recovered from a single tornb. The tombs principal occupant is given the T.153 !abel, while the other occupants (found in minor rooms, niches, or dissarticulated in the main chamber) were labeled as entierros within a tornb, such as T.153, ent. 3.

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Several tombs contained a large mass of disarticulated bones in which single individuals could not be

identified, and so the area was divided and exacavated in quadrants (1-1V). ui these instances, the bones

were labeled according to their quadrant (e.g. T.128, ent. sec. II) Since very little information is available regarding the entierros, it is not known if the entierros in tombs are temporally related to the principal

occupant, or if they were once principal occupants themselves, moved as a consequence of tomb re-use.

When Winter conducted his excavation of Monte Albn, he chose to use a new method of labeling

interments (Winter 1974; Winter and Payne 1976; Winter and Wilkenson n.d.). Whereas the tomb

designation was still used, Winter chose to use burial rather than entierro. In addition to the burial designation, Winter also assigned either house number or individual number, and the year of excavation to

each interment (e.g. Burial 25, M.A.1973B, House 3 or Burial 14, ind.1, M.A. 1972).

Many hours were spent deciphering the association between interment classjficatjon and actual burial

context. Since Wilkinson and Norelli (1981) used only a tomb/non tomb division to assign social status to the skeletal population at Monte Albn, I thought it necessary that the bur context of all interments

(entierros and tombs) be clearly defined befon status assumptions were posited. As suggested aboye, the

term entierr should not be used to identify social status (as Wilkinson and Norelli c Entierros can be

inhabitants of shallow graves, but they can also be fou in elaborate tombspossibly representing past principal occupants.

Consequently, in this research, the actual burial context, rather than the interment classification became the

important variable in selecting bone samples for analysis.

Interment Provenience

Burial location was also selected as a variable in determining social differentiation at Monte Albn

(Appendix 1). During Blantons survey of the area, he discovered 15 subdivisions of the site, each with its own cluster of elite residences and civic-ceremonial structures. He refered to these areas as barrios and

numbered them from 1 to 15 (Blanton 1978) (Figure 4.1). Due to the differential distribution of status-

related artifacts such as exotic rocks and minerals, marine shell, and exotic pottery type Blanton suggested

that several of the barrios closest to the Main Plaza had a stronger elite presence than barrios farther

downslope. Assuining a possible status differentiation by barrio, I attempted to assign each skeletat sample

to its appropriate barrio for analysis.

A thorough review of ah the published and unpublished data available was necessary to determine the

spatial distribution of the Monte Albn sample. Alrnost all of the tombs were located in the Main Plaza or

just north in an area known as the North Cemetery (see Figure 4.1). These spatial data were collected from a

variety of sources, including the published reports, letters and manuscripts of Caso and his associates (Caso

1934, 1935, 1937a, b), and the field notes and maps from Blantons survey of Monte Albn (1978). As illustrated in Appendices A and B, almost ah tornbs have been located according to the numbered terrace it

rests on, and the barrio in which it is found.

Caso and his fellow researchers placed a heavy emphasis on the elaborate tornbs of Monte Albn, while

making only cursory references to the entierros in rnost published articles and unpublished manuscripts.

Sorne of the entierros could be assigned to a specific terrace due in large part to the careful and detailed

field notes and maps of Blantons survey of the Monte Albn area (1978). When the specific locale of an entierro could not be obtained from the available data, a general barrio location was assigned (Appendices A

and B). Barrios were assigned on the basis of where excavations were conducted during a particular field

season. For example, during the second season of field work, Caso and associates excavated in and around

the Mam Plaza (Caso 1932). Although this area contams several different terraces, they are all located in

barrio 2. Thus, aH entierros and tombs recovered during Temporada II were placed in barrio 2. In this way, a majority of entierros whose provenience was previously unknown could be given a general barrio

affiliation, thereby expanding the scope of the skeletal population across space. Of the 83 entierros available

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for analysis, 5 were securely located to barrio, 23 had barrio Iocations that could be inferred, and 55 samples

had no provenience data present.

