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Isotopic Determination of Region of Origin in Modern Peoples: Applications for Identifying U.S. War-Dead from the Vietna...

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PAGE 1

ISOTOPIC DETERMINATION OF REGION OF ORIGIN IN MODERN PEOPLES: APPLICATIONS FOR IDENTIFICATI ON OF U.S. WAR-DEAD FROM THE VIETNAM CONFLICT By LAURA A. REGAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Laura A. Regan

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To Ronald D. Reed, Ph.D., Brigadier General, USAF 11 November 1948 20 April 2005 It is with bittersweet appreciation that I tha nk an incredible mentor for taking a risk and agreeing to this absolutely insane advent ure; for the opportunities he provided and his unwavering confidence in me. It is with great regret that I cannot share my success in this endeavor with him. I was fortunate to know him and I dedicate this work to his memory.

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iv ACKNOWLEDGMENTS I could not have met all the crazy dead lines of this breakneck-paced program without the assistance of a great many peopl e. First, I wish to extend my sincere appreciation to the members of my doctoral committee, Drs. Anthony B. Falsetti (Chair), David Daegling, Thomas Holland, Connie Mulligan, and David Steadman. Not one of you ever indicated you had any reservations about my ability to complete this program in a blistering 3 years. Your faith in me fueled me on when I was doubting myself. Boss, I have never encountered a professor who is so nurturing of his students, yet has no qualms about telling us when we’re being knuckleheads. I cannot express my gratitude for all you have taught me, your unwavering friendship, your constant confidence in me, and your personal support throughout this program. You’ve opened countless doors for me. I will never be ab le to repay you for all of your kindness, generosity, and all of the laughs. You take good care of your “kids.” Please take good care of yourself as well. No se tting yourself on fire any more. Dr. Daegling opened my mind and challenge d me to think critically on a whole new plane. The academic rigor of his courses was both mildly overwhelming and incredibly fulfilling. Receiving an “A” in hi s course was truly something to covet. A great thank you goes out to Dr. Thomas Holland, Joint POW/MIA Accounting Command-Central Identificati on Laboratory Scientific Director, for supporting me and this project, allowing me to intern with hi m for three incredible months, and putting me on that week-long C-17 ride to Vietnam. I have partaken in some once in a lifetime

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v experiences through your generosity, and those memories I will always cherish. Please don’t forget about me. I’ll be looki ng for a job in about 8 years. Dr. Mulligan broke down my internal block when it came to understanding genetics and taught me a great deal about attention to detail and organization. She held my feet to the fire and made me not only address but fu lly understand the flaws in my work, vastly improving the quality of my scientific work. The journey to enlightenment could sure be frustrating though. Dr. Steadman was a consta nt source of enthusiasm and energy. His positive attitude kept me going and allowed me to overcome a long seeded loathing of avian fauna that arose during my days in undergraduate Vertebrate Zoology. Birds are cool! I owe Dr. Andy Tyrell a great deal of gratitu de as well, for getting me se up at CIL and through his continued guidan ce and assistance. To Col. (ret) Thomas and Col. Merle Sprague, thank you for allowing me to mooch off of you for 3 months. You opened your hearts and home to me. I am truly gratef ul and a better person for knowing you. I would also like to pass along my appr eciation to LTC Mark Gleisner for showing me the basics of drilling teeth and along w ith the rest of the CIL dent al guys, answering my many, many questions. I owe a great deal to Col. Nancy Perry and Maj. Albert Ouellette, 10th Dental Squadron, U.S. Air Force Academy for agreei ng to assist me with this project and especially to Albert, who pr ovided over 1000 freshly extracted third molars to me during the course of this study (are you sure you guys do not have a quota?). Thanks also go out to Drs. Jack Meyer and Ray Berringer from the North Florida/South Georgia Veterans Health System, Veterans Affairs Dental Clinic..

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vi I am indebted to Dr. Bruce MacFadden, Florida Museum of Natural History, who really exposed me to the possibilities of isotope studies and in whose class this project all took shape. He graciously allowed me the use of his laboratory to prepare samples, took keen interest in my progress, and always greeted me with a smile, no matter what the circumstances. I also learned a great deal about the basics of isotope work from Dr. Joann Labs Hochstein and am grateful for her tutelage as I was starting out. I would like to thank Dr. J ohn Krigbaum for planting th e seed of awareness of stable isotope studies and bailing me out during a great time of need. Your genuine concern for your students is well known. I w ould like to acknowledge the contributions of George Kamenov, who showed me the rope s of heavy isotopes and gave me great insight into their power and Dr. Jason Curtis, who worked around my crazy schedule, even when his was just as bad, and always had time to answer my questions, even when he was out of the country. To the “Frogs,” I cannot wa it to rejoin your ranks. A special thank you goes out to Col (ret) James Kent. Sir, you have been there for the course of my journey in academia. I thank you for your patience, guidance, and gen tle pushes in the right direction. Look—I did not change my major once this degree program! I would like to thank my family and frie nds for all of their love and support over the years. You mean the world to me. Anna, you are the most selfless friend anyone could ever be blessed with. I don’t know what I would have done without you but I do know I can never repay your kindness nor the countless times you bailed me out of a difficult situation. Greg, you were a consta nt sounding board and helped me through some very difficult times. Hang in there my friend. There is light at the end of the

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vii tunnel, and no, it isn’t a train. Shanna and Erin, I can’t tell yo u how my stress melted away when I was in your company—and thanks to some apple juice-laced wine. I’ll miss our girls’ dinners more than you will ever know. Thanks to Laurel and her technical wizardry and extraordinary and often utilized dog sitting sk ills. I also wouldn’t have been able to launch this project if it hadn’t been for the assistance of Alicia during the 3 months I was away. I can’t tell you how much your help eased my mind. I owe much appreciation to Carlos for teaching me the basics of tooth iden tification and to Miss Shiela for assisting me with numerous mind-nu mbing tasks. Have a great Air Force day! I’d also like to thank the rest of the P ound Lab rats and lab ra ts by-proxy: Dr. Mike Warren, Shuala, Joe, Trey, Paul, Ron, Nicolett e, Kathy, Debbie, Pat, Melissa, Megan, and Jennifer; and my friends Chad, Laurie, and Erin. You added immeasurable levity to my life during a period of extrem e stress and thoroughly deprogr ammed me. I couldn’t ask for a better cohort to be associated with. It ’s going to be tough going back to the real world. I also owe a huge debt of gr atitude to two undergraduate assistants, Ursula Zipperer and Ana del Alamo, who spent countless hours helping me with the most mundane of tasks. Lastly, I must express my heartfelt appreciation to Calvin and Hobbes. I would not have survived this program, especially the first year, without you guys, but it would have been nice if you had not eaten the door . .twice. To all the men and women who have gone befo re me in service to our nation and to those who currently serve, I salute you. I am proud to be among your company. It has been an incredible honor to complete this project with the hopes of reuniting families with their long, lost, loved ones. Until they are home .

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viii My tuition was provided by the United States Air Force. This research was funded in part by the Joint POW/MIA Accounting Command-Central Iden tification Laboratory, the C.A. Pound Human Identification Laborat ory, a William R. Maples Scholarship, and my savings account.

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ix TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................xii LIST OF FIGURES.........................................................................................................xiv ABSTRACT.....................................................................................................................xv i CHAPTER 1 STABLE ISOTOPES....................................................................................................1 Study Isotopes...............................................................................................................6 Carbon...................................................................................................................6 Oxygen..................................................................................................................7 Strontium...............................................................................................................8 Lead.....................................................................................................................10 Fractionation...............................................................................................................11 Frequently Sampled Human Tissues..........................................................................13 Bone.....................................................................................................................14 Teeth....................................................................................................................15 Hair......................................................................................................................17 Fingernails and Toenails......................................................................................18 Skin......................................................................................................................19 Complications.............................................................................................................20 Diagenesis............................................................................................................20 Anthropogenic Contamination............................................................................27 Global Economy..................................................................................................28 2 APPLICATIONS OF ST ABLE ISOTOPE ANALYSES..........................................30 Tracing Studies...........................................................................................................30 Fractionation Studies..................................................................................................32 Zoology and Ecology..................................................................................................33 Archaeology................................................................................................................35 Diet Assessment..................................................................................................35 Introduction of maize...................................................................................35 Weaning practices........................................................................................37

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x Region of Origin..................................................................................................39 Material Culture...................................................................................................41 Forensic Investigations...............................................................................................42 3 HUMAN FORENSIC IDENTIFICATION................................................................50 Military Identification Measures................................................................................54 Present Study..............................................................................................................58 4 MATERIALS AND METHODS...............................................................................66 Dental Protocols..........................................................................................................67 Sampling.....................................................................................................................73 Central Identification Laboratory...............................................................................74 United States Air Force Acad emy and Veterans Affairs............................................77 Carbon and Oxygen Sample Preparation....................................................................80 Central Identification Laboratory Samples.........................................................80 United States Air Force Academy Samples........................................................83 Strontium and Lead Sample Preparation....................................................................84 Statistical Analyses.....................................................................................................90 5 ANALYTICAL COMPARISION OF EAST ASIAN AND AMERICAN SAMPLES..................................................................................................................93 Light Isotopes.............................................................................................................93 Carbon.................................................................................................................93 Oxygen..............................................................................................................108 Acetic Acid Test................................................................................................114 Heavy Isotopes..........................................................................................................118 Strontium...........................................................................................................118 Lead...................................................................................................................125 Multi-element Approach...........................................................................................133 6 VARIATION WITHIN USAFA SAMPLES...........................................................139 Year of Birth.............................................................................................................139 Sex............................................................................................................................ 141 Race..........................................................................................................................1 42 Tobacco Use.............................................................................................................143 Diet........................................................................................................................... 146 Residency..................................................................................................................146 Strontium..................................................................................................................146 Lead..........................................................................................................................1 50 Regionality................................................................................................................151 Relationship Between 18O Values and Latitude.....................................................156 Duplicate Residences................................................................................................159 Comparison to the Literature....................................................................................161

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xi 7 SUMMARY/CONCLUSION...................................................................................165 APPENDIX A REPLICATED VETER ANS AFFAIRS BINDER...................................................172 B CENTRAL IDENTIFICATION LABORATORY SAMPLING.............................203 C UNITED STATES AIR FORCE ACADEMY SURVEY RESULTS.....................210 D EXAMPLE PRISM LOAD SHEET.........................................................................248 E COLUMN CHEMISTRY VESSEL AND IMPLEMENT CLEANING INSTRUCTIONS.....................................................................................................249 F MISCELLANEOUS HEAVY ISOT OPE RESULTS WORKSHEETS..................251 LIST OF REFERENCES.................................................................................................258 BIOGRAPHICAL SKETCH...........................................................................................278

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xii LIST OF TABLES Table page 1-1 Stable isotope standard materials and calibrants........................................................5 1-2 Mean age of completion of permanent crown mineralization..................................16 2-1 Mean and standard deviations for sel ected groups of immigrant teeth (enamel).....49 3-1 Forms of forensic identification...............................................................................54 3-2 Numbers of unaccounted for U.S. pris oners of war and/or those missing in action........................................................................................................................5 9 3-3 United States casualties in Southeast Asia by race..................................................60 3-4 United States military listed as una ccounted for in Southeast Asia by race............60 4-1 Crown formation/tooth eruption...............................................................................74 4-2 Isotope sampling matrix...........................................................................................78 5-1 Summary statistics and ge neral linear model results of all isotopes examined for CIL samples compared to USAFA samples (CIL outlier excluded). All values are in ‰....................................................................................................................94 5-2 Carbon and oxygen isotope results. All values are in ‰........................................95 5-3 Central Identification La boratory outlier run data.................................................102 5-4 13C value comparison. Twelve most enriched CIL samples and 12 most depleted USAFA samples (CIL outlier excl uded). All values measured in ‰.....103 5-5 Summary statistics and ge neral linear model results of all isotopes examined for American and foreign USAF A comparison (CIL outlier excluded). All values are in ‰..................................................................................................................105 5-6 Summary statistics and ge neral linear model results of all isotopes examined for CONUS and overseas USAFA comparison (CIL outlier excluded). All values are in ‰..................................................................................................................105 5-7 East Asian 18O values, in ascending order...........................................................109

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xiii 5-8 Partial list of USAFA 18O values, in ascending order (30 most depleted and 30 most enriched)........................................................................................................110 5-9 Results of acetic acid test, with intertooth comparison when available.................116 5-10 Strontium isotope values for CIL a nd USAFA samples, in ascending order.........119 5-11 Comparison of the means for multiple runs of the GLM procedure for 87Sr/86Sr. (CIL outlier excluded.)...........................................................................................124 5-12 Lead isotope results for East Asia..........................................................................126 5-13 Lead isotope results for USAFA............................................................................127 5-14 Comparison of spiked lead concentration data (actual) with semi-quantitative data. (All values are in ppm.)................................................................................133 6-1 USAFA-provided sampling demogra phics, American natal region only..............140 6-2 Locations during amelogenesis re presented by sampled USAFA teeth................147 6-3 Strontium isotope values for Ameri can USAFA samples, in ascending order......148 6-4 Mean 207Pb/206Pb values for major U.S. lead ore deposits.....................................152 6-5 207Pb/206Pb values Americans reared in the United States, in ascending order......152 6-6 Region membership based on 18O values.............................................................153 6-7 Region-pair comparison for difference in 18O means..........................................155 6-8 Summary statistics for American USAFA 18O values based on latitude. All values are in ‰.......................................................................................................158 6-9 18O values corresponding to cities in which multiple participan ts resided. (All values in ‰.)..........................................................................................................160 6-10 Comparison of Alberta fu r trader lead values to USAFA donor from Alberta......164 B-1 Central Identification Laboratory sampling data...................................................204 C-1 United States Air For ce Academy survey data......................................................211 F-1 Semi-quantitative Sr concen tration calculation matrix..........................................252 F-2 Semi-quantitative Sr concen tration calculation matrix..........................................255 F-3 Comparison of the means for multiple r uns of the GLM procedure for Pb. (CIL outlier excluded).....................................................................................................257

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xiv LIST OF FIGURES Figure page 4-1 Joint POW/MIA Accounting Command survey......................................................70 4-2 Pre-drilling photo of CIL-033 #19 with data card. Note: the accession number is purposely, partially obscured...................................................................................76 4-3 Pre-drilling photo of AFA-093 #32..........................................................................80 4-4 Loaded tray for PRISM mass spectrometer analysis...............................................82 5-1 Carbon and oxygen isotope results with overlapping value overlay........................99 5-2 Carbon and oxygen isotope results for American and foreign USAFA comparison.............................................................................................................104 5-3 Carbon and oxygen isotope result s for CONUS and overseas USAFA comparison.............................................................................................................106 5-4 Latitudinal dispersion of major natal regions featured in this study. East Asia is on the right. Information drawn fr om Rand McNally Atlas (1998)......................111 5-5 Weighted Annual 18O for Asia. Map reproduced from IAEA (2001).................112 5-6 Weighted Annual 18O for North America. Map reproduced from IAEA (2001).112 5-7 Plot of strontium values compared to 208Pb/204Pb..................................................121 5-8 Plot of 87Sr/86Sr compared to 206Pb/204Pb...............................................................122 5-9 Box and whisker plot of 87Sr/86Sr values...............................................................123 5-10 Comparative histogram of CIL and USAFA sample Sr concentrations (semiquantitative)............................................................................................................125 5-11 Plot of 206Pb/204Pb compared to 208Pb/204Pb...........................................................128 5-12 Plot of 206Pb/204Pb compared to 207Pb/204Pb...........................................................128 5-13 Plot of 208Pb/206Pb compared to 207Pb/206Pb...........................................................129

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xv 5-14 Comparative histogram of CIL and USAFA sample Pb concentrations (semiquantitative)............................................................................................................131 6-1 Strontium isotope composition of the U.S. showing inferred 87Sr values, as calculated by age variations in basement rocks. Image reproduced from Beard and Johnson (2000), with permission.....................................................................149 6-2 Region map based on 18O values..........................................................................153 6-3 Plot of latitude compared to 18O values. Error bars equal 1 std dev...................157 6-4 Range of 18O values produced from this study for specific cities........................161 D-1 Example PRISM load sheet....................................................................................248

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xvi Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ISOTOPIC DETERMINATION OF REGION OF ORIGIN IN MODERN PEOPLES: APPLICATIONS FOR IDENTIFICATI ON OF U.S. WAR-DEAD FROM THE VIETNAM CONFLICT By Laura A. Regan August 2006 Chair: Anthony B. Falsetti Major Department: Anthropology This study is novel in that it is the first of its kind to co mpile a reference sample of isotopic values associated with known natal regions to be utilized in forensic work. Stable isotopes of carbon, oxygen, strontium, and lead were examined to determine if natal origins could be assessed isotopical ly between Southeast Asian and American dental remains as well as regionally within th e United States. Teeth believed to be of East Asian origin were compared to the extract ed third molars of recent American dental patients. Living subjects completed surv eys detailing physiological, behavioral, and residential information that affect isotope va lues. The least square s means for all isotope values examined exhibited significant differe nces between the East Asian and American cohorts. Based on this information, a discri minant function was cr eated that correctly classified individuals, through resubstitution an d cross-validation, as belonging to one of these two groups by 95% or better. The sexe s differed significantly as to their carbon ratios with females displaying more enriched va lues than males. Significant differences

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xvii were also noted for 13C means among those who have ne ver used tobacco products and those who partook of smokeless tobacco. Ameri can strontium values displayed a distinct trend toward homogenization, with the mean value for 87Sr/86Sr varying only slightly from that of seawater. In order to identif y natal origin among Americans, nine regions were created within the United States based on 18O values. Good discrimination was noted between the mountain states and the southern states. A discriminant function analysis proved disappointing though, and additi onal sampling from most states is needed to improve the statistical robus ticity of the model. The re sults of this study will have wide-reaching effects across the medico-legal spectrum. This body of research will serve as the foundation for a database of modern, hum an, geolocational isotope values that will assist not only in the identification of fallen servicemen and women, but in the identification of victims of mass fatality incidents, undocumented aliens who perish attempting entry into the U.S., and local skeletal “Jane and John Doe” cases.

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1 CHAPTER 1 STABLE ISOTOPES Ascertaining the national origin of unid entified human remains is problematic, especially with the passage of time. Often, the number of identifiable bony elements is so few, fragmentary and/or degraded by th e chemical properties of the soil, that estimating biological profiles and DNA analyses cannot effectively be performed. This challenge is particularly acute for the Joint POW/MIA Accounting Command’s Central Identification Laboratory (JPAC-CIL). Th e identification of unknown remains believed to be missing U.S. service personnel is freque ntly hampered by high levels of degradation and fragmentation as a result of circumst ances of loss and subsequent taphonomic regimes. If the geo-political region of origin for a set of remains could be established, it would facilitate the construction of identifica tion shortlists, especia lly from large, openended decedent populations. This, in turn, would provide a highly effective means of excluding possible candidates for identific ation, notably for human remains whose provenience is either unknown or suspect One potential tool in determining geolocational origins of skeletal material is that of st able isotope analyses. Developed primarily in the geochemical community (Fogel et al 1997), stable isotope work has revolutionized the anthropologi cal realm, beginning with pioneering, archaeological, dietary studies in the la te 1970s (DeNiro & Epstein 1978a and 1978b, van der Merwe & Vogel 1978). Isotopes of a particular element are atoms whose nuclei contain the same number of protons but differ in their number of neutrons (Hoefs 2004). It is the number of protons

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2 in the atom that determines what the element is as well as how many electrons the atom has (Herz & Garrison 1998). An atom at rest has a neutral charge; therefore, the normal state for an atom is to have the same numbe r of protons within th e nucleus as electrons outside of the nucleus. As stated previ ously, isotopes vary because of the differing number of neutrons within the nucleus. This neutron variation, will in turn, affect the atomic masses of different isotopes of the same element because the atomic mass is a measure of the sum of the number of protons and neutrons (Hoefs 2004). For example, carbon has an atomic number of “6,” meaning an atom of carbon contains 6 protons within the nucleus. Even though the number of protons is constant within a carbon atom, it can take on three isotopic forms: 12C, 13C, 14C. A carbon atom with a mass of 12 (denoted 12C) has 6 protons and 6 neutr ons, one less neutron than a carbon atom with a mass of 13 (13C) and two fewer neutrons than 14C. Since chemical reactions are largely dete rmined by the ionic or atomic electron configuration, the varying isotopes of an individual element will have the same chemical properties (Schwarcz & Schoeni nger 1991). Different isotopes of a single element will have different kinetic and thermodynamic properties when they undergo chemical reactions though, because of differences in r eaction rates and heat capacity influenced by their different atomic masses (Urey 1947). So, while isotopes of a like element will react the same chemically, they will react at different rates, due to their different atomic masses and sizes. Different metabolic and chemical processes therefore change the ratios between the isotopes in a characteristic manne r (van der Merwe 1982). It is also noted that as atomic weight incr eases, the differences in thermodynamic properties between isotopes generally decrease (Urey 1947). In ot her words, light isotop es such as those of

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3 hydrogen, carbon, and oxygen will have a much greater variation in their thermodynamic and kinetic characteristics than heavier isotopes such as strontium and lead. Stable isotopes are not radioactive (Hoe fs 2004), thus they do not spontaneously change into another atom or another isotope of the same element (Herz & Garrison 1998). Revisiting the carbon example, when c onsidering the three isotopic forms of carbon (12C, 13C, 14C), the former two are stable isotope s, while the latter is radioactive (van der Merwe 1982), and commonly utilized for archaeological dating purposes. Stable isotopes may also be characteri zed as radiogenic or nonradiogenic. A particular isotope is classifi ed as radiogenic if it is th e product of the decay of a “longlived” radioactive isotop e (Schwarcz & Schoeninger 1991). Strontium (87Sr) and lead (206Pb, 207Pb, 208Pb) are the primary radiogenic is otopes used in nutritional ecology studies. 87Sr forms from the radioactive decay of rubidium (87Rb) while 206Pb and 207Pb arise from the decay of uranium (238U in the case of 206Pb and 235U for 207Pb) and 208Pb results from the decay of thorium (232Th) (Herz & Garrison 1998). These radiogenic isotopes vary considerably in abundance with respect to their associated non radiogenic isotopes (86Sr and 204Pb) (Schwarcz & Schoeninger 1991) an d serve as useful analytical tools. Eighty-one elements have stable isotope s of varying numbers (Herz & Garrison 1998). All of the biochemically important elem ents, with the exception of fluorine, have more than one stable isotope (Schwarcz & Schoeninger 1991). Four of these; carbon, oxygen, strontium, and lead; were examined in this study will be discussed in detail being on page 6.

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4 Measurements of stable isotopic ratios are performed by a mass spectrometer, an instrument that determines the relative a bundances of different isotopic masses in a variety of elements (Thirlwall 1997). For carbon, the mass spectrometer determines the raw ratio of 13C/12C, which it then compares to the ra tio of a marine carbonate standard, known as Pee Dee belemnite (PDB, now refe rred to as V-PDB, based on the Vienna Convention; Hoefs 2004). The difference between the sample ratio and the V-PDB standard ratio is what is known as the relative 13C content and is the value reported and used for inferential purposes (van der Merwe 1982). The equation is as follows: element = (ratiosample/ratiostd -1) x 1000‰ = value in ‰ 13C = 13C/12Csample 1 x 1000‰ (1-1) 13C/12CV-PDB This measure is denoted by the symbol (delta) and measured in parts per mil (‰) (van der Merwe 1982). If the hypothetical 13C/12C ratio of a sample was calculated as 12 per mil less than the V-PDB standard, the 13C value would be -12‰ and considered depleted compared to the sample. It is im portant to note that th e V-PDB standard does not equal zero (it equals 2.0671 x 10-6; Hoefs 2004) and results s hould not be interpreted as deviations from the zero point. Oxygen values for 18O/16O are calculated similarly. When 18O is calculated in concert with 13C, the V-PDB standard is used along with a conversion factor (Dr. Jason Curtis, personal communication). When isot opic calculations are pe rformed singly or in combination with hydrogen, the internationally accepted standard of standard mean ocean water (SMOW or V-SMOW) is used (Hoefs 2004). The heavy isotopes of strontium and lead are not generally normalized to a c onventional standard, but instead, results are

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5 expressed directly as ratios (Herz & Garris on 1998) and the standards are used for mass spectrometer calibration adjustments. Stable isotope standards have been drawn from a variety of sources over the years. Some of the most commonly utilized in zool ogical and anthropologi cal studies are listed in Table 1-1. Table 1-1. Stable isotope sta ndard materials and calibrants. Element Ratio Standard (Std) Std Notation Std Value Hydrogen1 D/H (2H/1H) Standard Mean Ocean Water SMOW or V-SMOW 155.76 x 10-6 Carbon1 13C/12C Belemnitella Americana from the Cretaceous Peedee formation, South Carolina PDB or VPDB 2067.1 x 10-6 Nitrogen1 15N/14N Air nitrogen N2 (atm) 3676.5 x 10-6 Oxygen1 18O/16O Standard Mean Ocean Water also SMOW or VSMOW 2067.1 x 10-6 Belemnitella Americana from the Cretaceous Peedee formation, South Carolina PDB or VPDB 2067.1 x 10-6 Strontium2 87Sr/86Sr Strontium carbonate/bulk earth NBS-987 or NIST 987 0.7045 Lead3 208Pb/204Pb 207Pb/204Pb 206Pb/204Pb 207Pb/206Pb 208Pb/206Pb Lead metal wire NBS-981 or NIST 981 36.696 15.491 16.937 0.9146 2.1665 1 From Hoefs (2004) 2 From Beard and Johnson (2000) 3 George Kamenov (2006)

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6 Study Isotopes Carbon In 1968, Margaret Bender first reported th at the major photosynthetic pathways of plants manifest themselves in distinct carbon isotope ratios. This discovery served as the catalyst for the multitude of carbon isotope st udies documented in th e literature today. When interpreting carbon isotope signatures, on e must harken back to the days of basic biology class and discussions of the differences in the two major photosynthetic systems. C3 photosynthesis occurs in the majority of cultivated and wild plants in temperate regions (Schwarcz & Schoeninge r 1991), such as wheat, rice, and barley, and produces an initial three-carbon me tabolite (van der Merwe 1982, Schwarcz & Schoeninger 1991, MacFadden et al 1999b). C4 photosynthesis, found in mo re drought-resistant plants, produces an initial four-carbon compound in cu ltigens such as sugar cane, maize and millet (van der Merwe 1982, Schwarcz & Sc hoeninger 1991, MacFadden et al. 1999b). These different metabolic processes produce di fferent isotopic rati os, which are then incorporated into plant tissues. C4 plants exhibit more rapid carbon dioxide intake leading to values between -9‰ and -16‰. C3 plants on the other hand, have slow rates of carbon dioxide uptake leading to values from -20‰ to -35‰ (van der Merwe 1982). What makes carbon isotope analyses so pow erful is that these to ranges do not overlap. Intermediate values are found in pl ants utilizing a third photosynthetic pathway, CAM or crassulacean acid metabolism (van der Merwe 1982, MacFa dden et al. 1999b). These plants are primarily succulents such as cactus and pineapple, and as such, they neither factor significantly into most hu man diets nor the present research. Plants demonstrate preferential uptake of 12C to 13C, thus they are depleted in 13C compared to12C (Bender 1968). These two carbon speci es are differentially incorporated

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7 into body tissues (i.e., they are fractionated in a characteristic manner) during digestive processes (Durrance 1986). As a result of the differences in photosynthetic pathways in plants, it is also possible to dete rmine approximate proportions of C3 versus C4 plants in an individual’s diet based on the 13C value (Schwarcz & Schoeninger 1991). Carbon isotopes also convey information rega rding the use of marine foods in an organism’s diet. Marine animals presen t isotopic signatures intermediary to C3 and C4 food chains (Schoeninger & DeNiro 1984, Larsen et al. 1992). Marine mammals and fish display 13C values that are enriched by r oughly 6‰ over animals that feed on C3 foodstuffs, and depleted by about 7‰ compared to animals that feed on C4-based foods (Schoeninger & DeNiro 1984). The best indicato r of a reliance on marine food sources is the information provided through a joint 13C and 15N analyses (Schoeninger & DeNiro 1984, Ambrose & Norr 1993). Oxygen Oxygen is the most abundant elemental co mponent of the earth’s crust (Herz & Garrison 1998) and its isotopic ratios provide an indication of the point of origin of remains. Isotopes of oxygen take the form of 16O, 17O, and 18O (Mattey 1997). Oxygen is primarily incorporated into body tissues via atmospheric oxygen, water, and oxygen bound in food (Sponheimer & Lee-Thorp 1999b). Because the 18O value of atmospheric oxygen is relatively constant, it is believed th at oxygen isotopic signatures are primarily representative of imbibed water, and to a lesser extent, the m acronutrients found in foodstuffs (Sponheimer & Lee-Thorp 1999b). The oxygen isotopes in water are preserved in bone, teeth, and other tissues a nd are reflective of a pa rticular environment and climate, decreasing with increasing la titude, increasing altitude, and as you move inland (Dupras & Schwarcz 2001, Kendall & C oplen 2001, Rubenstein & Hobson 2004).