.

A majority of the samples come from barrio 2 (Table 4.3). The apparent emphasis on this barrio stems from

the numerous excavation conducted by Caso and his associates in and around the structures of the Main

Plaza. During their 18 field seasons at the site, most of their energy was focused on the recovery and

reconstruction of the buildings encircling the Main Plaza, and the associated burial tombs and chambers.

The only excavations outside of barrio 2 were conducted during Temporadas 1V, VIII and XII, when

interments were recovered from barrios 1, 4, 6, and 8 (Acosta n. d.; Caso 1935, 1942).

Following the last excavation in 1958 the site stood relatively untouched until 1971, when Richard Blanton

began his settlement pattern study of Monte Albn (Blanton 1976a, 1976b, 1978; Neeley 1972). In 1972 and

1973, Marc Winter carried out an excavation downslope from the Main Plaza, in what is considered to be

the residential zone of the city (Winter 1974; Winter and Payne 1976). He uncovered several small

residential units in barrio 7, and a number of human burials. With the help of Richard Wilkinson, Winter

recorded the sex, age, burial type and associated grave furniture for each individual (Winter & Wilkenson

n.d.) On the basis of burial information and residential type, size and complexity, Winter suggested that

these individuals represented the middle or low social segment of society at Monte Albn (Winter 1974;

Winter and Payne 1976).

Sample Preparation and Analytical Procedure

Trace Element Analysis

The preparation and analysis of all samples were done in the Laboratory for Archaeological Chemistry,

Department of Anthropology, at the University of Wisconsin-Madison. Prior to analysis, the samples were

cleaned usmg a variety of methods adapted from Siliens research (1986, 1989). Approximately 1-3 mm of outer bone cortex were mechanically removed from each sample by means of a hand-held sander with an

abrasive bit. Each sample was placed in a polyethylene vial containing 1N acetic acid and sonicated

(cleaned via somc waves) for 30 minutes.

According to SilIen (1986, 1989), the acid wash is necessary to remove diagenetic carbonates from the

mineral fraction, which can cause an artificial elevation of the calcium and strontium values. After

sonication, the samples were rinsed repeatedly in deionized water and transferred to a vacuum chamber,

where more 1N acetic acid was added. By using a vacuum chamber, acetic acid comes in contact with the

entire surface area of the bone, since air is drawn out of the bones microscopic pore space in exchange for acetic acid. All samples were left in vacuum for 2 hours, after which time they were left to soak in solution

for approximately 12 hours. Next, the samples were thoroughly rinsed in deionized water and placed in a

drying oven at 80C for 24 hours.

Once the samples had been cleaned and dried, the dry weight of each sample was recorded. They were then

placed m a porcelam crucible and reduced to ash in a muffle furnace for 8 hours at a temperature of 725C.

Once cooled, an ash weight was recorded, and the remaining ash percentage was calculated (Appendix E).

Next, each sample was ground to a fine powder and approximately 50 mg of this powdered bone ash was

placed in a test tube.

Digestion proceeded when 1 cc of concentrated nitric acid (HNO3) was added to each test tube and heated to

110C for 1 hour. Once cooled, 9.5 cc of 5% nitric acid was added to all samples. Following digestion, the

samples were analyzed for eleven elements (alummum, barium, calcium, iron, magnesium, manganese,

sodium, phosphorus, strontium, and zinc) by means of inductively coupled plasma emisson spectroscopy.

In addition, to the 227 Monte Albn samples analyzed, 4 bone standards were periodically analyzed to

20

assess the machines precision. One standard used was the Internal Atomic Energy Comrnission bone standard, H5. However, since the quantity of H5 was limited, three other internal laboroatory standards

(B0126, B5407 and CowRef) were used in conjunction with H5. The results of the elemental analysis of the

Monte Albn samples are provided in Appendix A, while those of the bone reference, H5, are provided in

Appendix F.