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8 Analytically available oxygen is present in both the phosphate and carbonate ions of hydroxyapatite in the mineral phase of skel etal tissues. Most studies have examined phosphate oxygen because the P-O chemical bond is much stronger than the C-O bone, suggesting that phosphate oxygen is less suscepti ble to diagenesis than carbonate oxygen (Iacumin et al. 1996, Sponheimer & Lee-Thorp 1999b). Lengthy and harsh chemical procedures are required to extract the phos phate oxygen from apatite however, while the carbonate portion is easily obtained from the CO2 produced during mass spectrometry for carbon isotopes (Sponheimer & Lee-Thorp 1999b) Bone carbonate has shown a strong positive correlation to local meteoric water with an r2 value = 0.98 (Iacumin et al. 1996). Additionally, both carbonate a nd phosphate are better pres erved by highly-mineralized tooth enamel versus more porous dentin and bone phosphate (Iacumin et al. 1996). Strontium Strontium has been used to characterize prehistoric mobility patterns since the mid 1980s (Budd et al. 2004, Millard et al. 2004). There are f our stable isotopes of strontium: 88Sr, 87Sr, 86Sr, and 84Sr. Only 87S is the product of radioactiv e decay (radiogenic), being a product of the beta decay of rubidi um 87. This radioactive decay pair, 87Rb 87Sr, has consequently produced di stinctively different 87Sr abundances in different parts of the earth over its history (Beard and Johnson 2000) that have proven quite valuable in tracing the origin of matter to a particular locale. Strontium signatures depend purely on local geology since they reflect the underlying bedrock of a particul ar area. Strontium isotopic ratios vary with the age and type of bedrock underlying the soil. So the qua ntity of strontium in a particular rock will depend not only on the amount of rubidium pare nt material found in the rock, but the age of the rock as well as the original amount of 87Sr present in the rock when it was formed.

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9 Strontium varies in plant tissu e with the age and type of geological substrate or bulk composition. Older soils are more enriched compared to younger soils as are calciumrich soils compared to calcium-poor soils. Additionally, atmospheric deposition or dry fall from natural sources can also affect strontium values (Beard and Johnson 2000). Anthropogenic factors that can influence isotope ratios include nuclear fallout, airborne pollution from fossil fuels, and land-use prac tices that expose bedr ock (Rubenstein and Hobson 2004). Strontium is incorporated into human tissue following the calcium pathway because this non-nutrient, non-toxic element ha s chemical properties similar to calcium (berg et al. 1998). During nutrient uptake strontium ofte n replaces calcium in bones and therefore can be used to trace the flow of minerals from the soil through the food web (Rubenstein and Hobson 2004). “Strontium con centrations in plants and animals are controlled by trophic position, but the isotopic composition is invariant; that is, Sr does not fractionate. Thus, bones and teeth in an individual will have di fferent Sr abundances but identical 87Sr/86Sr ratios” (Herz & Garrison 1998), w ith human enamel demonstrating lower strontium content than bone (Price et al. 1994, Grupe et al. 1997, Beard & Johnson 2000). If food sources are local then, all partic ipants in the food chain, regardless of what tissue is sampled, should reflect the same isotopic signature Additionally, strontium abundance has comm only been examined to discriminate between the meat and vegetable components in an organism’s diet. Toots and Voorhies (1965) published the seminal study in this area, discovering significant diffe rences (p-value <0.001) not only between the mean strontium concentrations for fossil Pliocene carnivores and herbivores, but among the herbiv orous grazers and browsers themselves.

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10 The basis for this is that for each trophic level above the soil, there is a metabolic discrimination against strontium in mammalian epithelium, as opposed to calcium (Radosevich 1993). As one increases in trophic levels among the consumers, the contribution from food sources to skeletal st rontium is decreased at each step (Toots & Voorhies 1965). Plants will retain 50-100% of the strontium found in the soil, with each progressive trophic level exhi biting a reduction of 33% stro ntium over the lower level (Radosevich 1993). Keep in mind that th is refers to strontium abundance (or concentration) and not the 87Sr value. So theoretically, so meone such as a vegan should have a higher strontium concentration than an ardent follower of the Adkins’ diet, by approximately 33%. Radosevich (1993) cautions against blindly accepting these measurements however, without first considerin g factors such as pare nt material and soil chemistry variation influencing plant uptake and physiological differentiation, as well as behavioral changes in feeding strategies trophic placement, and cultural practices. Lead “Lead is one of the most heavily utilized metals in human history” (Sangster et al. 2000). Lead has four naturall y occurring stable isotopes: 204Pb, 206Pb, 207Pb, and 208Pb. As previously discussed, the latter th ree isotopes are radiogenic. Because 204Pb is not radiogenic, it serves as stable reference isotope (Sangster et al. 2000). Similar to strontium, the isotopic compos ition of lead in a particular locale (or ore deposit) is dependent upon four factors: 1) the lengt h of time before lead was separated by geological processes in the source reservoir; 2) the decay rate of the parent isotopes; 3) the initial ratio of the abunda nce of the parent material to the abundance of lead in the source reservoir; and 4) the in itial isotopic constitution of th e reservoir lead (Sangster et al. 2000). The variations in parent isotope decay rates result in systema tic differentials in

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11 the ratios of 206Pb, 207Pb, and 208Pb to each other, as well as to 204Pb (Sangster et al. 2000). Most archaeological studi es are based on the ratios of the radiogenic isotopes to 204Pb, whereas environmental studies tend to also form ratios from only the radiogenic isotopes themselves (Dr. George Kamenov, pe rsonal communication). Additionally, lead is favored by many researchers because like st rontium, it does not exhibit fractionation in nature (Stille & Shields 1997). Lead is assimilated into skeletal elements in a similar manner to st rontium, in that it accumulates from the blood through calcium pathways and substitutes for calcium in the carbonate hydroxyapatite fraction of hard tissu es (Vogel et al. 1990). Juveniles exhibit a higher propensity to absorb ingested lead than adults (Reinhard & Ghazi 1992), likely due to the rapid modeling of bone occurring during the growth phase and because small children tend to frequently put objects in thei r mouths. Lead particles are thought to enter the body through ingestion, either through food stu ffs/fluids or lead objects, or inhalation (Gulson 1996). Environmental contam ination by lead is found through mining operations, waste dumps, emissions from l ead smelting, coal combustion, and leaded gasoline (berg et al. 1998). Furthermore, acid rain can transmit contamination from emissions/combustion over great distances. Fractionation Prior to drawing conclusions regarding the delta value of a material, additional issues such as fractionation e ffects must be factored in. Fractionation is the disparate partitioning of isotopes between two substanc es or tissues (Hoefs 2004). Without it, biological processes would be homogenous and some of the most powerful inferences in isotopic analyses would not be possible.

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12 Differential fractionation manifests itself in a variety of forms. One example is the different rates carbon is fracti onated as one progresses thr ough the food chain. Carbon found in the atmosphere is pr esent with a near constant 13C/12C ratio of about 1:99 (Chisholm 1989). As plants incorporate carbon into their tissues during photosynthesis, isotopic fractionation oc curs altering the 13C/12C ratio. Since C3 and C4 photosynthetic pathways differ chemically, they produce di fferent degrees of fractionation. This is beneficial, and in fact, essential in the case of carbon isotope studies, because the 13C values can be utilized to classify between C3 and C4 plants and diets based on a complete separation of approximately 14‰ between gr oups allowing for discrimination between them (DeNiro & Epstein 1978a, 1978b, Chisholm 1989, Ambrose & Norr 1993). The selective metabolism and recombinati on of plant chemicals within organisms feeding upon them, results in fractionation of el emental isotopes, leading to differences in 13C values between diet and bone collagen of primary consumers of +3‰ to +5.3‰. An additional fractionation factor of about +1‰ must be accounted for as you increase in trophic level (i.e., from primary to sec ondary consumer) (Schoeninger 1985, Chisolm 1989, Schoeninger 1989, Ambrose 1993). The 13C values of mammal hydroxyapatite trend even further from the whole diet, with rats on experimentally controlled diets showing an enrichment of +9.6‰ (DeNir o & Epstein 1978b) and other mammals displaying enrichments of +12‰ to +13‰ (Lee-Thorp et al. 1989). Additionally, preferential uptake among differe nt tissues within the same organism has been noted and can further complicate matters, with animal muscle generally showing 13C values 3‰ to 4‰ less positive (-3‰ to -4‰) compared to bone collagen (Schoeninger 1989).

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13 Oxygen undergoes fractionation due to envir onmental factors such as evaporation, condensation, and freezing and is also strongl y influenced by temperature and humidity (Stille & Shields 1997, Iacumin 1996, Hertz & Garrison 1998, Kendall & Coplen 2001). This leads to differential isotope incorporati on in plant tissues and is reflected in the differing values of herbivores thought to be a result of foraging habits. For instance, oxygen isotope ratios were found to vary by as much as 8‰ to 9‰ in herbivores based on whether they were browsers or grazers (Iacumin et al. 1996). A difference of approximately 9‰ has also been measured between the carbonate and phosphate fractions of bone and teeth fr om a variety of mammals (Iacumin et al. 1996) as well as marine invertebrate she lls (Longinelli & Nuti 1973). This consistent enrichment of carbonate 18O values, regardless of the animal, seems fairly constant as long as temperature remains within the range of 0O C to 37O C. Outside of this temperature range, the fractionation is not as predictable (Iacumin et al. 1996). One reason strontium and lead analyses appear so attractive is the general consensus that these elements do not undergo fr actionation in nature. Strontium and lead do not appear to exhibit this trend due to their significantly large atomic masses (Stille & Shields 1997) versus the lighter isotopes such as carbon and oxygen. Such being the case, comparisons can be drawn then utilizi ng organisms from differe nt trophic levels as well as between different tissues, without having to employ conversion factors. Frequently Sampled Human Tissues A variety of human tissues have proved useful in is otopic studies within the anthropological disciplin es in recent years. Tissues primarily available to forensic anthropologists include bone, teeth, hair, de siccated skin, and finger/toenails; each

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14 presenting its own benefits and drawbacks pot ential isotopic use a nd preserving records of residency and diet at different points of the individual’s life. Bone Bone is arguably the most utilized ti ssue in archaeologi cal isotope studies (Schwarcz & Schoeninger 1991). It is a co mposed of three primary constituents: 1) water; 2) an inorganic mine ral fraction (hydroxyapatite); and 3) an organic matrix (Schwarcz & Schoeninger 1991). Bone isot ope studies utilize both hydroxyapatite (apatite) and collagen, which is found in th e organic phase. Dry bone is composed of approximately 70% inorganics and 30% orga nics (Katzenberg 2000). The overwhelming majority of the inorganic phase is compri sed of the protein collagen (85% to 90%) (Katzenberg 2000). Bone has a turnover rate of between 10–30 years (Ambrose 1993) owing to the fact that different bone components remodel at diffe rent rates. On average, trabecular bone remodels much more rapidly than its dens er cortical counterpa rt (Teitelbaum 2000). Regardless of the speed of turnover, it is cl ear that bone delta values slowly change throughout an individual’s life as stable isotopes are constan tly incorporated into this continually remodeled tissue. Apatite is a calcium phosphate product of which the carbonate portion arises from dissolved carbon dioxide (CO2) in the blood plasma. Frac tionation does o ccur between these two reservoirs with the bone carbonate portion 13C value enriched by approximately 12‰ over plasma CO2 (DeNiro and Epstein 1978b). Bone carbonate therefore reflects the total metabolic carbon pool found in an individual’s diet, incorporating carbon equally from all dietary energy sources and representing the isotopic signature of the w hole diet (DeNiro and Epstei n 1978b, Ambrose & Norr 1993).

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15 Collagen is the most abundant protein in the body (Champe & Harvey 1987), constituting roughly one-quarter of all proteins occurring in mammals (Stryer 1975). The collagen found in bone, dentin, skin, and tendon is molecularly similar and falls under the category of Type I collagen (Schwarcz & Schoeninger 1991). Cont rolled experiments using rats demonstrated that collagen unde restimates the non-prot ein component of the diet, but is an excellent representative of th e protein portion because of its heavy nitrogen constituent (Ambrose & Norr 1993, Tieszen & Fagre 1993). One difficulty with bone collagen is that is does degrade over tim e, much more so than apatite. Teeth Teeth are especially useful in isotopic studies because of their robustness and ability to survive in environs where bone would normally degrade. Unlike bone, tooth enamel tends to be highly inert in terms of mineral exchange with the environment (Price et al. 2002, Lee-Thorp & Sponheimer 2003), consequent ly they represent small, closed systems. Because enamel is non-cellular and heavily mineralized with 96% or greater of the weight of the enamel comprised of the inorganic constituent (Hillson 1996), it withstands the effects of diagenesis very well and long preserves an accurate biogenic isotopic signal (Lee-Thorp & Sponheimer 2003). Dentin and cement, on the other hand, are much heavier in organics (roughly 20% and 25%, respectively) (Hillson 1996) and much more susceptible to contamination. Additionally, the inorganic natu re of enamel, and specifica lly the apatite, reflects the whole diet of the individual while the coll agen in dentin, because of its high nitrogen content, primarily mirrors the protein cont ent of the diet (van der Merwe 1982, Harrison and Katzenberg 2003). This is one of th e drawbacks of using enamel. You cannot analyze nitrogen isotopes.

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16 Moreover, since teeth are genetically conser vative, there is little variation in the development and specifically, the period of mineralization of the tooth, although females are slightly precocious in terms of dental formation, completing most stages of dental growth before males (Fanning & Brown 1971, Hillson 1996). This observation was confirmed by the 1976 study by Anderson et al of the mineralization in permanent dentition, although the authors state the degree of variability between the sexes has been reported to be similar. Their calculations of the mean age of attain ment of mineralization in the adult teeth are presen ted below in Table 1-2. Table 1-2. Mean age of completion of permanent crown mineralization. 1st Incisor 2nd Incisor Canine 1st Premolar 2nd Premolar 1st Molar 2nd Molar 3rd Molar Males Maxillary 3.70.28 4.00.48 4.90.53 5.81.0 6.30.65 3.80.30 6.70.72 13.31.58 Mandibular 3.6+0.21 4.00.46 4.80.59 5.61.21 6.30.70 3.70.14 6.70.71 13.31.51 Females Maxillary 3.60.14 3.80.40 4.10.49 5.10.56 5.90.65 no data 6.30.66 12.71.49 Mandibular 3.60.20 3.70.28 4.10.49 5.00.54 5.90.74 no data 6.30.66 12.81.63 Source: Anderson et al. (1976) With inand outflow of materials ce asing once amelogenesis is complete, examining the permanent enamel provides a sn apshot of the nutriti onal ecology of that individual during the period of crown mineralization for that specific tooth. Dentin primarily is laid down during and after ameloge nesis, thus the bulk of it is formed during childhood. Secondary dentin lines the pulp ch amber and has a slow, continued formation during adulthood, with turnover rate s similar to bone (Hillson 1996). Sampling can be done in bulk, which will average the isotope value for the entire tissue component, or serially. In serial analys es, very specific regions of the enamel or dentin, corresponding to even finer time periods are sampled and compared. This takes much greater skill in drilling and one must be sure what point in the individual’s life the

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17 area represents, but this method can also allo w even finer resolution of dietary studies over a period of years. Hair Several studies have turned to hair as an alternative sampling tissue (van der Merwe et al. 1993, Yoshingaga et al. 1996, O’Connell & Hedges 1999, White et al. 1999, Bonnichsen et al. 2001, Ayliffe et al. 2004 Cryan et al. 2004, West et al. 2004 Roy et al. 2005). Hair holds a great untappe d potential in forensic isot ope work. Often, hair masses are found in association with sk eletal remains. It is extr emely durable, proving insoluble to a variety of fluids, and can remain intact for thousands of years (Bonnichsen et al. 2001). The shaft is sheathed in a cuticle, a hard protective covering that is resistant to chemical and microbial insult (Lub ec et al. 1987, Macko et al. 1999b). Because of its hardiness and the fact th at the average human sheds 50-100 hairs a day (Macko et al. 1999b), sampling is easy and essentially non-invasive. Non-keratinous material, such as the root (bulb) is not normally sampled (Ayliffe et al. 2004) because of its signature is not reflective of the shaft. Hair is also re adily renewable, growing roughly 1 cm/month in humans (Yoshinaga et al. 1996), with isotope shifts demonstrating about an 8-day delay (in the case of beard hair) from change of diet to hair exposure from the follicle (Sharp et al. 2003). The isotopic composition of hair offers information concerning an individual’s diet and recent ge olocational background. Thus a section of hair provides a snapshot of an individual’s nutritional ecology at a particular point in time and a chronological record of the same al ong its length (White et al. 1999, Roy et al. 2005). Hair is easier to isotopically analyze th an bone and only very small samples (much less than bone) are required (Cryan et al. 2004, Roy et al. 2005). Hair is made of

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18 approximately 95% keratin, the proteinacious component (Taylor et al. 1995). Because of its high protein content, hair requires minimal chemical processing and no chemical purification. Conversely, bone and dentin collagen must be chemically extracted and purified before the protein fraction can be analyzed (Ambrose 1993). Roy et al. (2005) produced consistent carbon and nitrogen isotope values with human hair specimen weights as low as 100 g, corresponding to a length of 2 cm of hair. The authors expect that strand lengths as small as 5 mm could be analyzed without significant loss of precision when determining 13C alone, as nitrogen was the limiting factor in their study. The 13C values of hair keratin correlate well with that of total dietary protein, with keratin being enriched by +1‰ to +4.8‰ relative to protein in the diet and depleted by -2‰ to -3‰ compared to bone collagen (DeNiro & Epstein 1978b, Ambrose & Norr 1993, Tieszen & Fagre 1993, Yoshinaga 1996) in lab animals and contemporary humans. Carbon isotope signatures from hair keratin and bone collagen are related but cannot be directly equated (O’Connell & Hedges 1999). Fingernails and Toenails Like hair, fingerand toenai ls also offer a non-invasive way to examine isotopic values in both the living and dead. The keratin composition of human nail material makes them an excellent source of collagen values, as well as a variety of elemental isotopes. They also provide a recent geol ocational reference for a specific individual with a whole nail repres enting approximately 6 months of gr owth in adults (note: authors did not state the length of the nail) (Fraser et al. 2006) and 2 to 3 months growth from cuticle to fingertip in infants (Fuller et al. 2006a). While some state that hair and fingernail values are “similar” to each other (O’Connel et al. 2001, Fuller et al. 2006a) it

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19 has been noted that nails are depleted in 13C and 18O compared to hair by mean values of and -0.55‰ and -1.6‰, respectiv ely (Fraser et al. 2006). Fogel et al. (1989, 1997) published the details of a landmark study examining the weaning of modern infants as reflected in the differences in 15N between mothers and infants. Fingernails prove an excellent medium for studying diets in modern infants because they are metabolically inert, resist ant to degradation, and have such a fast synthesis rate (Fuller et al. 2006a). What the authors found was that the isotopic values of the babies’ fingerna ils were enriched in 15N by approximately +3‰ from that of their mothers, indicating that the infants were f eeding at a higher trophic level than their mothers. This was confirmed by Fulle r et al. (2006a), who found infant 13C values enriched by +1‰ over their mothers’ and 15N enrichment of +1.7‰ to +2.8‰ compared to maternal values (for more detail, see Chapter 2). Such conclusions have been extrapolated to bone and tooth isotopic anal yses of weaning practices of archeological populations (Schurr 1997, Herring et al 1998, Schurr 1998, Wright & Schwarcz 1998, Wright & Schwarcz 1999, Dupras et al. 2001, Mays et al. 2002, Clayton et al. 2006, Fuller et al. 2006b). Skin Often desiccated skin is adherent to bone on remains submitted for forensic analysis. This skin serves as an additional potential reservoir for isotopic values and can be relatively easily removed from associated bone. Carbon turnover rates for skin and hair are much faster than for bone, giving thes e tissues the ability to confer information regarding diet and provenance much closer to death than hard tissues. Skin has an estimated carbon turnover rate of roughly 15 days (Tieszen et al. 1983). The integrity of skin after the decomposition pr ocess takes hold however is suspect, as skin appears

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20 highly susceptible to contamination (White et al. 1999). For those studies in which viable skin samples were obtained, relative to hair sample 13C values, skin appears to be consistently depleted from -0.2‰ to -2.7‰ (White & Schwarcz 1994, White et al. 1999) Complications Radosevich (1993) aptly states that a reason for uncritical acceptance of methods or assumptions is often the simple desire for a new technique to wo rk. Stable isotope analyses have seemingly been hailed as near-omniscient and people may turn a blind eye to the limitations of such methods. On the other hand, modeling biological systems is an extremely complex undertaking. There are times when a reductionist approach can overwhelm the model in minutiae; where ac counting for all the potentials of error eclipses the actual data. All factors with the potential for confounding th e data need to be explored and understood, but of ten a relative weight can be assigned to them so the model is not overloaded. There are much poten tial for error in stab le isotope analyses; but, as long as they are recognized a priori and dealt with, isot ope ratios can provide valuable insight into past and present systems. Diagenesis After the initial glow wore off followi ng the popularization of stable isotope techniques, researchers began to find chinks in the analytical armor. Often confusing or contrary results were obtained leaving researchers to scratch their heads as to what it all meant and if isotope studies were really wo rth all the hype. In 1981, A. Sillen was one of the first to propose that perhaps post-depos itional contamination, or diagenesis, was responsible for at least a portion of this noise but the effects of di agenesis were largely ignored or dismissed in most studies (Price et al. 1992).

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21 Diagenesis is a subset of the study of the postmortem processes which can affect bone appearance and integrity, commonly know n as taphonomy (literally meaning the “laws of burial;” Sandford 1992). These proc esses take both physical and chemical forms. When diagenesis in an anthropological cont ext is discussed, it is in reference to the postmortem alterations in the chemical c onstituents and physical properties of bone following deposition in soil. Diagenesis ta kes the form of both contamination and leaching and arises from several di fferent mechanisms (Sandford 1992). The dense mineralization of enamel affo rds teeth a great measure of protection against effects, but it is important to keep in mind that no skeletal element is impervious to postmortem modification. The porous stru cture of bone however, makes it susceptible to infiltration by foreign elements, especially when it has been physically degraded. The intrinsic skeletal chemistry and microstructu re of osseous tissue therefore leads to a dynamic relationship between it and the enviro nment in which it is interred (Sandford 1992). Mary Sandford (1992) lists several diffe rent means by which the environment interacts with the structure of bone, leading to alteration: Elements may be precipitated as discre te “void-filling” mineral phases in the small cracks and pores of bone. Soluble ions present in soils may be exchanged for those that normally occupy lattice positions in bone hydroxyapatite. Bone apatite can “seed” formation of recrystallization thr ough a variety of means. Microorganisms break down bone colla gen releasing elements through its dissolution and the ac tion of acid metabolites on hydroxyapatite.

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22 Additional extrinsic factors such as the chemical environment of the burial sight and the properties of the e nveloping sediment influence th e incidence and rate of processes as well. Soil pH is one of the most important variables that affect change in bone. Gordon and Buikstra (1981) first quantified the relationsh ip, determining that it is strongly negatively correlated, thus as soil pH decreases, degradation of bone increases. The authors also noted that skeletal age was significant as well, with juvenile bone being more susceptible to decay. Temperature, microorganismal activity, gr oundwater, and precipitation also play a role, as does the local geochemical environm ent to include soil texture, mineralogy, and organic content. Sandford (1992) also men tions further intrinsic factors bearing on processes such as bone density, size, microstructure, and biochemistry. Recent investigations have shed light on bone alteration leading to several generalizations: 1) elements differ in thei r susceptibility to diagenesis; 2) certain categories of bones are more susceptible to diagenesis--less bone density, greater porosity, or large quantities of amorphous material may pr edispose certain classes of bones, such as immature bone, to taphonomi c processes; 3) denser cortical bone withstands diagenesis much better than th e lattice-like tr abecullar bone; 4) Direction and intensity of change is not necessarily tempora lly or spatially uniform (Sandford 1992); 5) the color and condition of skelet al material can be used as a general indicator of the degree of diagenesis (Carlson 1996). The more the color approximates the color of fresh bone, the less likely it is to have undergone change. The majority of changes seen in bone ar ise due to precipita tion of authigenic carbonate or other minerals, exchange reactions in original carbona te or phosphate, and

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23 uptake or loss of various trac e elements. Recrystallizati on can also occur, producing various phosphate-containing compounds with tr ace levels of elements often replacing calcium at higher concentrations than found in modern bone (Schoeninger et al. 2003). The same processes that bring about diagene tic change are ones that will eventually return bones the lithosphere. The overwhelming majority of all deposited skeletal material disappears relatively quickly, especi ally if exposed to taphonomic factors such as acidic soil, alternate wet/dry conditions strong solar radiati on, and/or injurious invasion by microorganisms (Lee-Thorp 2002). If we as anthropologists are fortunate to encounter remains in the first place, we shoul d not be discouraged from utilizing isotopic resources in attempting to uncove r clues about the lifes tyle of the individu al(s). We must keep in mind that these processes are not uni form over space and time, and thus even old remains can produce valuable results. Questions still remain however, as to what measures can be taken to minimize the impact of diagenesis on isotopic interpretation. So what is a researcher to do? The first step is to attempt to determine if processes ha ve occurred and to what extent. In reality, these processes are always o ccurring, but whether they ex act a measurable effect upon bone is another question. To begin with, a scientist should ask themselves several questions. The first is what is/are the element( s) of interest? Studies show that isotopes of such elements as strontium and lead are li ttle changed in bone due to diagenetic means (Beard and Johnson 2000, Carlson 2002), thus scient ists should have gr eater latitude in using bones that have been interred for any pe riod of time. Do the bones belong to an adult or child? Because smaller bones have greater surface area to volume ratios, they are more susceptible to change since there is more surface area for processes to act upon.

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24 The absolute volume of cortical bone is reduced in juveniles as well, as they are still growing, so bones are less shielded from e nvironmental assailants. What bones are available for sampling? Remains higher in co rtical bone preserve be tter, so if presented with a few cranial vault fragments, a resear cher may be wise to opt out of isotope analysis versus if a femoral shaft is available. Also, intact bone is always preferable to fragmentary bone. One should also assess the environment th e bones are interred in. Sandford (1992) believes chemical analysis of soil is a mandato ry requirement for gain ing insight as to the condition of bone. Soil samples should be re covered from feature fill in direct association with bone (Gordon and Buikstra 1981). Samples can be prepared and pH determined in situ utilizing a portable pH meter. Th ese values can then be applied to something similar to a regional variant of Gordon & Buikstra’s (1981) regression formulae for pH and state of preservation. (It is interesting that wh ile the authors provide several regression formulae, for example, in adult assemblages, preservation = -1.3(pH) +12.5, there is no scale provided in which to interpret the preservation value.) Further testing can compare total elementa l concentrations of bone and associated soil. Following the assumptions of the c oncentration gradient theory, significant contamination of bone by soil is considerably less likely if soil concentrations of a specific element are disproportiona tely different than those same elements in bone. If a more homogenous elemental state has been reached between bone a nd soil, it is a good indication that sign ificant change has transpir ed (Sandford 1992, Carlson 1996). Other factors such as temperature and expos ure to water should also be accounted for. It is well accepted that higher temperature leads to degr adation of collagen and that

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25 warm, moist habitats encourage microbial pro liferation. Exposure to water can also lead to increased rates of both contamination a nd leaching of minerals into the surrounding soil. The best environs for the preservation of DNA are those that ar e cool, dark, and dry (Smith 2005). That is because these same c onditions optimize the re silience of the whole bone complex, so isotopic fractions will be be st preserved as well. Heavy bone erosion, trauma, burning, associated human alterations such as boiling and internment/funeral practices, and carnivore and r odent activity compromise the structural integrity of the bone itself leaving it more vulnerable to processes. Instrumental analyses can be completed as well to include electron microprobes and x-ray diffraction (Sandford 1992), and backscatter scanning electron microscopy (Collins et al. 2002). These methods attempt to look at the structur e of bone and analyze it for changes in crystalline architecture, ch emical constituency, and microbial activity. Analyses of collagen content of bone may also provide insight, since some have observed low yield in collagen is often associated with aberrant stable isotope readings (Katzenberg 1992). Osteological comparisons can also be comple ted in conjunction with soil analyses examining constancy in values (Sandfor d 1992). Intrabone comparisons look for statistically significant correlations between elements and known contaminants or “indicator elements.” Interbone comparisons look for agreement with the assumption that different types of bone, such as ribs and femora, should reflect varying degrees of diagenesis. Interspecies comparisons can indicate activity when measured elemental values vary from those predicted on the basis of dietary patterns. Additionally, if

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26 interpopulational data were available as we are attempting to collect, congruency to published values could be ascertained (Sandford 1992). Further precautions are esse ntial during sample prepara tion in the laboratory. Standard protocols attempt to minimize ef fects of diagenesis through mechanical abrasion, to physically remove contaminants from outer bone surfaces, and acid washing. None of the aforementioned methods are fa il safe, but their use enhances overall understanding of the processes active in a ce rtain area and attempts to circumvent diagenetic effects by careful sa mpling selection and preparation. Many subscribe to the notion that the longe r a set of remains ha s been interred, the greater the alteration to the mate rial. It is unwise to use temp oral criterion in isolation in making a decision about employi ng isotopic analyses though. As in any scientific situation, you must take measure of as many variables as possible in order to make the most informed decision. Diagenesis is a co mplex mechanism and time is but one factor that comes into play. Cases in the literature abound detailing the su ccessful extraction of viable isotopic material from fossils that would have proved oppor tunities lost if the authors had decided against isotopic analyses simply because they were working with very old material. Studies have examined diets in ancient, human mummies (White & Schwarcz 1994, White et al. 1999) and Neolithic Icemen (Macko et al 1999a, 1999b; Mller et al. 2003), and the di et and paleoecology of Australopithecus africanus (van der Merwe et al. 2003) and 5 million-year-old horses (MacF adden et al. 1999b), to cite but a few. Differential preservation is a rule, rath er than an exception and thus each interment must be individually assessed for the appropriateness of isotopic analyses.

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27 Anthropogenic Contamination All organisms alter their environment. Hu man beings are unique though, in that we are the only species on the planet that is actu ally altering the basic conditions of life on Earth (Vitousek et al. 1997). We have al tered landscapes, climate, and biogeochemical cycles. Many of the wastes generated by our industrial metabolism play no useful role in nature, cannot be recycled (i.e., nuclear wa ste) or overwhelm the current processing capabilities of the biosphere (McMichael 2001 ). The ecological footprint of the human species is enormous. Ev erything we do leaves tra ces of our kind behind. This anthropogenic effect ex tends to isotopic signal va riation. Industrial pollution is implicated in the changing of isotopi c values when contemporary populations are compared to paleological assemblages and can outright alter or mask the isotopic signatures a researcher is attempting to interp ret. This can complicate analyses and lead to false conclusions if not id entified. To account for this, se veral correction factors have been established to ease temporal analyses. Because nearly all of the anthropological work done with stable isotopes has been in bioarchaeological contex ts, these corrections are essential in drawing conclusions. The carbon isotope ecology of terrestrial systems is controlled by atmospheric carbon dioxide (van der Merwe et al. 2000). Th is has changed dramatically in the years since the Industrial Revolution, with fossil fuel emissions altering the 13C/12C ratio of the atmosphere by -1.5‰ in the last 150 years. (van Klinken et al. 2000). To correct for this change in 13C values, “Industrial Effect” (van der Merwe et al. 2000) or “fossil fuel effect” (van Klinken et al. 2000) calibrations must be factored into results, normally by adding 1.5‰ to convert modern samples to pr e-industrial values (van Klinken et al. 2000).

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28 We also have significantly altered the lead content of certain environs. Budd et al. (2000) state, “It is widely believed that the contamination of the atmosphere by anthropogenic lead has led to far greater human exposure today than that which prevailed in the distant past, but this has proved diffi cult to quantify.” A marked increase in the mobilization of lead in Europe and North America occurred after industrialization. Drilling of Greenland ice-cores has revealed a ten-fold rise in lead concentration, with rates skyrocketing from roughly 10 parts per bi llion (ppb) to 100 ppb in the last 100 years (McMichael 2001). This is due primarily to environmental contamin ation due to the use of leaded gasoline (which is still utilized in many nations), lead-based pigments and compounds, lead-acid batteries, and through mining operations, soldering, and coal combustion (Sangster et al. 2000). Global Economy Today’s global economy has the pote ntial to homogenize biogeochemical signatures in contemporary people. Because of world-wide trade, especially when it comes to food importation, what people eat may not necessarily reflect where they came from. Strontium values are especially vulne rable to being washed out by the effects of the global food market. Archaeological rese arch does not usually concern itself with such matters because food tended to be loca lly grown and consumed. After the Industrial Revolution and the establishmen t of global trade networks, food in the U.S. was very rarely grown in the localities were people live d. So, on a trip to th e refrigerator one may find bananas from Guatemala, grapes from Chile, and free range, grass-fed beef from Argentina. Increasing consumption of bottled wa ter from non-local sources further complicates matters, affecting not only st rontium values, but oxygen and hydrogen as

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29 well. This situation may be further compli cated by the importation of fertilizer produced in foreign countries (Price et al. 2002). Such soil additives will affect not only plant intake but run-off will affect, and may significantly change, the isotopic values of groundwater (Bhlke & Horan 2000).

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30 CHAPTER 2 APPLICATIONS OF STABLE ISOTOPE ANALYSES Examples of the varied usages of isotopes in the literature abound. Stable isotope analyses are an extremely effective means of recreating paleoecology (e.g., Amundson et al. 1997, Cerling et al. 1997), tracking anim al movements (e.g., Burton and Koch, 1999 Rubenstein & Hobson 2004), assessing migrat ory patterns of humans (e.g., Beard and Johnson 2000, Dupras and Schwarcz 2001), and de termining diet (e.g., DeNiro & Epstein 1978, van der Merwe 1982, MacFadden et al. 1999b). Within anthropology, stable isotope analyses have been primarily rele gated to realm of archaeology, but by applying the technologies currently used in geol ogy, paleontological and modern zoology, and archaeology to forensic science, an eff ective means for presumptive identification emerges. Tracing Studies One exciting application of stable isotope s that transcends disciplinary bounds is that of tracing studies. In a tracing study, an element is introduced into a system with a known delta value and tracked through the system or at the termination of certain processes to see how that element normally m oves through the system. This approach is frequently used in clinical nutrition studies to understand the uptake of various nutrients (see Abrams 1999 for a review). Stable isotopes offer many benefits over more traditional radioactive approaches in that th ey present little of a safety concern for pregnant women or children and are less di fficult and less expensive to remove than radioactive wastes (Abrams 1999).

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31 For instance, isotopic tracer studies were used to measure the efficiency of zinc utilization at different doses. Patients were given labeled zinc solutions, and then urine samples were collected to determine absorpti on rates. Based on this approach the study concluded aqueous zinc doses greater than 20m g resulted in quite small and diminishing increases in absorptivity (Tran et al. 2004). Magnesium tracer studies demonstrated that absorption of isotopically labeled magnesium could be accurately monitored through urine sampling versus more invasive blood a nd fecal sampling methods (Sabatier 2003). Additionally, tracer studies in Nigerian children with rickets determined that those with the disease did not express impaired abiliti es to absorb calcium when compared to healthy counterparts, although fractional calc ium absorption did increase after resolution of the active disease (Graff et al. 2004.) Stab le isotopes were even utilized to measure calcium metabolism of two cosmonauts and one astronaut aboard the Mir space station prior to, during, and after a 3-m onth spaceflight (Smith et al. 1999). Further non-human trials utilized three diets of different isot opic compositions to dete rmine the turnover time of carbon isotopes in horse tail hair West et al. 2004) and tail hair and breath CO2 (Ayliffe et al. 2004). These baseline studies could then be applied to other wildlife studies in an attempt to understa nd the dietary history of mammals Ecological studies have also utilized isotope tracers to examine nutrient flow in various systems. One recent study added isotop ically-labeled nitrogen to a creek for 6 weeks and monitored 15N in dissolved, aquatic, and terrestrial riparian food web components. High levels of incorporation of the tracer into the tissues of resident organisms led researchers to believe that st reams within undisturbed primary forests may be highly efficient at uptake and retention of nitrogen (Ashkenas 2004). Another project

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32 examined root turnover in relation to fore st net primary production by fumigating a stand with labeled 13C in the form of 13CO2 over a 5-year period, then sampling fine roots. Their results suggest that root production and turnover in forests have likely been overestimated and that sequest ration of anthropogenic atmosphe ric carbon in forest soils may be lower than currently be lieved (Matamala et al. 2003). Fractionation Studies Fractionation studies have proven quite illust rative in a variety of genres as well. Examination of carbon and nitrogen stable isot opes has yielded great er understanding of the decompositional processes found within so il organic matter (Kramer et al. 2003). Fractionation studies have also proven useful in attempting to meas ure the contribution of gluconeogenesis to glucose production in hum ans. Here, body water was enriched with 2H2O and the ratio of 2H bound to carbon-5 versus carbon-2 of blood glucose was measured (Katanik et al. 2003). Oxygen isot ope fractionation has also been employed in niche separation studies of African rain forest primates occupying overlapping microhabitats. Oxygen isotope ratios from bone carbonate were positively correlated with relative dependence of leaves in the diet, a fact obscured by carbon isotope analyses (Carter 2003). A final study le d to the discovery of what is commonly known as the “canopy effect” (van der Merwe &Medina 1991). Van der Merwe and Medina discovered the re-use of plant-fractionated, respired CO2 in dense vegetation can cause systematic bias between plant and animal sp ecies living on the forest floor versus those living in the forest canopy and open environmen ts (also in van Klinken et al. 2000). Due to the “canopy effect,” the 13C value of atmospheric CO2 is lowest near the forest floor. “Leaves fixing this 13C-depleted CO2 have lower 13C values than those higher up in the canopy. Combined with the effects of low light intensity, high humidity and high CO2

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33 concentrations on water use efficiency, th is creates a vertical cline in leaf 13C values” (Ambrose 1993). Zoology and Ecology Within zoology and ecology, the examples of stable isotope use seem limitless. One of the first such studies examined carbon ratios of two sympatric fossil hyrax species, determining one a was browser, based on the C3-like signature these animals displayed, while the other was chiefly a grazer, feeding on tropical grasses, which utilized a C4 photosynthetic pathway (DeNiro & Epstein 1978a). Sim ilar studies have shed new light on the diet and ecology of 5-milli on-year-old horses (MacFadden et al. 1999b) and Cenozoic sirenians from Fl orida (MacFadden et al. 2004). Stable isotopes have proven especially insightful for scientists attempting to determine feeding strategies of marine organisms, and in fact, “Most work on ma mmal and reptile movements using stable isotopes has been done in the marine e nvironment” (Rubenstein & Hobson 2004). Carbon isotopes have been used to dete rmine food sources for Red Sea barnacles (Achituv et al. 1997) and examine photosymbiosis in fossil mollusks (Jones et al. 1988). Delta 13C and 15N were useful in assessing not only the foraging strategies of Pacific pinnipeds (Burton & Koch 1999), but tracking their migratory movements as well and have been used in dietary studies of Nort h Atlantic bottlenose dolphins (Walker et al. 1999). Moreover, adult female loggerhead tu rtles were sampled from around Japan to determine the relationship between body size and feeding habitats (Hat ase et al. 2002). Claws (Bearhop et al. 2003) and feathers (Rubenstein et al. 2002, Bowen et al. 2005) have also been utilized to determine di ets and habitat use of migratory birds whose summering and wintering grounds are separated by thousands of kilometers; so too has hair been examined in bats for evidence of seasonal molt and long-distance migration

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34 (Cryan et al. 2004). Wing membranes from monarch butterflies have been sampled for hydrogen and carbon isotopes to identify natal regions within the United States and revealed that 13 discrete wint ering colonies in Mexico were fairly well mixed as to the origins of the individuals (Wassenaar & Hobson 1998). Biogeochemi cal fingerprints of African elephant bone have been assessed to determine change in diet and habitat use (Koch et al. 1995). Isotopic analyses have even been extended to determining the allocation of reproductive resources in butte rflies (O’Brien et al 2004) and assessing prey quality in predatory sp iders (Oelbermann & Scheu 2002). Stable isotope ratios also allow us a glimpse into the past. Based on 13C enamel values of worldwide fossil mammals and modern endemic and zoo-housed African mammals, Cerling et al. (1997) has postulated that between 8 and 6 million years ago, there was a global shift to increased C4 plant biomass and a corresponding decrease in atmospheric carbon dioxide. This has imp lications today as in creasing levels of atmospheric carbon dioxide could bring about a major biotic alteration towards a world dominated by C3 plants, which would have widespread ecological consequences. Carbon values have also been analyzed from an cient pollen in attempts to reconstruct paleovegetational and paleocli matic conditions with the hop e of someday tracing the origin of the C4 photosynthetic pathway (Amundson et al. 1997). One further study has taken an innovative a pproach to tying stable isotopes to hominin evolution. Wynn (2004) examined paleosols of Turkana Basin, Kenya, and ascertained that modern hominins evol ved during a period of waxing and waning diversity of savanna-adapted fauna in an environment that trended towards increasing aridity. Those hominins best suited to genera lization of resources were the most capable

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35 of surviving through evolutionary “pruni ng events” as savanna ecosystems changed through time. Mention of these zoological and ecologi cal studies does not even scratch the surface as to the diversity of isotopic studies that have been and are continuing to be conducted within these disciplines. Isotope us e in these fields is gaining momentum and results generally enjoy widesp read acceptance, ensuring thei r continued use far into the future. Archaeology Within anthropology, stable isotope analyses have primarily been relegated to the realm of archaeology. Here, they have been used extensively to answer a litany of questions in a variety of contexts concerning the human experience. Diet Assessment Introduction of maize In archaeological contexts, stable isotopes ha ve been used extensively to infer diet, mobility patterns, and origins of material cult ure. When considering diet, a great amount of effort has been expended attempting to determine when exactly maize became prominent dietary component in various human populations (Vogel & van der Merwe 1977, van der Merwe & Vogel 1978, DeNiro & Epstein 1978b, Farnsworth et al. 1985, Norr 1995). In fact, the majority of early archaeological stable isotope studies were aimed at resolving the temporal and geographi c origins of maize in troduction, especially into North America (Schwarcz & Schoeninger 1991). Striking changes in 13C values of collagen resulted from the introduction of ma ize into human dietary patterns. These values markedly decreased from roughly -21.4‰ to -12.0‰ during the period of A.D. 1000–1200 indicating that proportion of carbon from C4 plants went from 0 to more than

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36 70% in some individuals (van der Merw e & Vogel 1978, van der Merwe 1982). This agrarian shift also had other implications, w ith the development of permanent settlements and an abandonment of the hunter/gatherer life strategy and all associated changes inherent in the transition to a sedentary lifestyle. Not all agree on the timing of the introduction of maize to North America though, with individuals such as Farnsworth et al. (1985) concluding that mai ze was incorporated into human diets much earlier than indicated in the fossil record. Today, the cons ensus seems to be that there is a temporal variation in the conversion to maize agriculture within North America (Schwarcz & Schoeninger 1991). Age effects in a prehisto ric maize horticultural population (Ontario Iroquois) have also been examined, with significantly higher 13C values found in infants and young children suggesting a weaning diet hi gh in maize (Katzenberg et al. 1993). Isotopic dietary studies have been applied to fossils as old as Australopithecus africanus, where individuals demonstrated an unus ually varied diet, a large portion of which was C4-based (Sponheimer & Lee-Thorp 1999a, van der Merwe et al. 2003). From this information, the authors speculate that by about 3 million years ago, hominins had become savanna foragers for a significan t part of their diet. Based on carbon and nitrogen stable isotope values, the diet of a Neolithic Alpine “Ice Man” was determined to likely be primarily vegetarian, at least in the period closest to his death, based upon hair values (Macko et al. 1999a). Stable isotope dietary studi es have also been perform ed on individuals from other Mesolithic/Neolithic sites (Krigbaum 2003, Richards et al. 2003, Milner et al. 2004), the Bronze Age in Northern Jordan (Al-Shorma n 2004), prehistoric Chile (Macko et al. 1999b), preclassic and historic Mayan Beli ze (White & Schwarcz 1989, Tykot et al.

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37 1996), ancient Egypt (Macko et al. 1999b, Wh ile et al. 1999) and Sudan (White & Schwarcz 1994), prehistoric South Africa (Sealy et al. 1992, Lee-Thorp et al. 1993), and prehistoric Micronesia (Ambrose et al. 1997). In addition, indige nous Easter Islanders (Fogel et al. 1997), additional Native North American groups throughout time (Price et al. 1985, Larsen et al. 1992, Fogel et al. 1997, Hedman et al. 2002, Roy et al. 2005, Yerkes 2005), and colonists from the Ch esapeake area (Ubelaker & Owsley 2003) have also been examined. Weaning practices One significant aspect of di et that has received much recent attention is that of infant feeding. Breastfeeding practices, to includ e weaning, have wide implications for population dynamics in earlier human groups (Mays et al. 2002). Breastfeeding is a major determinant of fecundity and interval be tween births in societies lacking reliable artificial contraceptive measur es, (Vitzthum 1994, as cited in Mays et al. 2002) and thus, can be a major factor in determining lif e histories of certain population groups. Ultimately, the success of infant feeding will have far-reaching impacts in terms of population health and growth, for it is the esse ntial first step for realizing adulthood. Bone chemistry has been critical in this area for archaeologi cal interpretation of remains. In 1989, Fogel et al. published a groundbreaking study comparing the fingernails of mothers and newborns from bi rth through weaning, to determine the utility of using isotopes for such analyses Fetuses and newborns have a 15N roughly equivalent to that of their mothers (He rring et al. 1998, Mays 2000) This makes sense because a fetus receives nutrition through ma terials exchanged across the maternal and fetal circulatory flows in the placenta. Once born and breastfeeding begins, neonates change their trophic stratigraphy. They effec tively become carnivores relative to their

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38 mother (Ambrose 1993). Nursing infants are feeding at one tr ophic level above their lactating mothers and hence, should show an enriched 15N level of +2‰ to +4‰ over their mothers (Fogel et al. 1989, Fogel et al. 1997). This is exactly what Fogel et al. found. During the period of breas tfeeding, they measured infant 15N ratios approximately +3‰ higher than their mothers (Fogel et al. 1989, Fogel et al. 1997). These results were further confirmed by Fuller et al. (2006a), who found infant 15N values enriched by +1.7‰ to +2.8‰ compared to maternal values. As a child is weaned from its mother’s breast, its 15N level will begin to fall back towards a standard adult average, because the shift from milk proteins to proteins obtained from solid foods registers as a decrease in 15N bone collagen values (Wright and Schwarcz 1998, Fuller et al. 2006b). Since Fogel et al. (1989), numerous researchers ha ve applied their findings to various archeological assemblages ranging from mid-Holocene South Africa (Clayton et al. 2006), the Roman period of Egypt (27 BC to AD 395) (Dupras et al. 2001), Mediaeval England (Mays et al. 2002), pre-contact North America (Schurr 1997) to 19th century Ontario (Herring et al. 1998). Wright and Schwarcz ( 1998, 1999) have taken a slightly different slant by including oxygen isotopes in thei r analyses of prehistoric Guatemalans. Their studies are based on the fact that, “H uman breast milk is formed from the body water pool and, thus, is heavier in 18O than the water imbibed by a lactating mother” (Wright & Schwarcz 1998). Infants who onl y breastfeed are enriched in their oxygen ratios compared to their mothers, because of the mothers’ metabolic processing of the water incorporated into breast milk (W right & Schwarcz 1998, Wright & Schwarcz 1999). Additionally, many studies have inco rporated carbon delta values with the standard nitrogen values to determine th e approximate ages of supplementary food

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39 consumption by children in their respective pop ulations, providing further validation of the conclusions drawn from nitrogen data (Wright & Schwarcz 1998, Clayton et al. 2005, Fuller et al. 2006b) Following in the footsteps of Fogel et al. (1989), many more contemporary studies have been carried out in the hopes of appl ying the results to ar chaeological work. O’Connell et al. (2001) compared pairings of hair keratin and bone collagen taken from patients undergoing orthopedic surg ery in the United Kingdom as well as pairings of nail and hair keratin from living subjects to examine the utility of applying similar 13C and 15N results to archaeological work. Lead is otopes in modern people have also been examined to determine comparative lead load s and digenetic effects in prehistoric teeth (Budd et al 1998, Budd et al. 2000), with Budd et al. (2000) concluding that Neolithic human enamel lead values were only an order of magnitude lower than modern juveniles. Region of Origin Paleodiet analyses have also been applie d to detect human mobility since the mid1980s (Sealy & van der Merwe 1985, 1986). Such studies are predicated upon the notion that individuals practicing seasonal migration from coastal to inland areas should have similar 13C values, while those permanently i nhabiting such diverse areas should demonstrate distinct carbon ratios (Sealy & van der Merwe 1985, 1986). In addition to carbon, there are a wide variety of isotopes that can be drawn on to infer information concerning human migration. Schwarcz et al. (1991) were th e first to demonstrate the use of bone phosphate 18O to in attempting to identify the geographical origin of 28 soldiers from the War of 1812, interred in the Snake Hill cemetery, New York. Their findings of uniformity among 18O values indicated the group all spent a major portion of their lives living in the same geographical area. These values differed however from

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40 oxygen isotope analyses performe d on interments in southwestern Ontario and Antietam, Maryland. Dupras and Schwarcz (2001) used o xygen isotopes to distinguish immigrants from native peoples from a third-century cem etery in the Dakhleh Oasis, Egypt, and both oxygen and strontium isotopes have been used to determine the geographic origin of remains found from Viking occupation-era gr aves in Great Britai n (Budd et al. 2004). Strontium isotope ratios have been used extensively in transhumance studies from Neolithic Europe (Grupe 1997, Budd et al. 2000, Bentley et al. 2002, Bentley et al. 2003, Mller et al. 2003, Bentley et al. 2004) as well as Bronze Age and Romano-British sites (Budd et al. 2000a, Montgomery et al. 2005, Fuller et al. 2006b), and prehistoric and historic South Africa (Sealy et al. 1995). Two studies used strontium isotopes to discriminate between immigrants and life-long residents of 14th century Grasshopper Pueblo, Arizona (Price et al. 1994, Beard & Johnson 2000). Beard and Johnson (2000) determined local strontium values by analyzi ng local field mice. I ndividuals outside of this range were deemed immigrants to the area, with those having the greatest 87Sr differences being the most recent additions to that area. ber g et al. (1998) demonstrated that strontium and lead isotopes could definitively distinguish between west coastal and rural inhabitants of Medieval Norway. Th e authors further conc luded that Medieval residents subsisted on local products while contemporary pe ople relied on imported or industrially processed food to a greater degree. Carlson (1996) discovered that lead is otope values corresponding to different sources of anthropogenic and natural lead can indicate cultural affinity among Native Americans and fur traders buried in a 19th century fur trade cemetery. Montgomery et al. (2005) found lead to be a bit ambiguous, with results suggesting that lead isotopes

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41 provide dissimilar types of information depe nding on what era is being examined. In some instances it seemed to serve as a geographical marker, while in others it served better as a cultural indicator. Material Culture Not all archaeological applications of is otopic signatures are anatomically-based. The origins of various forms of material culture have al so been traced using these techniques and well as additional dietary analyses. The earliest attempt to determine provenance through isotope use was attempte d in 1965 by Robert Brill and colleagues on lead and glass artifacts (Brill & Wample r 1967, Herz & Garrison 1998). Not only were lead objects associated with specific mining regions in antiquity, but samples separated by nearly a millennium in time were found to have virtually identical lead isotopic signatures and are believed to have come from the same mine (Brill & Wampler 1967). The source quarries of ancient marb les have been interpreted through 13C and 18O values (Craig & Craig 1972) and today, an extensive database exists for the isotope values of principle classical quarries so marble items can no w often be associated with the areas in which they originated (Herz & Garrison 1998). Oxygen values have traced emerald trade routes from the Gallo-Roman period through the 18th century (Giuliani et al. 2000) and the mining locations of lead artif acts, such as musket balls and coils, found among Omaha Native Americans have been identified (Reinhard & Ghazi 1992). Building materials such as the timbers fo r the prehistoric great houses of Chaco Canyon, New Mexico, have been traced to their individual mount ain growing areas (English et al. 2001). Major cons tituents of prehistoric and hi storic diet have also been accomplished through the analysis of cooki ng residues found on potsherds or within intact kitchenware (Hastorf & De Niro 1985, DeNiro 1987, Hart et al. 2003). When

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42 carbon and nitrogen analyses are combined for proven plant encrustations, they can distinguish among three plant groupings: 1) le gumes; 2) non-leguminous C3 plants; and C4 or CAM vegetation (Hastorf & DeNiro 1985). Forensic Investigations Stable isotope analyses have been applied to a wide variety of contexts within the forensic sciences. Within Europe two majo r organizations have emerged to advance the development and application of isotopic work in this field. The Forensic Isotope Ratio Mass Spectrometry (FIRMS) network and the Natural Isotopes and Trace Elements in Criminalistics and Environmental Forensics (NITECRIME) European Union Thematic Network both aim to raise awaren ess of the benefits of isotope s to forensic investigations, encourage collaboration, and develop and validate new methodologi es (Benson et al. 2006). Stable isotopes have shown great promise as an analytical asset in the war on drugs, specifically in determining the origin of illicit narcotics. The 2H (also denoted as D), 13C, 15N of components extracted from 3,4-me thylenedioxymethylamphetamine or “ecstasy” have shown that individual tablet s can be traced back to a common batch (Carter et al. 2002). Carbon and nitrogen isotopes have been further used to link heroin and cocaine samples to the four major ge ographic regions in wh ich they are grown (Mexico, Southwest Asia, Southeast Asia, a nd South America). Morphine, which is derived from heroin, demonstrated the mo st pronounced regional difference (Ehleringer et al. 1999). Further studies were able to determine the c ountry of origin in 90% of 200 coca-leaf samples, the source material for co caine, as deriving from Bolivia, Columbia, or Peru (Ehleringer et al. 2000).