Stable Isotope Analysis

A subset of 40 individuals was chosen for stable carbon and nitrogen isotope analysis. These individuals

were selected on the basis of thei, associated time period, since my research focuses on temporal shifts in

dietary behavior. Stratified random sampling was used to select the individuals for analysis. Since this

research aims to explore the diachronic nature of social inequality via dietary reconstruction, the samples

were divided by time period (MA II, IIIa, IIIb, IV, and V) and samples were then randomly chosen from

each time period. Equal numbers of individuals from time period was the ideal, however, due to the limited

availability of samples from sorne time periods, and badly preserved collagen from others, not ah time

periods are respresented in the isotope subset. Additionally, while it was hoped that ah barrios would be

represented, only samples from barrios 2, 6, 7, and 8 were available for analysis. Table 4.4 illustrates the

number of samples analyzed for each time period and barrio. Sample preparation and analysis were done

under the direction of Margaret Schoeninger, in the Paleodiet Laboratory, Department of Anthropology at

the University of Wisconsin-Madison. A variety of collagen contaminants can affect isotope ratios. Lipids,

post-depositional carbonates, biological carbonates in bone apatite, and even soil particles and other organic

matter adhering to the bone cam alter isotopic composition (Hassan & Ortner 1977; Hassan et al. 1977;

Hanson & Buikstra 1987; Kyle 1986; Piepenbrink 1986). In an attempt to control for these contaminants, all

samples were mechanically and chemically cleaned prior to analysis.

Approximately 1 g of bone was selected from which the first 1-3 mm outer cortex was removed to rid the

sample of any adhering contaminants. This was followed by an ultra-somc cleaning in distilled water for 30

minutes, after which time they were placed in a lyophilizer for 16 hours at a temperature of 80C.

Ah samples were placed in beakers containing .1M hydrochloric acid (HCI) for a period of approximately 2

weeks. The samples were checked daily and the acid was changed on a weekly basis. The use of a weak (1-

3%) hydrochloric acid not only removes the mineral bone fraction and any post-depositional carbonates, but

is better than strong acids (10%) at recovering collagen from poorly preserved samples (Schoerunger et al.

1989). Once the samples were demineralized, the collagen was rinsed to neutrality in deionized water and

left to soak in .125M sodium hydroxid (NaOH) for approximately 12 hours.

Treatment with sodium hydroxide removes any humic acid and lipid contamjna that can affect collagen

carbon isotope ratios. Whereas the isotopic composition of humic acid reflects the local plants rather than

diet, bone lipid isotope ratios can be 6-12% more negative than bone Collagen ratios (DeNiro and Epstein

1977; Ambrose 1990).

Upon rinsing in deionized water, the samples were dried in a lyophilizer for approximately 16 hours and a

dry weight was recorded. A percent weight of collagen was calculated for each sample; those with values

aboye 5% were considered acceptable for analysis, while those wit a collagen percentage below 5% were of

questionable composition and ha to be analyzed further using atomic C:N ratios (Schoeninger et al. 1989).

As research by DeNiro (1985) illustrates, reliable collagen samples use for isotopic analysis typically have

atomic ratios ranging from 2.9 to 3.6. Ratios higher than 3.6 can indicate collagen contamination by humic

acids, lipids, or carbonates, and should be rejected for analysis. As shown in Appendix G, of the forty-two

samples analyzed for carbon and nitroger isotopes, three had a C:N ratio higher than 3.6 and were

subsequently exciuded from this research.

Following collagen purification, the samples were completely converted to C02 and N2 gases via static

combustion (Sofer 1980). Between 3 and 5 mg of collagen were weighed into silver boats and loaded into a

21

heated quartz tube with copper oxide and diatomaceous earth. The quartz tubes were then placed under high

vacuum for several hours to remove all moisture, sealed with a torch and then placed m a furnace and heated