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43 Isotopic techniques have been used by food a nd spirit regulatory agencies as well to ensure quality control. There is an intern ational concern with not only simple validation of food label claims, but with food adulterati on as well. One applic ation is within the beer industry (Brooks et al. 2002). The prim ary ingredients in beer are water, malted barley, hops, and yeast. All ot her “non-essential” ingredients are called adjuncts. In many nations, the use of unlabelled adjuncts is forbidden by law. Carbon delta values have proven very effective at detecting adjunc ts and testing brewers’ claims as to the purity of their ingredients (Brooks et al. 2002). Additionally, 13C values have proven invaluable in determining whether forms of glycerol are animal or vegetal in origin (Fronza et al. 1998) and 13C and 15N values of eggs have been used to establish whether chickens were given animal or plant protein as feed (Rossmann 2001). Furthermore, oxygen values have been utilized to verify the regional origin of dairy products, especially certain chee ses, which must be produced from milk of a particular region (Rossmann 2001). Food adulteration is of con cern to authorities becaus e it is essentially the misrepresentation of an altered foodstuff as an authentic product. Here, a premium food product is extended or completely replaced with cheaper materials, yet fraudulently sold as a higher-end item (Parker et al. 1998). St able isotope analyses have established themselves as a particularly usefully anal ytical methodology in figh ting this trend. The most advanced applications of stable isotope analyses are within wine quality control, where the European Union has established an official wine stable isotope parameter database (Rossmann 2001). Carbon isot opes can detect the addition of exogenous glycerol deceptively added to wine to disguise poor quality (Calderone et al. 2004). They

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44 have also been used to differentiate between whiskies and assist in authenticating specific whisky products (Parker et al. 1998) and de termine the botanical origin of Brazilian brandies (Pissinatto et al. 1999). Isotopic fractionation of hydrogen and oxygen resulting from juice concentration processes have also been documented and utilized to quantify added sugars in orange and grape juice (Yunianta et al. 1995); while 13C values have been used for over 20 years to control for the authenticity of honey (Rossmann 2001). Stable isotope analysis has also been em ployed by criminal investigators in cases involving the use of firearms (Stupian et al. 2001). Bullet indi vidualization via lead isotope analysis was first re ported in 1975 (Stupian 1975). L ead isotopic information can indicate whether a fatal bullet shared a common origin with a box of ammunition collected from a suspect or provide a detective with a to ol independent of standard ballistic methods to potentially link bullets fr om multiple crime scenes (Stupian et al. 2001). In instances where there is a shoot-out with several types of firearms and/or ammunition, it may even be possible to conc lude which bullet and/or weapon caused a particular gunshot en try (Zeichner et al. 2006). Another forensic isotope breakthrou gh occurred in 1975, when Nissenbaum reported 13C could distinguish between trinitrotoluene (TNT) samples originating from different countries. Other areas of forensic isotope applications include connecting the sources of automobile (Deconinck et al. 2006) and architectural paints (Reidy et al. 2005), packaging tapes (Carter et al. 2004), and glass fragments (Trejos et al. 2003) to crime scenes. Similar measures have been drawn upon to detect environmental toxins in soils, waters, and plants. Isotopes can assist in identifying a geographica l relationship between

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45 a source and a spilled product, whether the c ontamination might be from an oil spill, illegal dumping, pipeline breaks, or leaking storage tanks (Philip et al. 2003). For instance, in the case of a crime scene, such a pplications may be able to link engine oil on the victim of a hit and run with a particular vehicle (Philip et al. 2003). Source identification of environmental perchlorat e contamination has been performed with chlorine and oxygen isotopes (Bhlke et al. 2005). Perchlorate, in even small amounts, can adversely affect thyroid function by inte rfering with iodine uptake (Bhlke et al. 2005), but hopefully, by identifying the source of such chemicals, this form of pollution can be stemmed. These techniques have further been extended to biowarfare defense efforts. Horita and Vass (2003) determined that cultured bacteria ( Bacillus globigii and Erwinia aglomerans ) faithfully inherit the isotopic sign ature of hydrogen, carbon, and nitrogen from the media waters and substrates th ey were grown on, proving “stable-isotope fingerprint” can be created for chemical and biological agents. Because of these properties, Kreuzer-Martin et al. (2003) were able to unde rtake sophisticated tracing studies involving oxygen and hydrog en isotopes. Culture medi a was prepared with water spiked with known isotopic quant ities of hydrogen and oxygen. The 18O and D found within strains of Bacilus subtilis spores grown on this media were then traced back to specific water sources establishing that the origin of microbes can be pinpointed to particular areas based on th e water content of the media on which they are grown. Stable isotopes are also prominent in wild life forensic issues. Several studies have used a trivariate approach, combining car bon, nitrogen, and strontium isotope ratios to create geolocational fingerprin ts for elephant ivory and bone (van der Merwe et al. 1990,

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46 Vogel et al. 1990). It is hoped this will aid in conservation e fforts by assisting in efforts to stem the illegal trade of ivory. Similar goals are also being a pplied to the bounty of information concerning animal migrations (Bowen et al. 2005). While great advances have been made in th e applications of stable isotope analyses to the forensic sciences, human stable isot ope studies in the medico-legal realm are relatively recent phenomena. To date, very fe w studies examining stable isotope ratios as they pertain to region of origin in contem porary human populations have been presented or published. When examining the literature, it appears that the bulk of isotopic research in modern humans is in the form of isot opic tracers for nutriti onal studies (see also Abrams and Wong 2003, Mellon and Sandstrm 1 996). Many, as previously discussed, are also used as proxies for archaeological comparison (Fogel et al. 1989, O’Connell et al. 2001, Fuller et al. 2006a). Several studies have been conducted to i nvestigate lead exposu re and identify the sources of lead absorbed in contemporary, living children by examin ing their deciduous teeth (Alexander and Heaven 1993, Gu lson & Wilson 1994, Gulson 1996) and other tissues and excretions (i.e., blood and urine, Angle et al. 1995). Alexander and Heaven (1993) measured 206Pb/207Pb ratios and lead abundance in teeth finding significant difference among the lead isotope ratios. Wh en compared against various environmental sources of lead, the authors we re able to identify differen ces in sources in northwest England. While these studies were not ut ilized for geoloca tional purposes, they nonetheless could be applied as such, (alt hough anthropological stud ies tend to utilize isotopes compared to 204Pb), and provide a good example of the multiple uses for isotope data.

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47 One weaning study went one step further th an those previously discussed and has exciting forensic potential. Fuller et al. (2006) analyzed bovine milk-based and soybased formulas to determine if unique isotopic signatures exist that could identify infants being fed different forms of supplementati on. The authors purchased seven different formulas sold within California and found that while the 13C values overlapped between formulas derived from cow’s milk and soy, the soy products demonstrated significantly lower 15N values. This again, is a reflection of trophic level effects in nitrogen values. Fraser et al. (2006) have begun compiling a database of modern human hair and nail values examining the stable isotopes of hydrogen, carbon, nitrogen and oxygen. The authors sampled hair and fingernails from 20 individuals living in Belfast, Northern Ireland for a minimum of 6 months as well as an additional 70 individuals from 9 countries representing 4 Europ ean nations, Syria, the United States, Australia, India, and Sudan. They did not report having yet applied the database results to a forensic situation, but preliminary data is at least at the ready should the need arise. Similarly, at the 3rd European Academy of Forens ic Science Meeting in Istanbul, Turkey, Cerling et al. (2003) presented results of a multi-element study of modern human hair. The authors discovered regional differences in the D, 13C, 15N, and 18O values of long-time residents of particular locations and a ppear to still be collecting samples. Beard and Johnson (2000) were the first to demonstrate the utility of strontium isotopes in a human forensic sett ing. In their paper, they determined the region of origin of an illegally harvested deer using the 87Sr/86Sr ratio of antler, then also applied this information in an attempt to differentiate between the teeth of three commingled Americans associated with the Vietnam conflict. They were able to match the natal area

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48 of one individual, but the two others presented overlapping va lues. If the study had also utilized alternative isotope comparisons, perh aps the authors might have been able to discern between the remaining two individuals. Also, preliminary data for a study using str ontium isotope values in an attempt to determine the geolocational fi ngerprints for Mexican-borne individuals residing in the U.S. was presented at the 2005 annual meeti ng of the American Academy of Forensic Sciences (Juarez 2005). Several bay-area de ntal clinics provided the author with 25 permanent 1st molars of individuals originating from four different Mexican states. Samples were accompanied by information as to the subjects’ regions of origin within Mexico, their ages, and sex. Initial results indicate four specific ranges of strontium isotope ratios, one for each of the four states involved in the study. Within-state variation proved too great however, to disc riminate location further. Additionally, a presentati on at the 2001 annual meeting of the American Association of Physical Anth ropologists addressed the use of strontium isotopes and its applications in forensic science (Schutkowsk i et al. 2001). The abstract makes reference to the presentation of a multi-regional sample demonstrating differences in regional and local strontium isotope ratios. Bone and tooth signatures were examined to determine if mismatches of individual values with local isotope ratios demonstrate changes in domicile. The areas of study were likely west ern European, as the authors at the time of publication practiced in the United Kingdom a nd Germany. Unfortunately, it appears this data has yet to be published in a western source. Gulson et al. (1997) detail a pilot study co mparing the lead isotope values in teeth of native Australians to those of Australian migrants from Eastern and Southern Europe

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49 (Table 2-1). While the actual data presented by Gulson et al. are not pa rticularly useful in cases of American service members this pape r does indicate lead is otope ratios have the potential to discriminate region of origin. As can be seen from this short review, isotopic analyses and applications serve a wide variety of functions. The incredible inferential value of isotopic analyses in anthropology is clear. Examples of the power of isotopic studies a bound in the literature and continued advances will only further solidif y how essential their inclusion is within an anthropologist’s analytical toolbox. Table 2-1. Mean and standard deviations for selected groups of immigrant teeth (enamel). Australia (n=29) CIS* (n=14) Yugoslavia (n=13) Lebanon (n=8) Poland (n=6) Mean 206Pb/204Pb 16.56 17.98 18.23 17.62 18.07 SD 0.17 0.06 0.15 0.29 0.20 Mean 207Pb/206Pb 0.9318 0.8664 0.8566 0.8825 0.8617 SD 0.0088 0.0033 0.0063 0.0136 0.0088 Source: Gulson et al. (1997) *CIS denotes the former Soviet Union

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50 CHAPTER 3 HUMAN FORENSIC IDENTIFICATION Assuming that isotopic analys es do prove fruitful for fo rensic practitioners, this technique will be added to a bounty of ava ilable measures for use in the personal identification process. Those specializing in the forensic ar ts acknowledge that there is stratification when it comes to the probative value of identification data. In attempting to tease a name from a body, certain characteristics of the person will be much more unique and individualizing than others. The most powerful measure of identification is a positive identification, the e ssential component of which is the possession by the decedent of unique characteristics (Ubelaker 20 00). Because these characteristics are not replicated in anyone else, they exclude a ll other individuals from consideration. Even with the high resolution of DNA, the method of choice today for positive identifications tends to be dental comparisons (Col. Brion Smith, personal communication). Dental records are still c onsulted when available. The ComputerAssisted Postmortem Identification system (CAPMI) is based on the presence of dental restorations and has increased the e fficiency of matching and comparing antemortem/postmortem records (Friedman et al. 1989), especially in the case of mass fatalities. Dental radiographic matches are much quicker and less costly than DNA evaluations, although the number of individuals with no dental anomalies (Friedman et al. 1989, Col. Brion Smith, personal communication) is rising due to advances in dental hygiene and medicine and mass fluorination of community water sources. In those cases

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51 where the skin of the fingers is still intact, fingerprints may establish a positive identification as well. When these measures prove inconclusive, genetic fingerprinting utilizing nuclear and mitochondrial DNA is another option. W ith the development of the polymerase chain reaction procedure, which enabled rapi d amplification of genetic material, and lowered costs, DNA analysis is much more pr actical (Herrero 2003) th an in days past. Nuclear DNA is known to be a unique identifier (unless the subjects are identical twins). Many investigators, including the Department of Defense (DoD), test 16 bands from the available microsatellite loci pool (Col. Br ion Smith, personal communication). From experimental observations, the average odds that one band will be shared by any two unrelated individuals is approximately 0.25 (S udbery 2002). So the resolution of a 16band testing procedure is 0.2516 = 2.33 x 10-10; that is, there is a 0.000000000233 chance that two unrelated individuals will share all 16 bands tested. Put another way, if you take the reciprocal of this figure you see that th ere is a 1 in nearly 4.3 trillion chance that someone unrelated has the same DNA profile. Since this number is considerably larger than the world’s population, nuclear DNA te sting is said to provide for unique identifications. This calculation is made with the assumpti ons that all individuals are unrelated and that the chance that bands will be shared is the same for all people. In truth, people are related and ethnic affinities may lead to higher rates of band sharing than among the general world populace. Even so, after acc ounting for such complications, nuclear DNA analyses are still considered positive and unambiguous identification (Sudbery 2002).

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52 The resolution of mtDNA, on the other hand, is not as fine. Because mtDNA is passed through maternal lineages only, recombination does not occur. Mutations aside, this accounts for the integrity of mtDNA as it is passed from mother to child. This constancy of code allows for familial tracing by comparing sequences of certain base pair lengths among those who are maternally related. This is a very powerful tool indeed, and allows for a distinctive disc riminating function from nucl ear DNA. The downside to it though is that is cannot disti nguish among relatives and can be preserved for generations, leading to populations of people with the same or similar mtDNA profile (Col. Brion Smith, personal communication). Many also consider various forms of radiographic comparison to equate to positive identification. This is especially true with the frontal sinus. The sinus becomes radiographically visible between 7 and 9 year s of age, and barring trauma or disease, remains relatively unchanged throughout lif e (Ubelaker 1999). In a comparison completed by Ubelaker (1999), the author noted than in a radiographic comparison of 35 radiographs (595 comparisons), no two frontal sinuses were alike. The number of differences between individuals average to a pproximately 8, with a range of 3 to 15. If additional antemortem radiographs exist doc umenting unique skeletal anomalies (i.e., pathology or trauma), these characteristics ma y also serve as a basis for positive ID. One further skeletal anomaly for considerati on is that of prosth etic devices (Burns 1999). While it may not be unique that an indi vidual has a total knee replacement, what will be unique is the serial number that is imprinted upon the prosthetic device along with the manufacturer’s emblem. Hospitals must doc ument these serial numbers. With a little detective work, the serial or lot numbers can be traced back to th e manufacturer who in

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53 turn, can direct an investigator to the hos pital to which the device was sold (Warren 2003). Some also consider the comparison of still photos and the skull at the same angle, or what is known as video superimposition, to be conclusive as well (Ubelaker 1999). Hope for a positive ID can often prove fr ustrating and futile though, when there are no reference samples on file for that individua l. An individual must have antemortem information available to compare against if a pos itive identification is to be achieved. So, for instance, while DNA may have successfully been extracted from a set of remains, an identification cannot be accomplished when there is no nuclear DNA on file or source material available and no relatives of mate rnal lineage for the decedent can be located. One step below a positive ID is exclusi onary evidence for identification. When remains are presented for identification, they wi ll arrive from one of two environs, either an open environment, or one which is clos ed (Warren 2003). Open environments are those in which the person laying before you could be anyone in the world who was up until recently, alive. For example, a body f ound in the woods could be an indigent, a local, or a tourist from another country. In th e case of a light airc raft crash however, the potential for identification is much higher. If a passenger manifest was filed listing two adult males and child of 12, and assuming it was correct, then there is the potential for an exclusionary identification. The child will be easy to distinguish from the adults due to developmental differences in the skeleton. If one of the adults is identified via antemortem radiographs and the other has no antemortem comparison data, then the latter would usually be identified by exclusionary methods, since ideally, in a closed system there is no one else it could be.

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54 Burns (1999) also lists iden tification by means of a pr eponderance of evidence. This is often linked with tentative identifica tions, or what are also known as presumptive identifications. There is much greater unc ertainty with presumptive identifications because they are based on evidence found a ssociated with the body, such as personal effects, and/or verbal te stimony of witnesses, last known whereabouts of the body, and familial recollections of undocumented condition s the individual may have suffered from (Burns 1999). See Table 3-1 for a recap of identification measures. Table 3-1. Forms of forensic identification. Type of I.D. Basis for I.D. Tentative identification Clothing Possessions Location of body Verbal testimony Identification by preponderance of evidence Anomalies known by family or friends, but without the existence of written records Identification by exclusion “Everyone else is identifie d and there is no evidence that this is not the only person still missing.” Positive identification Dental identification Radiographic identification Mummified fingerprints Prosthetic identification DNA analysis Unique skeletal anomalies Reproduced from Burns (1999) Military Identification Measures “Over the past 200 years, the United St ates has set the standard for the identification and return of its servicemem bers [sic] to their families” (AFIP 2004). Since as early as the American Revolution, e fforts have been made to recover, identify, and provide individual burial for American military personnel (AFIP 2004). As the years have progressed, standards and expectations for identification have increased and the technology with which to do it ha s made sweeping advances.

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55 The United States DoD employs all of the standard personnel identification measures previously mentioned. What is unique about the mili tary as a population however, is that their physic al attributes and markers ar e much better documented than the general populace (i.e., they have much better antemortem records). Members have fingerprints on file and flight crews have footprints docum ented as well. Meticulous medical and dental records ar e fairly centralized. With few exceptions, blood cards are on file for all current total fo rce members in the case their DNA needs to be sequenced. Individuating marks are noted such as scars, large birthmarks and moles, and tattoos as well as information such as hair and eye color, race, stature, weight, and age. Even so, such measures are not without their complications. Dental radiographs are commonly not available of military members unaccounted for from previous conflicts, especially World Wa r II and the Korean War (Ada ms 2003a). The Office of the Armed Forces Medical Examiner notes that greater than 5% of all service members have no dental restorations, the primary mean s for dental identifications, and the number is rising (AFIP 2004). In a study of 7030 living U.S. soldiers, it was revealed that 9% had a full complement of unres tored teeth (Friedman et al. 1989). In a pooled data set of over 29,000 individuals from the Third nati onal Health and Nutrition Examination Survey (NHANES III) and the Tri-Service Comprehensive Oral Health Survey (TSCOSO), Bradley Adams found that 12.77% had “perfect teet h” (2003b). Not only has the number of dental restorations declined in younger individuals, but the complexity of them has decreased as well (Friedman et al. 1989). Historically, only 70% of service personnel actually have their fingerprints on file with the Federal Bureau of Investigation w ith a further 15–30% of the fingerprint cards

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56 submitted by the services rejected as “unc lassifiable” (AFIP 2004). These numbers will likely be reduced significantly though, with the wide-sprea d implementation of digital fingerprinting DoD-wide, which instantly scans recorded images for acceptability immediately after each individua l print is taken. Additionall y, radiographic analyses may not be possible on highly fragmented remain s (AFIP 2004). Identifications made based on material evidence associated with rema ins can be very problematic as well. Traditional items such as dog tags are not n ecessarily accurate either. As an example, during current operations in Iraq and Afghanist an dog tags have been known to have been blown off one individual and burned into the chest of another (Dr William Rodriguez, personal communication). On the leading edge of identification efforts for the U.S. government is the Armed Forces DNA Identification Laboratory (AFDIL). AFDIL is the focal point for the DoD in all matters concerning DNA identificati on efforts for military personnel and special federal government projects. In addition to performing laboratory testing, AFDIL manages the DoD DNA Registry. This func tion is responsible for maintaining blood cards for DNA testing as well as providing admini strative oversight of the database of all sequenced data. Besides the Registry, AFDIL is also responsible for the DNA Repository, which administers the AFDIL Fa mily Reference Specimen database for mtDNA matching when nuclear DNA is unavailable (AFIP 2004). It is a common misconception that the m ilitary maintains DNA profiles on all its personnel. It does not. In stead, AFDIL houses nearly 4.5 million blood stain cards for active duty, reserve, guard component, re tired military members and additional specialized government personnel (Col. Brion Smith, personal communication).

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57 According to Colonel Brion Smith, Chief Deputy Medical Examiner for the Forensic DNA Division, Office of the Armed Forces Medical Examiner, there are two basic reasonings behind the logic of this The first is that it is cost prohibitive to perform DNA analyses for every member of the armed for ces. It is much less expensive to house blood stain cards and generate the same informati on on an “as needed” basis. The second is that storing the profiles of all who serve pres ents an ethical dilemma, especially when it comes to who should be permitted access to th e information and for what purposes. This is further complicated by th e fact that medical and dent al records, to included DNA information and blood cards, stay on file fo r 50 years after the service member retires (Col. Brion Smith, person al communication). An additional benefit of this system is it gives examiners the option a posteriori to decide which test is best suited based on the conditions of the remains. If the body is in good condition, nuclear DNA would be the prefe rred method. If the remains are charred and disassociated, mtDNA might be most appropriate. Furthermore, a fully utilized blood card can provide 30–40 punches, allowi ng less common tests such as Y short tandem repeats to be completed (Col. Brion Smith, personal communication) or providing the opportunity for future testing utilizing methods that have yet to be developed or hit the mainstream. The beauty of this practice then is that technicians are not restricted to only performing a form of an alysis that matches the information present in a data base so there is greater flexibility in analyses and hopefully the best method for the materials available. The utilization of both nuclear and mitochondrial DNA is dependent upon the situation and essential to m ilitary identification. All individuals who die in current

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58 combat, training, or in otherwise duty-relate d capacities are sampled for DNA analyses upon intake to the Dover Air Force Base Po rt Mortuary (Col. Brion Smith, personal communication), the DoD central receiving and processing center for all military deceased. Even when other conventional methods of positive identification are available such as radiographic dental comparisons, a DNA fingerprint will be generated. This will delay returning a casualty to their families unles s identity is questionable, but instead, is performed to prevent questions su rfacing at a later date as to correct identification and to reassure family members that the body being returned to them is kin. (Col. Brion Smith, personal communication). Present Study This project was established to test the utility of stable isotope analyses for identification of region of origin for moder n, unidentified, human skel etal material that has poorly-documented or unknow n provenience. Initial efforts have focused on the approximately 1,800 service members who remain unaccounted for from the Vietnam conflict. It is hoped however, that this inform ation will eventually be refined to use in the identification of all those who remain unaccounted for and for those potentially recoverable from previous conflicts (Table 3-2). Often, the true national origin of rema ins recovered by the Joint Prisoner of War/Missing in Action Accounti ng Command (JPAC) is uncerta in. In addition, it is not uncommon for de-contextualized, poorly preser ved and/or highly fragmented remains to be unilaterally turned over to the Central Identification Laboratory (CIL) by a foreign agency. CIL personnel attempt to determ ine whether remains are U.S. service

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59 Table 3-2. Numbers of unaccounted for U.S. prisoners of war and/or those missing in action. Conflict Number Unaccounted For World War II 78,000 (35,000 considered recoverable) Korean Conflict 8,100 Vietnam War 1,800 Cold War 120 First Gulf War 1 Source: JPAC (2006) personnel through a variety of means. The id entification of unknown remains believed to be missing U.S. service personnel is frequen tly hampered by high levels of degradation and fragmentation as a result of circumst ances of loss and subsequent taphonomic regimes. These effects often combine to prevent effective DNA sa mpling strategies. Teeth often prove excellent at distinguishing among the populat ions in questions. U.S. military personnel had access to regular dental ca re. In countries such as Vietnam, this was not the case for the majority of the popul ation. In addition to untreated dental insults, the occlusal surfaces of the molars and other teeth are frequently worn down from the grit present in native diet s exposing the underlying dentin (Mark Gleisner, personal communication). The teeth then of modern Vietnamese often present similarly to historical/prehistorical Native Americans. Every effort is also made to extract DNA from a set of remains, although such efforts are of ten unsuccessful because of the poor state of preservation. Additionally, the number of U.S. casualtie s during the Vietnam conflict of Asian ancestry was relatively small. In 1985, th e DoD reported the number of “Mongolian” fatalities in Southeast Asia occurring from the period of 1 January 1961 to 30 April 1975 or as a result of injuries sustained in op erations during said period was 114 or 0.002%.

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60 Those listed of “Malayan” an cestry who died under the same circumstances was 253 or 0.004% (Reports 1985). See Table 3-3 for a complete listing of casualties by race. More importantly for this study, only 5 servicemen of Asian ancestry remain unaccounted for out of 1,760 total (JPAC 2006) The complete racial breakdown for service members still listed as missing in Southeast Asia can be found in Table 3-4. Besides military members, 32 American civili ans are also listed as missing in Southeast Asia. The racial backgrounds of these individua ls were unavailable, but it is interesting to note that two missing civilians are female All of the military members unaccounted for are male. Because of the very low likelihood of a U.S. service member being a female or of Asian ancestry, biological profiles can be useful in excluding individuals from consideration. This is assuming enough of th e skeleton remains to create a biological profile. When the biological information is combined with documented information Table 3-3. United States casualt ies in Southeast Asia by race. Race (reported by DoD) Total U.S. Casualties “Caucasian” 49,951 “Black” 7,257 “Mongolian” 114 “American Indian” 226 “Malayan” 253 “Other/Unknown” 221 Total 58,022 Source: Reports (1985) Table 3-4. United States military listed as unaccounted for in Southeast Asia by race. Race (reported by JPAC) Total U.S. Military Missing “White” 1,653 “Black” 92 “Asian/Pacific Islander” 5 “American Indian/Alaska Native” 2 “Other” 8 Total 1,760 Source: JPAC (2006)

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61 concerning troop engagement and staging areas and locations of downed aircraft, remains may be returned to the originating nation if the evidence points overwhelmingly to the fact that the remains are not of an America n. Unfortunately, such an assessment is an extremely complicated venture and in a great many cases it is simply impossible to make such a distinction. This project was initiated in the hopes that the results will assist in resolving this dilemma. A two-pronged approach for this study has been utilized based on the operating hypotheses that: 1) discernable differences exist between the isotopic ratios incorporated into American and Southeast As ian tooth enamel and that these differences can be used to determine regi on of origin; and 2) regional di fferences in natal isotopic signatures are also discerna ble within populati ons raised within the U.S. Because of the paucity of data in contemporary studies, it is near impossible to predict the likelihood of the ab ility of this study to disti nguish natal Vietnamese from American-born individuals. It is encouraging that Juar ez (2005) found significant variation among the strontium isotope valu es for Mexican-born peoples from four different states, even with her limited samp le. If historical, hu man, migratory studies (Montgomery et al. 2005, Mller et al. 2003, Montgom ery et al. 2000, Dupras & Schwarcz 2001, berg et al. 1998) are any indicator though, there is a high probability that the chosen stable isotopes will be able to discriminate between these two populations. The reasoning behind this is that the geochemical properties of different continental systems should vary significantly and this difference will be further magnified by the fairly culturally distinct dietary practices of the two populations.

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62 None of the studies mentioned in Chapte r 2 examined stable isotope use in a forensic context in any great depth. The largest sample size was Gulson et al. (1997) with 68, but it was a combined pool of perman ent and deciduous teeth. This study will utilize approximately 300–600 total samples and thus will have greater power. Furthermore, all studies make mention of overlapping isotopic values which makes discrimination virtually impossible. It is hoped this tendency will be reduced by introducing multi-element analyses to forensic work. Theoretically, a multivariate approach should allow finer resolution, esp ecially since the deposition of the elements depends largely on very differe nt factors: carbon isotope rati os are based on cultural food preferences; oxygen on meteoric water, altitude and distance from major bodies of water; and strontium and lead reflect th e underlying bedrock and soil. Carbon isotope ratios reflect the photosynt hetic pathways of ingested plants and echo cultural food preferences. It is expect ed that individuals w ho have subsisted on a traditional, rice-based (C3 plant) Southeast Asian diet wi ll differ significantly in their carbon isotope signature from individuals who have subsisted on a heavier corn-and sugar-based (C4 plants) American diet. Wild rice in the U.S. has produced results ranging from -26.3‰ to -29.7‰ (Hart et al. 2 003) and purified rice starch has been averaged to -26.6‰ (Ambrose & Norr 1993). Th is contrasts markedly to maize (corn) values varying between -14.0‰ (van der Merwe 1982) and -11.84‰ (Hart et al. 2003) and purified cane sugar at -11.2‰ (Ambrose et al. 1997). American s also eat a large variety of wheat products. Wheat is a C3 plant, but it is enriched compared to rice, with bread wheat leaves measuring -23.7‰ (van der Merwe 1982 ). It stands to reason then that those relying on a rice-ba sed diet, such as the Viet namese, would exhibit more

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63 negative carbon isotope values than their Amer ican counterparts, whose corn and sugar constituents of the diet, w ill shift the carbon isotope values in a less negative direction. Due to the fractionation effects highlighted in Chapter 1, one must keep in mind that the reported values will not trend directly with plant values. Mammal hydroxyapatite will demonstrate an enrichment of +9.6% to +13% (DeNiro & Epstein 1978b, Lee-Thorp et al. 1989) over plant material. Mixtures of the dietary plant constituent will also affect an organism’s overall 13C value as well as dependency on marine food resources (Schoeninger & DeNiro 1984). Since the majority of state borders within the continental U.S. are not based on geomorphologic formations, it is unlikely th at regional identification will be as straightforward. This should be partially am eliorated through a multi-signature approach. Due to the novelty of this approach, it is di fficult to say with a ny certainty how precise regional identification of geopolitical origin will become. Based on the limited success of Beard and Johnson (2000) however, it appear s natal origin within the U.S. can be narrowed down to a regional level ba sed on major geological formations. Because 13C values represent dietary intake, they will not indicate regional origins, since modern diets are primarily culturally ba sed. The stable is otope ratios for oxygen, strontium, and lead on the other hand, are aptly suited for this task. It is assumed that individuals from Alaska, Hawaii, and the Amer ican territories will be identifiable. The geographical distances between these areas and the continental United States (CONUS) are vast, with a variety of different, but in terrelated, environmenta l factors influencing oxygen isotope distribution such as latitude, temperature, altitude, coastal affinity,

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64 precipitation patterns, and humidity (I acumin 1996, Hertz & Garrison 1998, Kendall & Coplen 2001). The geologic history of the major land masse s nearly represents the 4.5 billion-year history of the earth (Beard & Johnson 2000). Because of this, there are large differences in the isotope compositions of different parts of the planet. relative to the analytical error of the 87Sr/86Sr measurements (+ 0.00001 to +0.00003). Within the U.S., the ages of crust varies from under 1 million years old in Hawaii to nearly 4 billion years old in areas of Michigan and Minnesota (Beard & J ohnson 2000). This age effect produces significant variations in the strontium isotope composition within different regions of the U.S. and is the basis for analytical technique s attempting to discern region of origin in different peoples. Another strengt h of strontium is that its is otopes are thought to be little influenced by fractionation (Toots & V oorhies 1965, Ambrose 1993, Carlson 1996, Hertz & Garrison 1998, Beard & Johnson 2000, Budd et al 2000) thus the isotope ratio remains constant from soil to top carnivore as you m ove through the ecosystem. Soil samples can then be checked against values to determine the geolocational origin s of a tissue sample. It is difficult to speculate whether str ontium isotope analyses can identify natal geolocation to the regional le vel in contemporary peoples. Th e analyses may seem fairly straight forward on the surface, but there are underlying fact ors for modern man that may inhibit its deductive power. Of primary conc ern is homogenization of strontium values due to the global food trade. An array of geological process are also re sponsible for the formation of these areas, hence the bedrock composition is quite vari ed. Discerning among individuals reared within the CONUS will likely prove more diffi cult, and overlapping values are expected.

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65 By using the three different geologically-bas ed isotopes in concer t however, it is hoped that general patterns will emerge. In isolation, isotope delta values have lim ited evidentiary value and will rarely lead to any form of identification. The same could be said of other bases of identification. Clothing alone will not lead to a presumptiv e identification. Someone has to recognize the clothing as belonging to the decedent before it has any realized significance. If the geo-political region of origin fo r a set of remains could be ascertained however, it would provide a direction in which to concentrate iden tification efforts. In mass disasters and in closed environments, isotopes could be co mbined with other methods, leading to exclusionary identifications or directing where to focus further analyses for potential positive IDs. Such techniques are relatively inexpensive and quick. Isotopic analyses can be performed for under $100 at the University of Florida and in the case of enamel, can be completed in roughly 1 week’s time. If stable isotope analyses are performed at the onset of the identificati on process, it could save count less man-hours and dollars for the military, preventing unnecessary analytical efforts if the remains are not deemed American.

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66 CHAPTER 4 MATERIALS AND METHODS This study is groundbreaking in that it is th e first of its kind to compile a reference sample of isotopic values associated with know n natal regions to be utilized in forensic work. More importantly, the information gl eaned from this study will be applied in support of the Joint POW/MIA Accounting Co mmand’s mission to achieve the fullest possible accounting of all Ameri cans missing as a result of our nation’s past conflicts. A two-pronged approach for this project was utilized based on the operating hypotheses that: 1) discernable differences exist between the isotopic ratios incorporated into American and Southeast Asian tooth enamel and that these differences can be used to determine region of origin; and 2) regional di fferences in natal isotopic signatures are also discernable within populations raised within the U.S. Teeth were utilized for this project becau se they are much more robust than bone and little affected by diagenetic processes. This reduces the sample preparation time by several days to a week. By only examining th e enamel, isotopic values can be studies for a known period of the subject’s life, because in and outflow of materials in enamel cease at the termination of amelogenesis (Hillson 1996). It is also much easier to obtain modern teeth than modern bone for sampling. When teeth are extracted, the standard protocol is to dispose of them as biomedical waste, so there is littl e objection to obtaining them for study. It is much more difficult to acquire samples of contemporary bone for legal and cultural reasons. Obj ection is further fueled by the f act that isotopic sampling is a destructive process.

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67 Teeth are genetically conser vative displaying little va riation in the period of mineralization of the tooth, although females are slightly precoci ous (Fanning & Brown 1971, Anderson et al. 1976, Hillson 1996), with Garn et al. reporting that females were in advance of males by an average of 3% ( 1958). Different ethni c groups have shown slightly different timing patterns as well, but all differences whether sex-related or ethnic, equate to not much more than a few months between groups (Hillson 1996). This fact should not impact this study however, as all cr own mineralization is completed prior to individuals being eligible for military service. All teeth supplied for this study had fully completed amelogenesis. Materials utilized in this study were supplied by three different institutions. The Joint POW/MIA Accounting Command’s Cent ral Identification Laboratory (CIL), Hickam Air Force Base, HI, permitted access to their “Mongoloid hold” collection for the creation of an East Asian reference sample The “Mongoloid hold” collection contains remains of individuals recovered from East As ia or unilaterally turn ed over to the CIL, whose governments have refused repatriation, once the remains were determined not to belong to U.S. service personnel. Donated contemporary teeth and surveys completed by their donors were also provided by the 10th Dental Squadron, Unite d States Air Force Academy (USAFA), Colorado Springs, CO and the Malcolm Randall Veterans Affairs Medical Center (North Florid a/South Georgia Veterans Health System) Dental Clinic, henceforth referred to as the “VA,” Gainesville, FL. Dental Protocols Prior to utilizing live subjects in this research, all appropriate permissions were obtained and training complete d (Appendix A). The USAFA Institutional Review Board (IRB) granted IRB exempt stat us to this project (HQ US AFA IRB FAC2005026H). Prior

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68 to conducting this study with the VA, the pr otocol was approved by the University of Florida’s Health Center Institutional Re view Board (IRB-01 approval #474-2005), and both the VA’s Sub-Committee for Clinical Inve stigation and Research and Development Committee. Additionally, a research template had to be created for incorporation into each study participant’s electr onic medical record, via the VA’s Computerized Patient Record System (CPRS). To further assist all partie s engaged in this researc h, information binders were distributed to both dental f acilities. These packets incl uded copies of all IRB and committee approval letters, dental staff instructions, a subject identifier log, copies of all required forms, a blank and completed, example survey, background information related to this specific research project, pre-paid FedEx shipping forms (for USAFA), and a CD with all electronic media on it (see Appendix A for a reproduction of the VA binder). This project was essentially a piggy-back study attached to the normal patient dental care of those indivi duals who are selected by USAFA and the VA for tooth extraction(s) for valid medical reasons. Th e study, in and of itself, had no bearing on whether an individual was select ed for dental extraction(s). All patients scheduled for dental extraction(s) during the study period were queried as to their willingness to participate in the study. Complete inclusion of all consenting subjects cut down on bias that would be introduced with nonrandom, arb itrary sampling by the dental staff. Dental administration personnel proctored all form s. Upon receipt from the patient, they reviewed the forms for completeness and verifi ed birth date and sex with the subject’s dental records.

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69 Patients, to include Air Force Academy cadets, active-duty military, and military retirees and/or veterans, alre ady identified for tooth extraction for oral health reasons, were asked to participate in a brief survey (Figure 4-1) and donate their extracted teeth for analysis. The survey and, in the case of the VA subjects, associated combined Health Insurance Portability and Acc ountability Act/informed consent form (Appendix A) were administered upon initial intake while the patient filled out requisite preoperative paperwork. This paperwork was in additi on to the normal documentation required for dental procedures. The HIPAA form was compulsory to prot ect participant health information. Researchers must obtain patient authorizati on before they are allowed to disclose protected health information. It was required in this instance because we were requesting information such as location of residence and birth date, which cannot be ascertained from observation alone. The informed cons ent form was required to secure subject participation in the study. The form detail ed the background, procedures, benefits and risks of the research project, and obtained witnessed, signed c onsent of the individual that they knowingly and voluntarily participated in the study. Dental staff were available to answer any questions and an example of a completed questionnair e and study background information (Appendix A) was made availabl e. Survey completion and tooth donation were the only requirements of subjects. The data acquired from each subject included: Date of birth Sex Race Tobacco product use Childhood diet

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70 Location of residence, birth to age 18 Date of prior dental extraction at each facility (if applicable) Figure 4-1. Joint POW/MIA Accounting Command survey.

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71 Figure 4-1. Continued.

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72 The aim of the survey was to control for as many sources of error or variation as possible in the data as well as create an is otopic mapping capability for natal region. All questions, with the exception of the prior dent al surgery question, were pertinent to the study in that they account for factors that ma y possibly lead to differential maturation in teeth or absorption/deposition of the various isotopes being studied. The first item, date of birth, allowed for temporal comparison of specimens between JPAC and VA samples versus USAFA sa mples. While dental development is relatively genetically conservati ve, there is some minor vari ation in dental development rates between sexes and major ethnic groupings. This information; date of birth, sex, and self-perceived race; served as potential blocking factors during data analysis. The effects tobacco use upon isotope analyses for teeth, have thus far not been addressed in the literature. While enamel is otopic fates are locked in after amelogenesis terminates, it is unknown whether tobacco use may trigger diagenetic changes within teeth that may affect isotope values. It is commonly known to stain teeth, and may need to be accounted for in preparation protocol s and in interpretation of results. Dates and locations of childhood residence were critical for making sense of the oxygen, strontium, and lead stable isotope results. The validit y of the residence information was confirmed by individuals vi sually approximating these areas on a map. It was also useful if someone could not reme mber the exact name of a town/city in which they lived but did know approximately where it was in relation to neighboring areas. The other questions served to control for potentially confounding variables. Dental extraction history was necessary to prev ent counting one individual who underwent

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73 multiple extractions over multiple days as more than one subject. Survey questions were limited to one page with the map encompassing a second page. Pertinent information corresponding to each patient was also recorded by the dental staff on each survey. Here, each facility assi gned a unique subject identifier number to each patient (i.e., VA-001). Additionally, the po sition in the arcade that each tooth came from (tooth number) was noted according to the Universal/National System dental numbering scheme for permanent dentition as well as the date of extraction. Teeth were extracted following standard de ntal protocols for each facility. Care was taken to preserve as much of the crown as possible. Each tooth was placed into its own vial, which was labeled with the subj ect identifier number and tooth number, All vials from a particular individual were then placed in a resealable bag and the bag stapled to the associated survey. The surveys and teeth from USAFA were shipped via FedEx to the C.A. Pound Human Identification Laborator y (CAPHIL). Surveys and teeth from the VA were picked up weekly and CPRS updated by the author. Teeth provided by both facilities were not stored in any solution or fixative. Sampling Teeth were selected for sampling using th e following hierarchy. Those teeth whose cessation of amelogenesis was most similarly ti med with the third molars were preferred, with other teeth chosen on a decreasing s liding scale (Table 4-1). The younger in an individual’s life crown comple tion occurred for a specific tooth, the less desirable the tooth was for sampling. Additionally, molars we re preferable as they have the largest surface area available for enamel removal. As a matter of course, mandibular teeth were chosen over maxillary teeth and right over left. Furthermore, for the East Asian reference samples from the CIL, teeth still present in the alveoli were selected over loose teeth,

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74 Table 4-1. Crown form ation/tooth eruption. Tooth Crown Initiation1 (upper/lower) in yrs Crown Completion2 (upper/lower) in yrs Tooth Eruption3 (upper/lower) in yrs 3rd molar 7.0–10.0/7.0–10.0 13.3/13.3 17–21/17–21 2nd molar 2.5–3.0/2.5–3.0 6.7/6.7 12–13/11–13 2nd premolar 2.0–2.5/2.0–2.5 6.3/6.3 10–12/11–12 1st premolar 1.5–2.0/1.5–2.0 5.8/5.6 10–11/10–12 Canine 0.3–0.4/0.3–0.4 4.9/4.8 11–12/9–10 2nd incisor 0.8–1.0/0.25–0.3 4.0/4.0 8–9/7–8 1st incisor 0.25–0.3/0.25–0.3 3.7/3.6 7–8/6–7 1st molar 0.0/0.0 3.8/3.7 6–7/6–7 1 range in Shour & Massler (1940) 2 mean values in Anderson et al. (1976) 3 range in ADA (1999) since the actual tooth number could be veri fied more easily with the presence of the associated bone. In nearly all CIL cases how ever, a full arcade was not present to choose from, thus the best option according to the aforementioned sampling scheme was selected based on the resources available. Each individual was assigned a unique identi fier, not each tooth. Therefore, if two teeth were utilized from the same indivi dual, the samples would be given the same identifier, with an additional tooth number id entifier. This numbering scheme prevented inflation of actual individual numbers. Furthe rmore, due to potential intertooth variation in stable isotope values, enamel was not co mbined from multiple teeth to achieve the desired weight of enamel powder. Central Identification Laboratory The CIL is an American Society of Cr ime Laboratory Directors (ASCLD)-certified crime lab. As a result, all sampling conducte d at the CIL conformed to their standard operating procedures, to ensure complianc e with ASCLD requirements. Prior to

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75 sampling, potential specimens were research ed utilizing a list of “Mongoloid holds” provided by Dr. Andrew Tyrrell, the Casualty Automated Recovery and Identification System (CARIS) database, and a thorough person al investigation of the entire evidence storage area. Once suitable specimens had b een identified, individual accessions were checked out from the evidence manager and tr ansferred to the CIL autopsy suite for the actual sampling. Study identifiers with a “CIL” prefix and a three-digit suffix were associated with lab accession numbers from the CIL Mongoloid hold collection. Two teeth, if available, were selected from each accession following the above procedures. In all cases, at least one intact and undisturbed t ooth was left with the case in the event that future identification efforts, such as DNA sequencing, were required. Each tooth selected for sampling was assigned an additional sample number (01A or 02A ) mirroring the lab’s DNA sampling procedures. Information cards for each tooth were created for photo cataloging and provided the followi ng information (Figure 4-2): CIL accession number Individual designator (if applicable) Tooth number Sample identifier Date “Isotope study” Subject identifier number Researcher’s initials From 14 June 2005 to 06 July 2005, a total of 112 teeth were sampled from 61 individuals believed to have or iginated from or been recove red from the following areas: Vietnam (48 individuals); Cam bodia (4 individuals); Laos (3 individuals); the Korean

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76 Figure 4-2. Pre-drilling phot o of CIL-033 #19 with data card. Note: the accession number is purposely, partially obscured. peninsula (3 individuals); the Solomon Isla nds (2 individuals); and the Philippines (1 individual). Teeth were eased out of their respective alveo li by hand or drilled out, when necessary, using an NSK UM50 TM slow-speed de ntal drill with either a #2 or #4 carbide dental drill bur, taking care to minimize damage to each alveolus. A photo, to include an information card, reference scale (ruler), a nd empty collection vial was taken of each tooth to document what each element looked lik e prior to drilling (Figure 4-2). Separate photos of the buccal or lingual and occlusal surfaces were taken. (In cases where the teeth had to be drilled out of their respective alveoli, pictur es of the unaltered arcades or portions thereof were taken us ing the same format.) Each tooth was then placed into a vial of 3%, household-use hydrogen peroxide and cleaned via a Branson Bransonic 2510 ta bletop ultrasonic cleaner for 30 minutes. When finished, teeth were removed from the solution and manually cleaned with a toothbrush. The enamel surface of the teet h was prepared for drilling by cleaning off excess calculus, soil, and/or staining using the same appara tus and a #8 carbide dental drill bur.

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77 Samples of approximately 100 mg of pristine enamel were drilled off of each tooth using the same set-up (see Appendix B for drilling data). Care was taken not to drill into the dentine. Enamel powder was collected on creased weighing paper and transferred to labeled 1.5 mL microcentrifuge tubes. The dr illed tooth, collection vi al, scale (ruler), and information card were again photographed to document the end-stage condition of the tooth and for chain of custody purposes. The t eeth were then returned to their original storage bag along with the information cards with the associated elements for that particular accession number. The bag was re sealed with evidence tape, and the tape initialed and dated on both sides. The rema ins were then turned in to the evidence manager. Drill burs and weighing paper we re discarded after each use and the drill cleaned of adherent enamel powder. Chain of custody forms were completed for all specimens, transferring possession of the enamel powder and any associated enam el chips to the author (Appendix B). The microcentrifuge tubes containing the enamel specimens were then transported from CIL to CAPHIL through the services of FedEx. The author also attempted to gain access to human teeth from native populations while performing duty-related activities in Vietnam from July and into August 2005. Such efforts were abandoned however, when pr ovincial officials stat ed that regional and higher government officials woul d be required to approve an y request to procure human samples. United States Air Force Academy and Veterans Affairs The Air Force Academy collected survey s and a total of 948 teeth from 274 individuals between la te August 2005 and late April 2006 (Table 4-2; see also Appendix C for a list of survey results). Of these, one third molar was selected from each

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78 Table 4-2. Isotope sampling matrix. # # Total # Individuals Run To tal # Teeth Run Total # Runs Source Inds Teeth C O Sr Pb C O Sr Pb C O Sr Pb CIL 61 112 61 61 36 36 64 64 3636 65 65 3642 USAFA 274 948 230 230 36 36 2382383636 279 279 3636 Total 335 1060 291 291 72 72 302 302 72 72 344 344 72 78 of 228 individuals for inclusion in the primary study. One tooth each from two individuals of unknown natal region were uti lized for additional testing examining the necessity of using acetic acid to process th e enamel. Samples originating from three different individuals were not used because of experimenter error in labeling the samples and erroneous or missing information provided by the subject. Furthermore, after sample AFA-185 from USAFA, samples were selectivel y chosen to fill in the geographic gaps until optimally, each state had a minimum of fi ve individuals represented. This approach was chosen to reduce costs. Additionally, individuals from duplic ate cities or those people born prior to 1980 were sampled as we ll. Collection of specimens from the VA began in mid-February 2006 and is ongoing. Unfortunately, because of the low number of teeth provided by the facility and the poor condition of these teeth (i.e., little to no enamel present) no samples were run for th e current study. Sample collection is still ongoing though, with the hope that the te eth can be used at a later date. Upon receipt at CAPHIL, teeth were soak ed in 3% hydrogen peroxide in their original vials for 2 days. Teeth were then rinsed of the hydrogen peroxide with tap water and scrubbed with a toothbrush to remove surface contaminants, such as blood. Any adherent periodontal tissue or accessible neurovascular bundles we re also removed. Clean teeth were allowed to air dry overnig ht in a ventilation hood and each tooth was stored in a separate, clean, la beled, resealable, plastic bag.

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79 All USAFA samples contained at least one thir d molar, with the majority of individuals providing all four. Only third molars were run from this facility. W hole teeth, in the best overall condition were preferentially selected for drilling. If mu ltiple teeth from an individual were of the same quality, sampling selection was based on the same criteria as mentioned for the CIL samples: mandibular teeth were chosen over maxillary teeth and right over left. Teeth exhibiting unusual crown anomalies or staining patterns and/or teeth in which the author disagreed with the dental staff numb ering were photographed prior to drilling only. The remaining teeth were not photo-documented. Photo content consisted of the tooth, subject identifier numbe r, tooth number, and a scale (Figure 4-3). Two photos, one of the buccal or lingual surf ace, and one of the occlusal surface were taken. Teeth were cleaned in distilled wa ter within individual capped vials with a Branson Bransonic 1510 tabletop ultrasonic cleaner for 30 minutes. After air-drying, teeth were cleaned of any surface contaminants to include alveolar bone remnants using a NSK UM50 TM slow-speed dental drill with a #8 carbide dental drill bur. Samples of approximately 100-200 mg of pristine enamel were drilled off of each tooth using the same set-up. Care was taken not to drill into the dentine. Enamel powder was collected on creased weighing paper and transferred to labeled 1.5 mL microcentrifuge tubes. Drill bits, weighing paper, and latex gloves were discarded after drilling each tooth and the drill cleaned of adhe rent enamel powder.

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80 Figure 4-3. Pre-drilli ng photo of AFA-093 #32. Carbon and Oxygen Sample Preparation Central Identification Laboratory Samples Chemical preparation of the enamel powde r was performed in the stable isotope laboratory at the Florida Museum of Natura l History, Gainesville, FL, according to the protocol developed by Dr. Pe nnilynn Higgins, museum postdoctoral fellow. The powder of one tooth from each individual was selected based on the integrity of the sample (i.e., whether there was the possibili ty of dentin or other contaminants mixed in with the enamel) and greatest mass of powder availabl e for analysis. Organic residues were removed from the sample powder by adding 1 mL 30% hydrogen peroxide (H2O2) to each microcentrifuge tube. Tubes were sh aken utilizing a Thermolyne Maxi-Mix 1, 16700 mixer and the lids lifted up to prevent gas pressure build-up inside the tubes. The opened vials were stored in a closed reaction cabinet. Samples were periodically shaken with the mixer, every 1 to 2 days, to re-sus pend the enamel powder that had settled at the bottom of the vial. On a weekly basis, the H2O2 was removed by centrifuging samples for 20 minutes at 10,000 RPM in an Eppendorf 5415D microc entrifuge and pipetting off the H2O2.

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81 Pipette tips were discarded between each samp le to prevent cross-contamination. Fresh H2O2 was then added following the same protoc ol. Samples were reshaken, lids opened, and placed back in the reaction cabinet. Th e absence of escaping air bubbles from the solution usually indicates the sample powder is finished reacting and ready for the next phase of treatment. After consulting with Drs. Bruce M acFadden, Florida Museum of Natural History, and John Krigbaum, Universi ty of Florida Department of Anthropology, the samples were decanted of all H2O2 after 51 days in soluti on, even though nearly half still appeared to be reacting. This was lik ely due to the large quantity of powder being processed, with most enamel samples measur ing 100 mg or greater. Samples were then twice rinsed with 1 mL deionized water utilizing the same procedure for removing H2O2 (i.e., water added, then tubes shaken, centrifuged down, and decanted). After rinsing the samples with deionized water and decanting all water from them, secondary carbonates were removed via an acet ic acid bath. Rinsed samples, free from water, were bathed in 1 mL 0.1 N acetic aci d, shaken, and allowed to sit for 30 minutes. The acetic acid was pipetted off after centr ifuging the microcentrifuge tubes at 10,000 RPM for 5 minutes. Samples were then twice rinsed with deionized water and the water removed in the same manner as previously disc ussed. Samples were allowed to air-dry in their open microcentrifuge tubes inside of a desiccator for 2 weeks. This ensured all liquid had evaporated from the enamel powder. An additional side test examining the necessity of performing the acetic acid step was performed. Theoretically, teeth extracte d from living subjects should not have to undergo the acetic acid bath, b ecause teeth in the living are not subject to diagenetic changes associated with the build-up of sec ondary carbonates due to taphonomic factors.

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82 The acetic acid bath was performed on all samples because there is no know precedence to do without acetic acid for forensic is otope purposes. Eight USAFA and two CIL samples were split in half with one half undergoing the full protocol previously outlined and the second sample of each pair undergoing the H2O2 bath and rinses only. Values were then compared to determine if this step of the protocol is indeed required. Portions of the dried enamel measuri ng between 1.2 mg and 1.5 mg were then loaded into stainless steel boats at the Univ ersity of Florida, Department of Geological Sciences, Light Isotope Laboratory. Each boa t was placed into 1 of 44 numbered slots on a brass tray (Figure 4-4) and the tray placed into a desiccator until they were run on the laboratory’s VG/Micromass (now GV Instruments) PRISM Series II isotope ratio mass spectrometer with an Isocarb common acid ba th preparation device. Load sheets were accomplished for each tray listing the sample name and weight for each position in the tray (Appendix D). All samples were load ed by the author. The PRISM was operated by Dr. Jason Curtis and Kathy Curtis. The first run was organized as follows: slots 1–4, standards of NBS-19 measuring between 60 g and 120 g; slots 5–20, altern ating enamel powder and blank positions; Figure 4-4. Loaded tray for PR ISM mass spectrometer analysis.

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83 slots 21–22, NBS-19 standard; slots 23–42, a lternating enamel powder and blank positions; slots 43–44, NBS-19 standard. This a rrangement allowed for the analysis of 18 samples. Empty or blank positions were included in the first run to check for contaminants within the samples. Mass spec trometer readings for the blank positions, indicate leaching of slow-re acting sample into these positions and hence likely contamination. Contamination tends to be much more of an issue with fossilized samples versus modern or historical (Koch et a l 1997). Because the firs t run ran clean with no indication of contamination, the blanks were replaced with sample for all subsequent runs. The sample line-up for all subseque nt runs therefore was as follows: slots 1–4, NBS-19 standard; slots 5–20, sample; sl ots 21–22, NBS-19 standard; slots 23–42, sample; slots 43–44, NBS-19 standard. This ar rangement allowed for the analysis of 36 samples for each run of the mass spectrometer. United States Air Force Academy Samples The Academy samples were prepared in th e same manner as the CIL samples with three exceptions. The first change to th e processing protocol entailed reducing H2O2 exposure time to 24 hours. This change was made upon the recommendation of Dr. Bruce MacFadden, Florida Museum of Natu ral History, and Dr. Pennilynn Higgins, Stable Isotope Ratios in the Environment Anal ytical Laboratory, Department of Earth and Environmental Sciences, University of Roch ester. It was implemented because whole teeth were cleaned for 2–3 days using a 3% H2O2 solution to remove external organics, but more importantly, because teeth were co llected directly from living subjects. Organics associated with diagenetic transf er due to burial were not encountered with these samples as they were with the CIL samples.

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84 Additionally, a GCA Corporation mechanical convection oven was procured at the beginning of the Academy sample preparation pr ocess. Instead of air-drying the samples in a desiccator, chemically processed sample s were placed in a covered microcentrifuge tray with their tops open, and allowed to eva porate to dryness over th e course of 4 days. The oven was set on a setting of “3,” which equated to a circulating temperature of 52O C as measured by an Ever Ready Thermometer Co., Inc. oven thermometer. Samples were loaded for analysis by the PRISM for a 36 sample run in the aforementioned format, except enamel wei ght was reduced to between 1.0 mg and 1.2 mg. Larger sample size was found to slow machine processing time because excess gas produced by the larger weights required multip le rounds of evacuation from the system before readings could be made (Dr. Jason Curtis, personal communication). Strontium and Lead Sample Preparation Strontium and lead isotope ratios were measured for 72 total individuals, evenly split between the CIL and USAFA samples. Additional sampling was not performed due to the financial constraints of this project. Individual CIL samples were chosen based on the purity of sample and highest overall powder weight. All samples believed to originate from outside of Vietnam were sampled.” For the USAFA samples, the author and Dr. George Kamenov constructed a crude geologic regional map based on underlying baseme nt rocks. All individuals residing in a foreign country or outside of the CONUS for the entirety or majority of amelogenesis were sampled. The remainder of the USAFA samples was drawn from a list composed of individuals who had resided only in one city for the entirety of amelogenesis. The list was split into regions and individuals chosen based on sample purity and highest powder weights. Additionally, multiple individuals from Arizona, California, and Colorado, and

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85 Florida were selected to determine if any obvious trends emerged for strontium and lead values within the states. Sampling was comp leted prior to receipt of all of the USAFA samples in order to comply with program deadlines. Sample preparation and column chemistry to extract strontium and lead from the CIL and USAFA samples was the same with one exception: the CIL samples run were those that underwent the carbon/oxygen pretreatment proc ess, while the USAFA samples were untreated. Prior to pretreatment of the USAFA samples for carbon/oxygen stable isotope analyses, the pristine enamel power was split, with approximately 100 mg of the enamel powder being transferred to a sec ond clean, labeled vial. This second, 100 mg portion of enamel powder was what was utili zed for strontium and lead isotope ratio analysis. All column chemistry was performed in a class-1000 rated clean lab facility within the Department of Geological Sciences, Univ ersity of Florida. Strontium and lead extraction was a 5-day process once all ma terials had been cleaned (for cleaning procedures, Appendix E). For the CIL samples, a maximum of 12 samples could be processed during the 5-day cycle. An additio nal 6 lead columns were located and tested prior to running the USAFA samples, raising the number of American samples processed during the 5-day cycle to 18. Day 1: Clean 6 mL Teflon vials were refl uxed utilizing approximately 1.5 mL 6N hydrochloric acid (HCl). Vial s were tightly capped and placed on a hot plate set at “3” overnight. Day 2: Refluxed vials were triple rinsed of the HCl utilizing 4-times distilled water (4X DI H2O) and labeled with the subject identifie r number. Each microcentrifuge tube

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86 containing enamel powder was wiped externally with a Kimwipe prior to enamel transfer. One drop of 4X DI H2O was placed in each Teflon vial to reduce static migration of the powder. A clean microspatula was used to loosen the enamel powder in the microcentrifuge tube and the entire contents of the mircocentrifuge tube were poured into the Teflon vials. The microspatula was cleaned between each transfer utilizing 4X DI H2O and a clean, lint-free wipe. The enam el powder within each Teflon vial was dissolved in 3 mL 50% nitric acid (HNO3) (optima). The vials were then capped and placed overnight on the hotplate set at “3.” An additional set of vials was refluxed utilizing the procedures listed in the “Day 1” protocol. Additionally, 6 CIL samples were spiked to determine both strontium and lead concentrations. Prior to spiking, the enamel powder for each sample was partitioned into thirds and each quantity of powder was plac ed into a dry, refluxed Teflon vial. Water was not utilized to reduce static migration of the powder, because precise mass values for each solution were required to calculate the concentration of the heavy isotopes. An RS95A 85Rb/84Sr spike was added to 1/3 of the en amel powder to measure strontium concentration. A UF-1A 208Pb spike was added to a second third of the enamel powder to determine the lead concentration. In order for the lead concentra tion to be calculated however, a third unspiked portion of enam el powder had to be run as well for comparative purposes. The spiked and uns piked, enamel powder counterparts were dissolved in 3 mL 50% HNO3 (optima) and all subsequent steps followed the normal extraction protocol. Day 3: The additional set of refluxed vials was triple rinsed of the HCl utilizing 4X DI H2O and labeled with the subject identifier number. Backup reserves of the samples

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87 were then created by transferring 1 mL of th e dissolved powder to the new vials. The original and back-up samples were then allo wed to evaporate with the caps off on the hotplate until the samples we re dry (usually 3–4 hours). Day 4 (lead extraction): The lead fraction was separated from the dissolved tooth enamel following a protocol modified after Manhes et al. (1978). A new set of vials was set to reflux utilizing the procedures listed in the “Day 1” protocol. Short, filtered Teflon columns packed in 6N HCl acid were shak en dry and twice rinsed with 4X DI H2O. The stem of the column and a sma ll portion of the chamber were then filled with 4X DI H2O, taking care to avoid introduci ng air bubbles into the column. Dowex 1X-8 lead resin was shaken, poured into the column chamber, a nd allowed to filter through until the stem of the column was packed with the resin (appr oximately 100L of resin), creating a “resin bed.” Excess resin was pipetted out. The re sin in each column was then washed with 2 mL 6N HCl (optima grade). While the acid was filtering through the colu mns, the original, evaporated samples were dissolved in 300 L 1N hydrogen brom ide acid (HBr) (seasta r), capped, and placed on a hotplate set at “3” for approximately 4 minutes. Dissolved samples were removed from the hotplate and set aside until needed. Once all of the acid had passed through the column, 200 L of the dissolved sample was loaded into the column. The collection beaker under the column was exchanged for the or iginal vial to collect the sample for the next day’s strontium collection. Sample numbe rs were written on paper towels in front of each column to prevent confusion. Once the sample had filtered through, the resins were ba thed in three subsequent washes of 1 mL 1N HBr (seasta r). After the second wash, the original sample vials were

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88 exchanged for the previously used collec tion beakers and the vials were placed on a hotplate set at “3” until completely evaporate d. During completion of the third wash, the refluxed vials were triple rinsed of the HCl utilizing 4X DI H2O and labeled with the subject identifier number. After the third wa sh, the collection beakers were switched out for the newly-refluxed, labeled, final collecti on vials. The lead fraction was collected utilizing 1 mL 20% HNO3 (optima grade). The vials containing the lead solution were then evaporated to dryness on a hotplate set at “3.” The columns were cleaned using a flushing bottle of 4X DI H2O and placed in a jar of 6N HCl for a minimum of 12 hours. Day 5 (strontium extraction): The strontium fraction was separated out following the strontium-specific procedure designed by Pin and Bassin (1992). A new set of vials was set to reflux utilizing the procedures list ed in the “Day 1” protocol. Long, filtered Teflon columns packed in 6 N HCl acid were shaken dry and twice rinsed with 4X DI H2O. The stem of the column and a small portion of the chamber were then filled with 4X DI H2O, taking care to avoid introducing air b ubbles into the column. EI Chrom Part #-B100-S strontium resin was shaken, poured in to the column chamber, and allowed to filter through until the stem of the column was packed with the resin (approximately 100L of resin), creating a “resin bed.” Excess resin was pipetted out. The resin in each column was then equilibra ted with 2 mL 3.5N HNO3. While the acid was filtering through the colu mns, the dried samples were dissolved in 350 L 3.5N HNO3. Once all of the acid had passe d through the co lumn, 100 L of the dissolved sample was loaded into the column. Sample numbers were written on paper towels in front of each column to prev ent confusion. Once the sample had filtered through, the resins were bath ed in four subsequent washes of 100 L 3.5N HNO3. A

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89 final wash of 1 mL 3.5N HNO3 was performed prior to collection. During completion of the final wash, the refluxed vials were triple rinsed of HCl utilizing 4X DI H2O and labeled with the subject identif ier number. After completion of this step, the collection beakers were switched out for the newly-refl uxed, labeled, final collection vials. The strontium fraction was collected by rins ing the columns in 1.5 mL 4X DI H2O. The vials containing the strontium solution were then ev aporated to dryness on a hotplate set at “3.” The columns were cleaned us ing a flushing bottle of 4X DI H2O and placed in a jar of 6N HCl for a minimum of 12 hours. Mass spectrometry: Both the lead and strontium frac tions were analyzed utilizing the University of Florida, Department of Geological Science’s, Nu-Plasma, MultiCollector, Inductively-Coupled Plasma Mass Spectrometer (MC-ICP-MS). Evaporated samples were dissolved in 500 L 2% HNO3 (optima). Initial analyses for both lead and strontium utilized 50 L of dissolved sample and an additional 950 L 2% HNO3. Ratios of sample to 2% HNO3 were then adjusted accordingly so the voltage read by the MCICP-MS for each sample fell idea lly between 2.0 and 8.0 volts. For mass-bias correction, lead solutions were spiked with thallium just before the analyses in order to avoid thallium oxid ation (Kamenov et al. 2004). Sample and standard solutions were aspi rated either through a Nu Inst ruments desolvating nebulizer (DSN-100) (“dry plasma” mode) or directly into the plasma source through a Micromist nebulizer with GE spray chamber (“wet plas ma” mode). Measured uptake rate for both sample introduction methods was about 100 l min-1. The instrument settings were carefully tuned to maximize the signal inte nsities on a daily basis (Kamenov personal communication). Preamplifier gain calibrations were run before the beginning of each

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90 analytical session. The analyses reported he re were conducted in static Time-Resolved Analysis (TRA) mode. All lead isotope rati os are relative to the following values for NBS 981: 206Pb/204Pb = 16.937 (+/-0.004 2 ), 207Pb/204Pb = 15.490 (+/-0.003 2 ), and 208Pb/204Pb = 36.695 (+/-0.009 2 ) (Kamenov et al. 2005). According to Dr. George Kamenov, stront ium isotopic ratios were acquired in static mode using 5 Faraday coll ectors (personal communication). 87Sr/86Sr was corrected for mass-bias using exponential law and 86Sr/88Sr=0.1194. 87Sr was corrected for presence of rubidium by monitoring the intensity of 85Rb and subtracting the intensity of 87Rb from the intensity of 87Sr, using 87Rb/85Rb=0.386. All analyses were done in TRA mode by using previous measured zeros determined on clean 2% HNO3 solution in order to correct for isobaric interf erences from the presence of impurities of krypton in the argon gas. Analyses of the NBS 987 strontium standard during the course of this study gave 87Sr/86Sr=0.71025 (+/-0.00004, 2 ) (Dr. George Kamenov personal communication). The machine was purged of any residual sample volume by introducing a pre-wash of 5% HNO3 and then a wash of 2% HNO3 through the MC-ICP-MS between each sample run. A solution of the NBS 981 sta ndard for lead and NBS-987 for strontium, was run every six samples to ensure proper calibration of the mach ine. The author operated the MC-ICP-MS under the s upervision of Dr. George Kamenov. Statistical Analyses Sample sizes of at least 30 in each p opulation satisfied the requirement for approximate normality, the variances were assumed to be homogenous and the samples independent, and random sampling was assume d since everyone presenting for dental

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91 extraction was queried to participate in the study (Ott and Longnecker 2004). (Note: it was realized that this was not a truly random sample because subjects utilized were only those who consented to participate). The nu ll hypotheses that no si gnificant differences exist for the mean values of each isotope be tween individuals from East Asia and the United States, nor between diffe rent regions of the United St ates, were tested via the general linear model (GLM) procedure in SA S version 9.1. The GLM procedure was in the form of a multivariate analysis of variance (MANOVA). This approach was chosen because of the presence of several dependent variables measured for multiple samples. The GLM function was utilized over a straight MANOVA becau se of unbalanced sample sizes between populations (SAS 9.1). Exploratory data analyses in cluded various plots to iden tify data trends and outliers and the calculation of summary statistics. Ge neral linear models we re employed to assess the differences between population means due to the populations having different numbers of observations. When three popul ations were compared simultaneously, a Tukey studentized range test to control for Type I experiment-wise error was included in the analysis. Paired t-tests were performe d to determine if the acetic acid step was necessary in the chemical preparation of teet h from living subjects. Linear discriminant function analyses were conducted in an attempt to create an equation that would correctly classify an individual as eith er being of Southeast Asian or igin or of American, based on the isotopes studied. In an attempt to identify potentially confounding variable s addressed in the survey such as age, sex, race, tobacco use, and ch ildhood diet, the general linear model was also performed to assess the differences between variable means. When greater than two

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92 subsets of a particular variable were comp ared, a Tukey-Kramer adjustment for multiple comparisons was included to control for Type I error. A discriminant function analysis was also accomplished to determine if an equa tion could be created utilizing the isotopes studies that would successfully categorize an individual as or iginating from a particular region within the United States. In additi on, a test for correla tion was conducted to determine the relationship between the 18O values and the corresponding latitudinal coordinates of the natal regions of the donors.

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93 CHAPTER 5 ANALYTICAL COMPARISION OF EAST ASIAN AND AMERICAN SAMPLES The first question this project sought to answer was whether the isotopic composition of enamel could be differentiate d between East Asians and Americans. Significant differences were found between the least squares means of the Central Identification Laboratory (CIL) and United St ates Air Force Academy (USAFA) samples for all isotope values examined (Table 5-1), demonstrating that all isotopes examined are potentially useful in distinguishi ng between these two populations. Light Isotopes The results from the PRISM analysis are pr esented in Table 5-2 and represented in Figure 5-1. Location was assessed as be lieved provenance for the CIL samples and locations of residence during amelogenesis of the third molar (ages 7-18 [Fanning & Brown 1971]) for the USAFA samples. Precision, as determined by replicate analyses of 86 separate NBS 19 standards over 13 mach ine-days from 01 December 2005 through 01 June 2006, was measured at 0.08‰ for car bon and 0.14‰ for oxygen. Similar numbers were calculated based on re plicate measures of homoge nous sample material with average standard deviations of 0.04‰ for carbon and 0.13‰ for oxygen. Carbon Carbon isotope ratios appear to be an excellent discriminato r of natal origin between East Asian and American populations based on cultural dietar y preferences. The least squares means of the two groups were significantly different, with a p-value of

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94Table 5-1. Summary statistics a nd general linear model results of all isotopes examined for CIL samples compared to USAFA samples (CIL outlier excluded). All values are in ‰. Population N Variable Min Max Mean Std Dev P-val CIL 60 13C -17.25 -11.60 -14.25 1.00 <0.0001 USAFA 228 13C -12.88 -7.77 -9.97 0.81 CIL 60 18O -10.61 -5.04 -7.45 0.90 0.0092 USAFA 228 18O -12.57 -3.14 -6.88 1.63 CIL 35 87Sr/86Sr 0.706811 0.721172 0.710995 0.002938 0.0013 USAFA 36 87Sr/86Sr 0.707449 0.711186 0.709273 0.000883 CIL 35 208Pb/204Pb 37.176100 38.883700 38.074157 0.355069 0.0073 USAFA 36 208Pb/204Pb 37.398300 38.616500 38.268931 0.225698 CIL 35 207Pb/204Pb 15.528800 15.695800 15.603740 0.040095 0.0006 USAFA 36 207Pb/204Pb 15.556800 15.674900 15.631292 0.022565 CIL 35 206Pb/204Pb 16.991700 19.620500 18.090809 0.435832 <0.0001 USAFA 36 206Pb/204Pb 17.681000 19.049300 18.595094 0.280822 CIL 35 208Pb/206Pb 1.958010 2.254450 2.105517 0.040400 <0.0001 USAFA 36 208Pb/206Pb 2.027260 2.115030 2.058318 0.020901 CIL 35 207Pb/206Pb 0.799982 0.914750 0.862969 0.018726 <0.0001 USAFA 36 207Pb/206Pb 0.822855 0.879814 0.840787 0.011796

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95 <0.0001. The predominance of rice in East Asian diets appears to lead to more depleted 13C values compared to young American adults who had less negative 13C values, due to a high preponderance of corn and sugar in their diet. Table 5-2. Carbon and oxygen isotope results. All values are in ‰. Identifier 13C 18O Location Identifier 13C 18O Location CIL-001 -13.09 -7.73 Vietnam CIL-032 -14.58 -6.78 Cambodia CIL-002 -13.72 -6.54 Vietnam CIL-033 -14.79 -8.11 Cambodia CIL-003 -15.17 -7.33 Vietnam CIL-034 -16.17 -10.25 Cambodia CIL-004 -14.47 -7.34 Vietnam CIL-035 -14.01 -7.83 Vietnam CIL-005 -14.69 -5.95 Vietnam CIL-036 -13.45 -7.25 Vietnam CIL-006 -13.03 -7.47 Vietnam CIL-037 -13.45 -7.82 Cambodia CIL-007 -13.64 -7.71 Vietnam CIL-038 -13.74 -6.68 Vietnam CIL-008 -14.32 -9.27 Solomon Isl. CIL-039 -13.66 -8.50 Philippines CIL-009 -14.43 -7.84 Vietnam CI L-040 -15.08 -8.56 Solomon Isl. CIL-010 -11.60 -7.76 Vietnam CIL-041 -15.03 -7.11 Vietnam CIL-011 -14.99 -8.23 Vietnam CIL-042 -14.20 -6.90 Vietnam CIL-012 -14.68 -7.24 Vietnam CIL-043 -14.63 -6.90 Vietnam CIL-013 -14.52 -7.75 Laos CIL-044 -14.43 -7.69 Vietnam CIL-014 -14.47 -7.65 Vietnam CIL-045 -14.10 -6.83 Vietnam CIL-015 -15.16 -7.89 Laos CIL-046 -14.44 -7.16 Vietnam CIL-016 -15.02 -7.97 Vietnam CIL-047 -14.72 -6.92 Vietnam CIL-017 -12.17 -8.07 Vietnam CIL-048 -13.83 -8.90 Vietnam CIL-018 -14.27 -6.96 Vietnam CIL-049 -15.31 -7.77 Vietnam CIL-019 -15.45 -6.52 Vietnam CIL-050 -13.90 -7.04 Vietnam CIL-020 -12.64 -7.40 Vietnam CIL-051 -15.45 -6.57 Vietnam CIL-021 -13.57 -7.42 Laos CIL-052 -12.20 -7.88 Vietnam CIL-022 -14.52 -8.06 Vietnam CIL-053 -14.45 -6.60 Vietnam CIL-023 -15.22 -7.75 Vietnam CIL-054 -14.58 -7.41 Vietnam CIL-024 -14.29 -7.50 Vietnam CIL-055 -15.43 -6.22 Vietnam CIL-025 -12.90 -7.07 Vietnam CIL-056 -14.90 -7.28 Vietnam CIL-026 -12.59 -7.25 Korea CIL-057 -14.72 -6.80 Vietnam CIL-027 -14.05 -6.95 Vietnam CIL-058 -17.25 -10.61 Vietnam CIL-028 -14.07 -7.07 Korea CIL-059 -14.53 -6.64 Vietnam CIL-029 -6.32 -8.24 Korea CIL-060 -14.88 -6.75 Vietnam CIL-030 -14.65 -6.94 Vietnam CIL-061 -12.74 -7.60 Vietnam CIL-031 -12.96 -5.04 Vietnam

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96 Table 5-2. Continued. Identifier 13C 18O Location Identifier 13C 18O Location AFA-001 -10.43 -7.49 OR AFA-044 -9.96 -8.91 MN AFA-002 -10.17 -7.48 US/intl-mix AFA-045 -10.49 -7.67 US-mix AFA-003 -9.80 -4.22 US-mix AFA-046 -10.45 -6.28 IL AFA-004 -11.25 -6.53 TN AFA-047 -11.47 -7.47 Philippines AFA-005 -11.10 -9.22 NY AFA-048 -9.05 -5.38 TX AFA-006 -10.78 -8.27 CA AFA-049 -9.63 -6.88 OH AFA-007 -9.92 -7.56 US-mix AFA-050 -10.45 -6.97 US/intl-mix AFA-008 -9.77 -6.92 TX AFA-051 -9.98 -10.21 CO AFA-009 -10.45 -5.91 SC AFA-052 -10.45 -7.39 CA AFA-010 -9.20 -6.73 US-mix AFA-053 -9.11 -4.49 US-mix AFA-011 -9.29 -6.50 AR AFA-054 -9.64 -6.85 US/intl-mix AFA-012 -10.58 -10.52 US-mix AFA-055 -10.30 -7.46 US-mix AFA-013 -9.90 -8.00 NM AFA-056 -10.63 -7.18 MN AFA-014 -9.92 -4.98 TX AFA-057 -9.61 -8.48 CA AFA-016 -11.31 -5.69 US-mix AFA-058 -9.75 -6.50 VA AFA-017 -9.64 -7.83 AZ AFA-059 -8.86 -6.99 WI AFA-018 -9.88 -5.22 US-mix AFA-060 -9.81 -8.98 NE AFA-019 -9.13 -6.48 MA AFA-061 -10.41 -7.57 CA AFA-020 -9.39 -5.19 US-mix AFA-062 -9.35 -4.71 TX AFA-021 -9.69 -8.52 AZ AFA-063 -11.23 -12.57 AK AFA-022 -10.10 -3.14 US-mix AFA-064 -11.33 -7.61 US-mix AFA-023 -9.80 -5.98 Guam AFA-065 -10.17 -6.79 CO AFA-024 -10.19 -10.16 CO AFA-066 -8.48 -6.41 IL AFA-025 -10.40 -6.81 MI AFA-067 -10.82 -6.99 US/intl-mix AFA-026 -10.76 -9.02 US-mix AFA-068 -9.51 -7.29 US/intl-mix AFA-027 -9.32 -7.95 US-mix AFA-069 -10.45 -5.67 NJ AFA-028 -9.82 -6.41 TX AFA-070 -10.16 -4.54 FL AFA-029 -9.73 -5.75 TX AFA-071 -9.81 -7.81 US-mix AFA-030 -11.57 -6.38 CA AFA-072 -10.16 -5.83 US-mix AFA-031 -10.87 -9.52 CO AFA-073 -10.80 -5.01 TX AFA-032 -12.52 -12.40 US/intl-mix AFA-074 -9.51 -5.58 AR AFA-033 -10.36 -7.25 MA AFA-075 -12.30 -5.47 US/intl-mix AFA-034 -10.66 -8.69 WA AFA-076B -9.91 -7.52 CA AFA-037 -10.33 -6.41 TN AFA-077 -9.72 -4.37 US-mix AFA-038 -10.05 -6.36 US-mix AFA-078 -9.98 -7.28 VT AFA-039 -9.49 -5.72 TX AFA-079 -9.28 -8.93 US-mix AFA-040 -9.10 -5.98 SC AFA-080 -10.14 -6.61 OH AFA-041 -10.65 -7.03 US/intl-mix AFA-081 -10.65 -8.05 US-mix AFA-042 -9.00 -5.75 US-mix AFA-082 -9.59 -6.58 IA AFA-043 -10.28 -5.83 GA AFA-083 -9.94 -5.12 TX

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97 Table 5-2. Continued. Identifier 13C 18O Location Identifier 13C 18O Location AFA-084 -8.92 -5.23 US-mix AFA-124 -8.33 -6.40 NE AFA-085 -9.76 -6.74 CT AFA-125 -10.54 -7.38 OR AFA-086 -10.43 -5.52 HI AFA-126 -10.32 -7.37 CA AFA-087 -9.49 -4.90 GA AFA-127 -9.95 -5.60 US-mix AFA-088 -9.47 -6.34 OH AFA-128 -9.71 -4.39 US-mix AFA-089 -9.66 -5.20 FL AFA-129 -8.91 -6.03 US-mix AFA-090 -10.41 -6.46 MA AFA-130 -9.44 -6.23 US-mix AFA-091 -9.75 -6.02 TX AFA-131 -9.63 -7.96 US-mix AFA-092 -10.72 -5.33 NC AFA-132 -11.14 -6.99 US/intl-mix AFA-093 -10.02 -7.13 OH AFA-133 -10.01 -5.41 AL AFA-094 -11.08 -6.35 SC AFA-134 -9.96 -6.09 MS AFA-095 -8.44 -7.17 PA AFA-135 -10.71 -5.43 US-mix AFA-096 -9.20 -6.71 VA AFA-136 -11.76 -11.46 ID AFA-097 -10.38 -6.15 TX AFA-137 -9.50 -6.10 US-mix AFA-098 -9.34 -6.43 IA AFA-138 -9.11 -5.45 NC AFA-099 -8.72 -9.88 CO AFA-139 -10.29 -4.79 US/intl-mix AFA-100 -9.99 -7.23 US/intl-mix AFA-140 -9.78 -6.20 US-mix AFA-101 -9.36 -6.74 MA AFA-141 -11.52 -6.59 US/intl-mix AFA-102 -9.95 -6.59 NJ AFA-142 -10.42 -5.47 US/intl-mix AFA-103 -8.51 -10.16 CO AFA-143 -10.58 -7.36 intl-mix AFA-104 -8.97 -7.42 US-mix AFA-144 -9.85 -7.97 US-mix AFA-105 -11.02 -6.07 NJ AFA-145 -10.38 -9.85 WY AFA-106 -9.71 -8.54 US-mix AFA-146 -10.02 -9.76 ID AFA-107 -10.39 -9.03 OR AFA-147 -9.64 -5.53 TN AFA-108 -10.34 -7.46 OR AFA-148 -12.44 -7.34 Korea AFA-109 -9.42 -9.73 Peru AFA-149 -10.30 -6.47 MO AFA-110 -9.71 -4.51 TX AFA-150 -9.20 -6.41 US/intl-mix AFA-111 -10.53 -7.87 CA AFA-151 -10.04 -4.55 TX AFA-112 -9.63 -8.22 NM AFA-152 -10.97 -7.45 US/intl-mix AFA-113 -9.85 -5.36 GA AFA-153 -9.43 -6.76 PA AFA-114 -8.40 -4.88 US-mix AFA-154 -10.03 -6.21 US-mix AFA-115 -9.12 -6.42 TN AFA-155 -9.25 -4.73 TX AFA-116 -10.74 -8.84 CA AFA-156 -10.88 -4.11 TX AFA-117 -11.06 -8.51 WA AFA-157 -9.56 -7.25 NE AFA-118 -7.77 -6.02 GA AFA-158 -10.33 -6.80 MD AFA-119 -8.87 -6.87 VT AFA-159 -10.32 -7.40 US-mix AFA-120 -10.43 -7.14 US/intl-mix AFA-160 -9.77 -4.13 FL AFA-121 -10.59 -11.20 MT AFA-161 -9.56 -8.41 MI AFA-122 -10.51 -6.44 US-mix AFA-162 -10.11 -6.76 US-mix AFA-123 -10.47 -8.75 US-mix AFA-163 -9.51 -5.36 TX

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98 Table 5-2. Continued. Identifier 13C 18O Location Identifier 13C 18O Location AFA-164 -8.91 -5.62 GA AFA-205 -7.98 -4.82 GA AFA-165 -9.41 -5.57 US-mix AFA-211 -10.14 -9.83 UT AFA-166 -10.07 -10.18 ND AFA-212 -10.57 -10.71 MT AFA-167 -9.98 -5.06 TX AFA-217 -10.51 -5.78 TN AFA-168 -11.09 -7.49 PA AFA-218 -9.68 -7.20 NY AFA-169 -8.85 -7.00 US-mix AFA-220 -12.88 -4.61 Suriname AFA-170 -9.83 -7.30 WI AFA-221 -9.88 -5.85 WV AFA-171 -9.08 -7.34 NJ AFA-222 -9.51 -6.00 OK AFA-172 -9.89 -4.76 TX AFA-223 -10.04 -8.58 CA AFA-173 -11.17 -7.71 CA AFA-225 -8.03 -7.14 AZ AFA-174 -9.39 -5.88 KY AFA-226 -10.10 -11.60 MT AFA-175 -10.49 -6.27 US-mix AFA-227 -9.08 -5.84 SC AFA-176 -9.40 -5.46 FL AFA-228 -9.77 -7.49 WV AFA-177 -9.78 -6.53 US/intl-mix AFA-230 -9.54 -6.82 IL AFA-178 -9.22 -5.80 US/intl-mix AFA-231 -10.22 -6.90 MI AFA-179 -9.70 -7.46 OH AFA-235 -9.99 -6.28 OH AFA-180 -9.96 -4.64 FL AFA-237 -9.76 -7.98 SD AFA-181 -10.17 -5.44 TX AFA-240 -8.69 -4.96 US-mix AFA-182 -8.47 -6.91 PA AFA-241 -9.91 -9.13 CO AFA-183 -10.35 -8.19 CA AFA-246 -10.67 -9.70 MT AFA-184 -12.69 -6.37 intl-mix AFA-250 -9.97 -6.73 CA AFA-186 -9.18 -4.75 OK AFA-251 -10.03 -4.92 AL AFA-187 -9.64 -5.85 IN AFA-252 -11.48 -10.30 UT AFA-188 -10.60 -7.30 MI AFA-254 -9.56 -5.75 VA AFA-192 -9.96 -8.26 OR AFA-257 -8.89 -6.29 PA AFA-194 -9.54 -9.79 CO AFA-263 -9.73 -6.74 US/intl-mix AFA-195 -9.88 -6.62 NJ AFA-264 -9.01 -7.17 WI AFA-196 -8.37 -5.09 GA AFA-265 -9.83 -8.25 CO AFA-197 -9.91 -6.63 MI AFA-267 -9.95 -6.77 AFA-198 -10.41 -6.93 MN AFA-270 -9.88 -9.06 CO AFA-200 -10.04 -6.83 NM AFA-272 -8.72 -4.35 GA AFA-201 -10.99 -6.42 IN AFA-273 -8.69 -5.55 MD AFA-203 -9.25 -7.09 MN AFA-274 -9.88 -3.75 FL AFA-204 -10.69 -6.88 IL AFA-276 -9.37 -5.82 NY One extreme outlier was present in the CIL sample, representing an individual disinterred from Korea, near the de-milita rized zone. While the oxygen value does not appear out of the ordinary, the carbon va lue of -6.32‰ for CIL-029 exceeds the range of

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99 -18.00 -16.00 -14.00 -12.00 -10.00 -8.00 -6.00 -4.00 -14.00-12.00-10.00-8.00-6.00-4.00-2.00 CIL USAFA Figure 5-1. Carbon and oxygen isotope re sults with overlapping value overlay.18O Value ( ‰ ) 13C Value ( ‰ ) Korea Korea mostly Bulgaria Ger/Belg until age 14 Suriname Alberta until age 14 ID

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100 both the CIL and USAFA samples. The nearest CIL sample to this value is an individual from Vietnam, whose 13C value was measured at -11.6‰. In fact, the least negative USAFA sample was -7.77‰, still nowhere close to the outlying value. A second sample of the same tooth was rerun with a 13C of -6.20‰. This is even more enriched than the first value. While the difference of 0.12‰ for carbon exceeds the precision of the machine by 0.04‰, it is possible that additional variation arose from contaminants or through the bulk sampling process itself. If you take the upper limit for C4 plants at 13C = -9‰ (van der Merwe 1982) and add in a fractionation factor of +9.6‰ (DeN iro & Epstein 1978b) fo r bone apatite, it is apparent than an organism feeding on a monotonous diet of the most enriched C4 plants could theoretically display a bone 13C value of approximately +0.04‰. A 13C value of -6.32‰ for apatite is not unheard of in the lite rature. Lee-Thorp et al. (1993) reported three human 13C values dating from the Iron Age as being near or above CIL-029, the highest of which was -5.5‰. The site was fr om southern Africa a nd the results pointed to an overwhelming dependence upon C4 foods or livestock grazing upon C4 grasses. High collagen values were also found among infants from a protohistoric Amerindian village in southern Ontari o, the most enriched of which was -6.8‰ (equivalent to approximately 2‰ for apatite) and attributed to a weaning diet almost exclusively of maize. Finally, such high va lues have also been reported in the tooth enamel of a hominid cousin, Australopithecus africanus Two separate studies have measured 13C values as high as -5.6‰ (Sponheimer & Lee-Thorp 1999a) and -4.4‰ (van der Merwe et al. 2003). If CIL-029 trul y was of Korean origin though, it is highly unlikely they subsisted on an exclusively C4-based diet.

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101 One possible explanation for this seem ingly errant value is the issue of contamination. Another though, is that this pe rson is not truly of Korean origin. The Korean War was a global conflict, with the Unit ed Nations steering the efforts to liberate the Republic of Korea from the invading communist forces from the north (Drew & Snow 2003). The United Nations command wa s manned by combat troops from not only the United States, but Australia, Belgium, Ca nada, Colombia, Ethiopia, France, Greece, Luxembourg, the Netherlands, New Zealand, the Philippines, Thailand, and Turkey. South Africa provided airpower; Denmark, Indi a, Norway, and Sweden supplied medical units; and Italy provided a hospi tal. The North Koreans also had assistance in the form of foot-soldiers from China and Soviet pilots engaged the United Nations air forces (Snow & Drew 2003, Evanhoe 2006). The potential exis ts then for a body disinterred from an unmarked grave to be an indi vidual from any one of these nations. In light of these factors, the isotope values for CIL-029 were not included in the statistical analyses, due to suspect provenance. A second tooth from the same individual was drilled with 13C and 18O values of -7.63‰ and -7.50‰ respectively. Th ese values are markedly different from the values from the first tooth (Table 5-3), but are most likely explained by inte rtooth variation. The raw data from Beard and Johnson (2000) indicate their range of same individual intertooth values was as large as 24.7‰ for st rontium. Because different teeth mineralize at different points during childhood, each will give a slightly different still-frame value of diet due to the progression of dietary intake from breast milk to adult foods during an individual’s formative years. The same can be said of geolocation as the person changes residence or is exposed to different environmental conditi ons during their childhood. The

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102 Table 5-3. Central Identificati on Laboratory outlier run data. Identifier Tooth # 13C (‰) 18O (‰)Date CIL-029 17 -6.32 -8.24 10 Jan 06 CIL-029 17 -6.20 -8.18 5 Feb 06 CIL-029 18 -7.63 -7.50 1 May 06 period of amelogenesis of tooth #17 is from ag es 7-18 in Caucasians while the period of amelogenesis of tooth #18 is roughly from ag es 3-8 in Caucasians (Fanning & Brown 1971). This should be applicable for Asian teeth as well, since variation at age of amelogenesis only varies by a few mont hs between populations (Hillson 1996). When the CIL outlier is excluded from comparison, only six USAFA values overlapped with the main cluster of East Asia n values (Table 5-4), the most enriched of which measured -11.60‰. It is interesting to note that all but one of these values correspond to individuals reared primarily outside of the United States. The most depleted 13C USAFA value was from an individu al reared in Suriname. Of the remaining overlapping USAFA valu es, the individuals originated from primarily Bulgaria (AFA-184); Alberta, Canada until age 14 (AFA-032); Ko rea (AFA-148); Germany and Belgium until age 14 (AFA-075); and a Caucas ian male from Idaho (AFA-136). The next two closest values are within 0.03‰ and 0.08‰ but do not overlap. They respectively belong to a self-reported Filipi no raised in California and a self-reported Asian who was raised primarily abroad in Ko rea, Germany, and England with one 3-year tour in New York. Only one out of the next four closest values belonged to an Asian individual, who was raised in the Philippine s, while the other three values are all associated with Caucasians raised in vari ous locations throughout the United States. To examine how individuals reared outsid e the continental United States (CONUS, which excludes Alaska) influenced the USAFA-CIL delta value comparisons, three

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103 Table 5-4. 13C value comparison. Twelve most enriched CIL samples and 12 most depleted USAFA samples (CIL outlier ex cluded). All values measured in ‰. East Asian USAFA Identifier 13C (‰) Location Identifier 13C(‰) Location CIL-010 -11.60 Vietnam AFA-220 -12.88 Suriname CIL-017 -12.17 Vietnam AFA-184 -12.69 intl-mix CIL-052 -12.20 Vietnam AFA-032 -12.52 US/intl-mix CIL-026 -12.59 Korea AFA-148 -12.44 Korea CIL-020 -12.64 Vietnam AFA-075 -12.30 US/intl-mix CIL-061 -12.74 Vietnam Overlap AFA-136 -11.76 ID CIL-025 -12.90 Vietnam AFA-030 -11.57 CA CIL-031 -12.96 Vietnam AFA-141 -11.52 US/intl-mix CIL-006 -13.03 Vietnam AFA-252 -11.48 UT CIL-001 -13.09 Vietnam AFA-047 -11.47 Philippines CIL-036 -13.45 Vietnam AFA-064 -11.33 US-mix CIL-037 -13.45 Laos AFA-016 -11.31 US-mix iterations of the general linear model proce dure were run. The first was the standard comparison between all CIL samples minus the outlier and all USAFA samples, which found the 13C means were significantly different at a p-value of <0.0001 (Table 5-1 and Figure 5-1). A second statistic al run weighed the CIL valu es against not only USAFA samples from individuals residi ng for at least a portion of amelogenesis within the United States and its territories but against those solely raised in a foreign locale (two mixed international locations, and 1 each Suriname Peru, Korea, and the Philippines) and among the two USAFA populations (Figure 5-2) The means for the three groups were compared and a Tukey’s studentized range test performed to control for Type I experiment-wise error rate. Results indi cate that the means for carbon for all three groups are significan tly different at =0.05 (Table 5-5). One last iteration compared the CIL sample s, USAFA individuals spending at least a portion of their natal resi dence within the CONUS, and those USAFA samples arising from donors who were raised outside of th e CONUS. Statistics were computed in

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104 -18.00 -16.00 -14.00 -12.00 -10.00 -8.00 -6.00 -4.00 -14.00-12.00-10.00-8.00-6.00-4.00-2.00 CIL USAFA US USAFA Foreign Figure 5-2. Carbon and oxygen isotope results for Am erican and foreign USAFA compar ison. 18O Value ( ‰ ) 13C Value ( ‰ ) Lima Korea Philippines mostly Bulgaria Surinam mostly Korea

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105Table 5-5. Summary statistics and general linear model results of all isotopes examined fo r American and foreign USAFA comparison (CIL outlier exclude d). All values are in ‰. Population N Variable Min Max Mean Std Dev CIL 60 13C -17.25 -11.60 -14.25 1.00 *† USAFA (US) 222 13C -12.52 -7.77 -9.92 0.75 ‡ USAFA (foreign) 6 13C -12.88 -9.42 -11.58 1.37 †‡ CIL 60 18O -10.61 -5.04 -7.45 0.90 USAFA (US) 222 18O -12.57 -3.14 -6.87 1.63 USAFA (foreign) 6 18O -9.73 -4.61 -7.17 1.67 *, †, or ‡ indicates signifi cant difference between means Table 5-6. Summary statistics and genera l linear model results of all isotopes ex amined for CONUS and overseas USAFA comparison (CIL outlier exclude d). All values are in ‰. Population N Variable Min Max Mean Std Dev CIL 60 13C -17.25 -11.60 -14.25 1.00 *† USAFA (CONUS) 219 13C -12.52 -7.77 -9.92 0.75 ‡ USAFA (OS) 9 13C -12.88 -9.42 -11.21 1.26 †‡ CIL 60 18O -10.61 -5.04 -7.45 0.90 USAFA (CONUS) 219 18O -12.40 -3.14 -6.85 1.60 USAFA (OS) 9 18O -12.57 -4.61 -7.44 2.41 *, †, or ‡ indicates signifi cant difference between means

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106 -18.00 -16.00 -14.00 -12.00 -10.00 -8.00 -6.00 -4.00 -14.00-12.00-10.00-8.00-6.00-4.00-2.00 CIL USAFA CONUS USAFA OS Figure 5-3. Carbon and oxygen isotope result s for CONUS and overseas USAFA comparison. 18O Value ( ‰ ) 13C Value ( ‰ ) Lima Korea Philippines mostly Bulgaria Suriname mostly Korea HI Guam AK

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107 the same fashion as the second run and the out come was the same in that all group means for carbon were significantly different at =0.05 (Table 5-6 and Figure 5-3). As only three individuals were added to the foreign group to create the new overseas (OS) group, (one individual each from Alaska, Guam, and Hawaii), the summary statistics did not change much between statistical runs two and three. The CIL samples represent the extreme end of the spectrum for carbon depletion, with a mean 13C value of -14.25‰. The heavy dependence of rice in the diets of humans and livestock raised for meat greatly influences the 13C values of the CIL samples, so they represent primarily a C3 plant signature. The modern foreign and OS individuals ar e all young and represent a distinct group from both the Vietnam-era CIL teeth and the American and CONUS USAFA donors. These individuals are also intermediate with a mean 13C of -11.58‰ for the foreign grouping and -11.21‰ for the OS grouping. The 13C values of -9.92‰ for both the American and CONUS participan t groups are indicative of indi viduals with a much larger corn constituent in the diet. The influence of corn-based products have become pervasive in modern American carbon isotope ratios. Not only have Americans’ consumption of whole corn increased, but co rn-based snacks such as popc orn, chips, and sodas/colas sweetened with high-fructose corn syrup ar e essentially a staple of contemporary American diets and consumed at ever increas ing rates. American consumption of meat has steadily increased over the years as well (Kantor 1998) Domestic livestock are primarily fed corn products (USDA 2006), and this base corn isotopic signature is eventually incorporated into the human tissue.

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108 Oxygen The CIL and USAFA samples also significantly differed with respect to their mean 18O values. The mean oxygen measure for the CIL cohort was -7.45‰ (CIL outlier excluded) while the mean for the USAFA donors was -6.88‰, with a p-value from the GLM procedure of 0.0092. No outlier from either sample group was noted. While the values between the two groups patently ove rlap, the CIL samples cluster much more tightly, ranging from -10.61‰ to -5.04‰ (Table 5-7). The USAFA samples completely encompass this distribution and extend out roughly 2‰ on either side, ranging from -12.57‰ to -3.14‰ (Table 5-8). Oxygen values are dependent upon a variety of interrelated environmental factors such as latitude, temperature, altitude distance inland, precipitation patterns, and humidity (Iacumin 1996, Hertz & Garri son 1998, Kendall & Coplen 2001). A rough latitudinal cline does appear to have emerged for the USAFA 18O values, with the higher latitudes trending more negativ ely and the lower latitudes de monstrating more enriched values (this will be discussed in greater de tail in Chapter 6). If latitude were the dominant factor contributing to an organism’s oxygen isotope signature, a similar cline should be observed for the CIL samples and ar eas that overlap latit udinally (Figure 5-4) should exhibit similar values. These trends do hold true to an extent, but latitude does not appear to be the dominant factor reflected in an organism’s 18O value. For instance, in looking at the East Asian geographical disp ersion, one would assume indi viduals from Korea would have the most negative oxygen isotope ratios be cause the Korean peninsula is the furthest north of the natal regions sampled. The three Korean values actually fall in the middle of

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109 Table 5-7. East Asian 18O values, in ascending order. Identifier 18O Location Identifier 18O Location CIL-058 -10.61 Vietnam CIL-004 -7.34 Vietnam CIL-034 -10.25 Cambodia CIL-003 -7.33 Vietnam CIL-008 -9.27 Solomon Isl. CIL-056 -7.28 Vietnam CIL-048 -8.90 Vietnam CIL-036 -7.25 Vietnam CIL-040 -8.56 Solomon Isl. CIL-026 -7.25 Korea CIL-039 -8.50 Philippines CIL-012 -7.24 Vietnam CIL-029 -8.24 Korea CIL-046 -7.16 Vietnam CIL-011 -8.23 Vietnam CIL-041 -7.11 Vietnam CIL-033 -8.11 Cambodia CIL-025 -7.07 Vietnam CIL-017 -8.07 Vietnam CIL-028 -7.07 Korea CIL-022 -8.06 Vietnam CIL-050 -7.04 Vietnam CIL-016 -7.97 Vietnam CIL-018 -6.96 Vietnam CIL-015 -7.89 Laos CIL-027 -6.95 Vietnam CIL-052 -7.88 Vietnam CIL-030 -6.94 Vietnam CIL-009 -7.84 Vietnam CIL-047 -6.92 Vietnam CIL-035 -7.83 Vietnam CIL-043 -6.90 Vietnam CIL-037 -7.82 Cambodia CIL-042 -6.90 Vietnam CIL-049 -7.77 Vietnam CIL-045 -6.83 Vietnam CIL-010 -7.76 Vietnam CIL-057 -6.80 Vietnam CIL-013 -7.75 Laos CIL-032 -6.78 Cambodia CIL-023 -7.75 Vietnam CIL-060 -6.75 Vietnam CIL-001 -7.73 Vietnam CIL-038 -6.68 Vietnam CIL-007 -7.71 Vietnam CIL-059 -6.64 Vietnam CIL-044 -7.69 Vietnam CIL-053 -6.60 Vietnam CIL-014 -7.65 Vietnam CIL-051 -6.57 Vietnam CIL-061 -7.60 Vietnam CIL-002 -6.54 Vietnam CIL-024 -7.50 Vietnam CIL-019 -6.52 Vietnam CIL-006 -7.47 Vietnam CIL-055 -6.22 Vietnam CIL-021 -7.42 Laos CIL-005 -5.95 Vietnam CIL-054 -7.41 Vietnam CIL-031 -5.04 Vietnam CIL-020 -7.40 Vietnam the CIL range. The two individuals disint erred from the Solomon Islands, located just south of the equator, are among the most depleted, but based on latitude, one would assume they would be among the most enriched. The oxygen values derived from bone carbona te correlate well to local meteoric water at an r2=0.98 (Iacumin et al. 1996). When the International Atomic Energy Agency overlays for weighted annual 18O for Asia and North America (Figures 5-5 and 5-6) were consulted, it became apparent why the range for East Asia is completely

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110 Table 5-8. Partia l list of USAFA 18O values, in ascending orde r (30 most depleted and 30 most enriched). 30 Most Depleted Values 30 Most Enriched Values Identifier 18O Location Identifier 18O Location AFA-063 -12.57 AK AFA-020 -5.19 AFA-032 -12.40 Alberta/OR AFA-083 -5.12 TX AFA-226 -11.60 MT AFA-196 -5.09 GA AFA-136 -11.46 ID AFA-167 -5.06 TX AFA-121 -11.20 MT CIL Overlap AFA-212 -10.71 MT AFA-073 -5.01 TX CIL Overlap AFA-014 -4.98 TX AFA-012 -10.52 VT/ID/VT AFA-240 -4.96 TX/CO AFA-252 -10.30 UT AFA-251 -4.92 AL AFA-051 -10.21 CO AFA-087 -4.90 GA AFA-166 -10.18 ND AFA-114 -4.88 TX/GA AFA-103 -10.16 CO AFA-205 -4.82 GA AFA-024 -10.16 CO AFA-139 -4.79 CO/Panama/HI/TX/Ger AFA-099 -9.88 CO AFA-172 -4.76 TX AFA-145 -9.85 WY AFA-186 -4.75 OK AFA-211 -9.83 UT AFA-155 -4.73 TX AFA-194 -9.79 CO AFA-062 -4.71 TX AFA-146 -9.76 ID AFA-180 -4.64 FL AFA-109 -9.73 Peru AFA-220 -4.61 Suriname AFA-246 -9.70 MT AFA-151 -4.55 TX AFA-031 -9.52 CO AFA-070 -4.54 FL AFA-005 -9.22 NY AFA-110 -4.51 TX AFA-241 -9.13 CO AFA-053 -4.49 FL/MN AFA-270 -9.06 CO AFA-128 -4.39 IL/OK AFA-107 -9.03 OR AFA-077 -4.37 TX/NH AFA-026 -9.02 PA/NC/TX AFA-272 -4.35 GA AFA-060 -8.98 NE AFA-003 -4.22 PR/FL/VA AFA-079 -8.93 MT/WA AFA-160 -4.13 FL AFA-044 -8.91 MN AFA-156 -4.11 TX AFA-116 -8.84 CA AFA-274 -3.75 FL AFA-123 -8.75 MS/WA/AL/FL/PA AFA-022 -3.14 CA/FL contained within the range for the USAFA valu es. Even though the latitudinal gradient for the two regions is fairly disparate, the annual 18O for precipitation is quite similar for East Asia and the CONUS, with the excepti on perhaps of the southeast U.S. So many different factors inter act to influence the 18O values for organisms that it appears local ecosystems have a much more overarching effect than generalized global features such as latitude.

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111 One aspect that likely is confounding thes e results is the unknown true provenance for many of the CIL accessions or uncertainty as to the natal origins of individuals disinterred from these regions. According to the CIL’s Casualty Automated Recovery and Identification System (CARIS) database, several sets of remains purported to be American servicemen were turned over to U.S. authorities by Vietnamese refugees seeking asylum in the U.S. After evaluati on by CIL anthropologists, the remains were determined to be Mongoloid of foreign origi n. Remains were surrendered by refugees in locations such as France, Hong Kong, Thaila nd, Malaysia, and Singapor e, thus teeth used from these skeletons could have originated from these nations, from Vietnam itself, or elsewhere. Further complicating matters is th e fact that the Pacific theater has been a hotbed of military conflict over th e past century. It is quite po ssible, for instance, that the remains disinterred from the Solomon Islands are not of native islanders, but of fallen Figure 5-4. Latitudinal dispersion of major natal regions featured in this study. East Asia is on the right. Information drawn from Rand McNally Atlas (1998). -20 -10 0 10 20 30 40 50 60 70 80AK Alberta Bulgaria CONUS Germany/Belgium Guam HI Lima Puerto Rico Suriname Cambodia Korea Laos Phillipines Solomon Isl. Vietnam Location Latitude (oN) (oS)

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112 Figure 5-5. Weighted Annual 18O for Asia. Map reproduced from IAEA (2001). Figure 5-6. Weighted Annual 18O for North America. Map reproduced from IAEA (2001). SEA Phili pp ines Korea

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113 Philippines, in that these areas were occupi ed by the Japanese during World War II. The difficulties with the true origin of remains disinterred from Korea have already been addressed. A latitudinal cline is much more appare nt in the USAFA samples, although those from the European nations were much more enriched than expected, with the individual primarily from Bulgaria exhibiting a 18O value of -6.37‰ while the American raised largely in Germany and Belgium measured -5.47 ‰. The altitude of Lima, Peru, seems to have overshadowed its southern latitude. The 18O value of the individual from there was -9.73‰, positioning them at the lower end of the U.S. mountain states. While Guam and Hawaii were fairly enriched overall, they we re not at the end of the U.S. spectrum of oxygen ratios as one would expect based on their more equatorial latitude. Their 18O values were likely influenced by island effects. One individual each from the CIL and US AFA cohorts was purported to be from the Philippines. The CIL sample measur ed -8.50‰ and the USAFA sample -7.45%, more than one standard deviation apart as calculated from the East Asian pool. The enamel 18O of two USAFA individuals rais ed in Korea varied only by 0.02‰, measuring -7.36‰ and -7.34‰. These ratios were very similar to one CIL Korean sample with a 18O of -7.25‰. The other CIL Koreans had values of -7.07‰ and 8.24%. The individual measuring -7.07‰ is we ll within the standard deviation of 0.89 for the CIL oxygen values when compared to the USAFA Koreans. The -8.24‰ individual (CIL-029) is at the very cusp of the standard deviation; CIL-029 is the extreme outlier discussed in the carbon se ction of this chapter. The oxygen data further reinforces the notion that Korea is probably not the native origin of this individual.

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114 Similar to the carbon series, three iterati ons of the GLM procedure were run to determine the influences of foreign and overseas natal regions upon the USAFA isotope ratios were run and a Tukey’ s studentized range test perf ormed to control for type I experiment-wise error rate (Table s 5-1, 5-2, and 5-3). It is ap parent that the removal of 6 and 9 individuals, respectively, out of a tota l of 228 sampled, does not notably affect the mean or standard deviation for the Ameri can group in either instance. The mean 18O for the six foreign USAFA particip ants was -7.15‰, a value that wa s intermediate to the CIL samples mean and the American USAFA sample s. When the Americans reared outside of the CONUS were added to the foreign gr oup to create the overseas assemblage, the mean 18O shifted to -7.44‰, just +0.01‰ from th e East Asian mean. The standard deviation for the overseas group jumped dram atically though to 2.41‰. When the means then are compared between the three groups for the two additional iterations, only the American USAFA samples in one run and th e CONUS USAFA samples in the other, are significantly different from the CIL samples at =0.05. The foreign USAFA samples are statistically indistinguishable from the CI L and American USAFA samples as was the overseas USAFA group from the CIL and CONUS USAFA groups. For a visual representation of these three gr oups, see Figures 5-1, 5-2, and 5.3. Acetic Acid Test In addition to the main project, a side study was conducted to assess the necessity of including acetic acid in the chemical processing of sample s for light isotope analysis. Acetic acid is normally used on archaeologica l specimens to reduce secondary carbonates within the samples resulting from interaction with the interment environment. Theoretically, acetic acid should not be requi red for samples extracted directly from a living subject. There is some debate as to whether the outer enam el surface does undergo

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115 exchange with the oral environment, but there is nothing in the literature to indicate even if it does, that secondary carbona te contamination would result. New teeth from 10 USAFA donors were drille d and the enamel powder chemically processed. Half of the powder for each t ooth was processed following the standard protocol while the other half omitted the step s in which the enamel powder was bathed in acetic acid for 30 minutes and washed twice with distilled water. The differences in each set of isotope values displayed no real trend with some pairs for each isotope showing positive differences and others, negative differences (Table 5-9). Based on two-tailed, paired t-tests, both the 13C and the 18O values were found not to be significantly different between samples treated with acetic acid and those without. The carbon values had a mean difference of -0.15‰ between treated and untreated specimens, with a p-value of 0.300. The oxygen values had a mean difference of 0.19‰ between treated and untreated specimens, with a p-value of 0.311. Based on the p-values however, it should not automatically be assumed that an acetic acid wash is not necessary in treating samples from living subjects. The machine precision for the carbon and oxygen analyses for this study averaged 0.08‰ and 0.14‰, respectively. If there truly were no difference between the treated and untreated values then the average differe nce should be near or below the precision value, not nearly twice the value of it, as in the case of carbon. The mean difference also exceeds the precision value for oxygen. It is di fficult to say why this may be the case, since the exposure time was limited to only 30 minutes. It may be if there are no secondary carbonates to act upon, the acetic acid may instead be interacting with the

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116 Table 5-9. Results of acetic acid test, with intertooth comparison when available. Subject Tooth ID 13C 18O Date AFA-015 No comparative data 32 ACE-001 -10.53 -10.30 10-Jan ACE-002 -9.73 -9.64 10-Jan w/o acetic AFA-035 No comparative data ACE-003 -11.33 -6.15 10-Jan ACE-004 -11.10 -6.18 10-Jan w/o acetic AFA-043 16 -10.28 -5.83 5-Dec 17 ACE-005 -10.56 -6.86 10-Jan ACE-006 -10.29 -6.86 10-Jan w/o acetic AFA-068 1 -9.51 -7.29 7-Dec 16 ACE-007 -9.53 -7.46 10-Jan ACE-008 -9.81 -8.10 10-Jan w/o acetic AFA-083 17 -9.94 -5.12 10-Jan 32 ACE-009 -9.97 -5.64 10-Jan ACE-010 -10.49 -6.23 10-Jan w/o acetic AFA-085 16 -9.76 -6.74 10-Jan 17 ACE-011 -9.68 -6.45 10-Jan ACE-012 -9.56 -6.97 10-Jan w/o acetic AFA-088 17 -9.47 -6.34 8-Jan 16 ACE-013 -9.45 -6.72 10-Jan ACE-014 -9.81 -7.81 10-Jan w/o acetic AFA-089 17 -9.66 -5.20 8-Jan 32 ACE-015 -9.96 -5.27 10-Jan ACE-016 -9.63 -5.26 10-Jan w/o acetic AFA-090 32 -10.41 -6.46 8-Jan 16 ACE-017 -10.54 -6.67 10-Jan ACE-018 -10.33 -7.02 10-Jan w/o acetic AFA-105 1 -10.85 -5.81 8-Jan 17 ACE-019 -11.58 -6.86 10-Jan ACE-020 -10.89 -6.22 10-Jan w/o acetic hydroxyapatite lattice of the teeth, affecting the oxygen values or that there is some other explanation that is not yet understood. Because there is a precision measure to co mpare against, a better way to calculate the true difference of the means is not to ta ke the mean of the real differences of the 13C and 18O values for each treatment set, but inst ead, to take the mean of the absolute differences between each treatment set. Si nce there is no overall directionality in differences, when averaging a positive differe nce and a negative difference, it will drive

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117 the mean inappropriately towards zero in this case. What we are more concerned with is the overall variation between those samples wa shed in acetic acid and those which were not. When this approach is taken, the mean of the absolute differences for 13C increases to 0.38‰ and for 18O jumps to 0.45‰, giving a better indi cation of just how disparate the acetic acid effects really are. Thus said, the results appear inconclusive at this time. This issue bears further examination, with perhaps a larger sample size shedding more light upon the situation. Different teeth were used for the acetic acid test than for the bulk of the study. It therefore also presents an opportunity, although limited because of small sample size, to examine the effects of intertooth variati on. As evidenced in Table 5-9, there were comparative values for eight individuals. Two individuals utilized for the acetic acid test were of unknown natal region and hence, were not used in the main study. All samples compared underwent the acetic acid step in pr ocessing. Like the acetic acid tests, the pairs of values showed no definite directional trends. The mean 13C difference was 0.18‰ while the mean 18O difference was calculated at 0.39‰. The results of twotailed, paired t-tests were p-value = 0.100 for carbon and 0.047 for oxygen, indicating there were intertooth variations among third molars for oxygen at =0.05, but not for carbon. The same argument can be made though for the carbon and oxygen values as was presented for the acetic acid test. The a ssessment of no statistical difference may not in fact hold true since the mean difference between teeth is greater than the precision values for the PRISM. When the means for the absolute differences of 13C and 18O are computed, they increase to 0.19‰ and 0.46‰, respectively.

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118 It also bears mentioning that some variat ion of isotope ratios will arise from the bulk sampling process. Enamel is laid down sequentially in layers (Hillson 1996). When a tooth is bulk sampled, one must consider that the bulk value is itse lf an average figure corresponding to dietary intake and envi ronmental conditions over the period of amelogenesis for that specific tooth. This period of crown formation can range from nearly 4 years postpartum in first molars to over 13 years postpartum in third molars in Caucasian males (Fanning & Brown 1971). This indicates that intr atooth variation is something that must be considered when pe rforming isotope analyses. This value can be fairly large, with values cited in the lite rature of up to 8.1‰ for the same third molar, likely arising from the bulk sampling process (B eard and Johnson 2000). It is difficult to speculate just what the degree of variation is as, “…dental tissues have not been well characterized with respect to compos itional heterogeneity” (Budd et al. 2004). Heavy Isotopes Strontium and lead analyses of the NBS 987 strontium standard during the course of this study gave 87Sr/86Sr = 0.71025 (+/-0.00004, 2 ) (Dr. George Kamenov, personal communication). All lead isot ope ratios were relative to the following values for NBS 981: 206Pb/204Pb = 16.937 (+/-0.004 2 ), 207Pb/204Pb = 15.490 (+/-0.003 2 ), and 208Pb/204Pb = 36.695 (+/-0.009 2 ) (Kamenov et al. 2005). Strontium The means of the strontiu m ratios for the CIL (outlier excluded) and USAFA pools differed significantly, with a calculated p-value of 0.0013 (Table 5-1). The mean strontium value for the CIL teeth was 0.710995 while the mean for the USAFA teeth was 0.709273. For this isotope ratio, the CIL samples have a much broader range with values

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119 flanking either side of the USAFA samples (Table 5-10). The USAFA samples displayed a great deal of uniformity, even though the United States varies considerably geomorphologically. The mean 87Sr/86Sr ratio for the Air Force Academy was similar to Table 5-10. Strontium isotope values for CI L and USAFA samples, in ascending order. Identifier Location 87Sr/86Sr 87Sr Identifier Location 87Sr/86Sr 87Sr CIL-032 Cambodia 0.706811 +33 AFA-109 Lima 0.707449 +42 CIL-020 Vietnam 0.708047 +50 AFA-047 Philippines 0.707483 +42 CIL-025 Vietnam 0.708568 +58 AFA-173 CA 0.707969 +49 CIL-047 Vietnam 0.708581 +58 AFA-086 HI 0.708247 +53 CIL-039 Philippines 0.708864 +62 AFA-003 Puerto Rico/FL 0.708348 +55 CIL-037 Cambodia 0.708943 +63 AFA-111 CA 0.708464 +56 CIL-033 Cambodia 0.709033 +64 AFA-184 Bulgaria 0.708569 +58 CIL-052 Vietnam 0.709052 +65 AFA-063 AK 0.708616 +58 CIL-060 Vietnam 0.709283 +68 AFA-032 Alberta/OR 0.708770 +61 CIL-001 Vietnam 0.709534 +71 AFA-004 TN 0.708783 +61 CIL-043 Vietnam 0.709577 +72 AFA-075 Ger/Belg/GA 0.708820 +61 CIL-058 Vietnam 0.709657 +73 AFA-174 KY 0.708919 +63 CIL-049 Vietnam 0.709716 +74 AFA-176 FL 0.708938 +63 CIL-034 Cambodia 0.709750 +75 AFA-163 TX 0.708971 +63 CIL-042 Vietnam 0.709872 +76 AFA-023 Guam 0.708984 +64 CIL-055 Vietnam 0.709885 +76 AFA-089 FL 0.709012 +64 CIL-054 Vietnam 0.709989 +78 AFA-025 MI 0.709149 +66 CIL-021 Laos 0.710185 +81 AFA-164 GA 0.709188 +67 CIL-005 Vietnam 0.710193 +81 AFA-134 MS 0.709264 +68 CIL-046 Vietnam 0.710207 +81 AFA-056 MN 0.709333 +69 CIL-050 Vietnam 0.710336 +83 AFA-021 AZ 0.709355 +69 CIL-004 Vietnam 0.710645 +87 AFA-116 CA 0.709388 +69 CIL-040 Solomon Isl. 0.710747 +89 AFA-133 AL 0.709439 +70 CIL-003 Vietnam 0.710781 +89 AFA-146 ID 0.709499 +71 CIL-009 Solomon Isl. 0.710798 +89 AFA-096 VA 0.709574 +72 CIL-011 Vietnam 0.710954 +92 AFA-006 CA 0.709597 +72 CIL-048 Vietnam 0.711485 +99 AFA-060 NE 0.709994 +78 CIL-012 Vietnam 0.712397 +112 AFA-085 CT 0.710012 +78 CIL-013 Laos 0.712672 +116 AFA-220 Suriname 0.710045 +79 CIL-029 Korea 0.712772 +117 AFA-017 AZ 0.710052 +79 CIL-044 Vietnam 0.713442 +127 AFA-078 VT 0.710055 +79 CIL-015 Laos 0.714369 +140 AFA-031 CO 0.710130 +80 CIL-059 Vietnam 0.714642 +144 AFA-051 CO 0.710463 +85 CIL-028 Korea 0.716412 +169 AFA-103 CO 0.710606 +87 CIL-007 Vietnam 0.718229 +195 AFA-143 Korea 0.711171 +95 CIL-026 Korea 0.721172 +237 AFA-148 Korea 0.711186 +95

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120 the value for open ocean water (0.709165), wher e the isotopic concentration of Sr does not change detectably from place to place (S tille & Shields 1997). This phenomenon will be explored in greater detail in the following chapter. Beard and Johnson (2000) state, “In or der to facilitate comparison of the numerically small differences in 87Sr/86Sr ratios, Sr isotop e compositions may be presented in 87 notation,” defined as: 87Sr = ([87Sr/86Sr]MEASURED/[87Sr/86Sr]BULK EARTH 1) 10,000 (5-2) where the [87Sr/86Sr]MEASURED is the measured 87Sr/86Sr and the [87Sr/86Sr]BULK EARTH is equal to 0.7045. The analytical uncertainty in terms of 87Sr values are 0.2 to 0.4 87Sr units (Beard & Johnson 2000). When examined this way, it is a bit easier to visualize the range of values for East Asia, from a Cambodian 87Sr value of +33 to a high of +237 from an individual thought to be of Korean origin. This contrasts marked ly to the USAFA samples which ranged from +42 from someone from Lima, Peru, to + 95 for the two individuals who grew up in Korea. The consistency of the USAFA Korean values is interes ting, although they are not quite congruent with the CIL Korean values, which ranged from 87Sr = +117 to +237. It is interesting to not e though, that both sets of va lues were at the high end for their respective sampling groups, while the valu es for the individuals thought to be and known to be of Filipino origin were both at the low end of the spectrum. These two individuals displayed a 87Sr difference of 20 for the CIL and USAFA value from the Philippines, although compared to the other i ndividuals sampled, they still trend fairly

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121 closely as represented in Fi gures 5-7 and 5-8. Furthermor e, while the two individuals disinterred from the Solomon Islands overlap with the Laotian and Vietnamese values, they cluster very tightly, both with 87Sr values of +89 (87Sr/86Sr = 0.710798 and 0.710747). These disparities in 87Sr values do not however, mean that the subset values of each population do not necessarily agree with ea ch other. One must be careful when inferring information from point values for stro ntium isotope ratios. National or regional borders, by and large, do not conform to the delineations of geological formations. The result is that one nation or component thereo f, may have several very diverse strontium values that are all equally valid. For instan ce, in mountainous regions there is often very young rock abutting very old rock, due to the geological upheava l that caused the 37 37.2 37.4 37.6 37.8 38 38.2 38.4 38.6 38.8 39 0.7060.7080.710.7120.7140.7160.7180.720.722 87Sr/86Sr208Pb/204P b CIL USAFA Figure 5-7. Plot of strontium values compared to 208Pb/204Pb. Solomon Islands Korea Suriname Philippines Vietnam Philippines Korea Korea Korea Laos Vietnam Cambodia Vietnam 206Pb / 204Pb

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122 16.5 17 17.5 18 18.5 19 19.5 20 0.7060.7080.710.7120.7140.7160.7180.720.722 87Sr/86Sr206Pb/204P b CIL USAFA Figure 5-8. Plot of 87Sr/86Sr compared to 206Pb/204Pb. mountain formation and erosional factors. This can lead to very diverse strontium values within a very small area (Dr. Brian Beard, personal communication). This concern is echoed by Budd et al. (2004),who maintain this “…also means that simplified geological mapping and a knowledge of strontium isotope geochemistry of rocks cannot always be reliably used to estimate the expected range of food 87Sr/86Sr for any particular locality.” When examining strontium values then, it is unwise to eliminate an area simply because the strontium isotope ratios differ, unless one is certain of the geological formations underlying that specific locale and its associated 87Sr/86Sr value. If a specific area is of interest, often the best comparativ e method is to determine reference values for strontium via soil leachates, wate r sampling, or through testing the 87Sr/86Sr of fauna feeding on local resources, due to strontium’s apparent lack of frac tionation in biological 206Pb / 204Pb

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123 systems (Toots & Voorhies 1965, Ambros e 1993, Carlson 1996, Hertz & Garrison 1998, Beard & Johnson 2000, Budd et al. 2004). Looking at the box and whisker plot (Figur e 5-9), it appears th at strontium does a good job of distinguishing among most of the East Asian nationalities. Cambodia and Laos are distinct groups with no overlap, ev en though they share a common border. The CIL Korean samples present a third discrete group if CIL-029, the outlier of questionable origin, is removed from consideration (although this is the closest value to the USAFA Korean values). While the number of indivi duals in each of these groups was small, the results do look promising. Twenty-three individu als believed to be of Vietnamese origin, comprised the group with the widest spread, although as previously mentioned, the true provenance in many cases is questionable. 0.705 0.707 0.709 0.711 0.713 0.715 0.717 0.719 0.721AK Al berta Bulgaria CO N US Ger m any/ Bel gi um Guam H I Li ma Puerto Ri c o Surinam e Phi l lipines (U S AFA) P hi llipines (CIL) S olom o n Isl. K or ea ( U S A F A ) Korea ( CIL) Cambodia Laos Vi etn amLocation87Sr/86Sr Figure 5-9. Box and whisker plot of 87Sr/86Sr values.

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124 Table 5-11. Comparison of the means for multiple runs of the GLM procedure for 87Sr/86Sr. (CIL outlier excluded.) Population N LS MeanStd Dev CIL 350.7109950.002938 USAFA 360.7092730.000883 CIL 350.7109950.002938 USAFA (US) 300.7092650.000654 USAFA (foreign) 60.7093170.001725 CIL 350.7109950.002938 USAFA (CONUS) 270.7093370.000642 USAFA (OS) 90.7090830.001420 *, †, or ‡ indicates signifi cant difference between means Three iterations of the GLM procedure were also run on the strontium analysis with a Tukey’s studentized range test to control fo r Type I experiment-wise error (Table 5-11). When the USAFA cohort was split into its Amer ican and foreign constituents, it did not markedly change the mean of the USAF A group, although it did reduce the standard deviation from 0.00088 to 0.00065. A comparison of the means of these two sample sets with the East Asians revealed the only si gnificant difference between means was among the East Asian teeth and th e American teeth, at an =0.05. When the nonCONUS Americans were combined with the foreign individuals however, the pattern remained unchanged. Again, significant diffe rences were only cal culated between the means for the CIL and CONUS groups. Attempts at spiking six samples to determine strontium concentrations were unsuccessful and further tests were not complete d due to time and monetary constraints. Crude concentrations of the heavy isotopes however, were derived based on the solution concentration aspirated into the MC-ICP-M S, the resultant voltage generated by the solution, and the NBS-987 standard. The data is semi-quantitative at best and true values

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125 could vary by as much as 30% to 40% (Dr. George Kamenov, pe rsonal communication) so the calculated strontium abundances hold little value themselves (see Appendix F for the raw data). They can be used comparativ ely however, to assess any major trends that may occur between the study populations. As ca n be seen from Figure 5-10, there is little difference between the strontium concentratio ns for the East Asians and the Academy personnel, although it does appear that the East Asians may have incorporated slightly more strontium into their tissues overall. Lead The MC-ICP-MS utilized for this study calcu lated five different lead isotope ratios (208Pb/204Pb, 207Pb/204Pb, 206Pb/204Pb, 208Pb/206Pb, and 207Pb/206Pb) and all five ratio means varied significantly between the CIL (out lier excluded) and USAFA sample groups (Table 5-1). The lead values may be found in Tables 5-12 and 5-13. The ranges of each of the lead isotope ratios for the CIL sa mples encompassed and extended beyond the 1 10 100 1000 Individual samplesSr (ppm) Figure 5-10. Comparative hi stogram of CIL and USAFA sa mple Sr concentrations (semi-quantitative). CIL USAFA

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126 Table 5-12. Lead isotope results for East Asia. Identifier Location 208Pb/204Pb 207Pb/204Pb 206Pb/204Pb 208Pb/206Pb 207Pb/206Pb Pb (ppm) CIL-001 Vietnam 37.9252 15. 5873 18.0996 2.09536 0.861194 1.20 CIL-003 Vietnam 38.0377 15.6045 18.1128 2.09999 0.861511 10.95 CIL-004 Vietnam 37.9448 15.5940 17.9290 2.11644 0.869753 4.23 CIL-005 Vietnam 38.0687 15. 6119 18.0142 2.11324 0.866669 6.63 CIL-007 Vietnam 38.1584 15. 6223 18.1142 2.10659 0.862426 4.62 CIL-009 Solomon Isl. 37. 2440 15.5288 17.4072 2.13968 0.892138 3.80 CIL-011 Vietnam 38.3545 15. 6395 18.1923 2.10838 0.859685 1.06 CIL-012 Vietnam 38.1442 15. 6135 18.1051 2.10681 0.862370 4.27 CIL-013 Laos 38.0637 15. 6050 18.0478 2.10912 0.864676 26.11 CIL-015 Laos 38.4167 15. 6958 19.6205 1.95801 0.799982 7.17 CIL-020 Vietnam 38.0458 15. 5897 18.0863 2.10365 0.862003 0.38 CIL-021 Laos 38.3389 15. 6322 18.1981 2.10679 0.859001 15.40 CIL-025 Vietnam 38.1107 15. 5941 18.2642 2.08661 0.853807 0.77 CIL-026 Korea 37.8117 15. 5544 17.3830 2.17529 0.894798 29.76 CIL-028 Korea 38.3060 15. 5431 16.9917 2.25445 0.914750 154.29 CIL-029 Korea 38.6656 15. 6467 18.0841 2.13804 0.865201 15.19 CIL-032 Cambodia 38.6853 15.6604 18.6979 2.06905 0.837586 42.92 CIL-033 Cambodia 38.3004 15.6251 18.2273 2.10127 0.857229 3.88 CIL-034 Cambodia 38.1621 15.6191 18.1597 2.10145 0.860097 248.03 CIL-037 Cambodia 38.1068 15.5942 18.1265 2.10229 0.860297 1.48 CIL-039 Philippines 37.1761 15.5292 17.4327 2.13262 0.890823 16.19 CIL-040 Solomon Isl. 37. 2649 15.5358 17.4544 2.13498 0.890065 0.68 CIL-042 Vietnam 38.5029 15. 6681 18.3566 2.09778 0.853647 1.73 CIL-043 Vietnam 37.9709 15. 5875 18.1291 2.09451 0.859806 0.28 CIL-044 Vietnam 38.1647 15. 6248 18.0883 2.10989 0.863783 9.48 CIL-046 Vietnam 38.0230 15.6015 18.0714 2.10403 0.863311 6.76 CIL-047 Vietnam 37.5495 15.5523 17.7935 2.11018 0.874050 3.24 CIL-048 Vietnam 38.2292 15.6148 18.2441 2.09542 0.855873 9.48 CIL-049 Vietnam 38.3071 15.6244 18.3712 2.08515 0.850476 14.40 CIL-050 Vietnam 38.1379 15. 6038 18.1940 2.09623 0.857674 15.43 CIL-052 Vietnam 38.0652 15.5983 18.1806 2.09378 0.857982 4.17 CIL-054 Vietnam 38.1141 15.6007 18.1955 2.09474 0.857416 5.24 CIL-055 Vietnam 38.1058 15.5968 18.2384 2.08929 0.855162 20.07 CIL-058 Vietnam 37.9591 15.5998 17.9428 2.11556 0.869424 7.28 CIL-059 Vietnam 38.8837 15. 6956 18.6266 2.08757 0.842655 4.71 CIL-060 Vietnam 37.9158 15. 5826 18.0817 2.09690 0.861787 1.73 USAFA samples in both directions, but when gr aphed on a scatter plot, it is evident that the two populations cluster along different sl opes (Figures 5-11 through 5-13). The CIL Koreans tended to separate out from the main cluster of CIL ratios and did not align with the USAFA Koreans. The values from the So lomon Islands always paired tightly and

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127 Table 5-13. Lead isotop e results for USAFA. Identifier Location 208Pb/204Pb 207Pb/204Pb 206Pb/204Pb 208Pb/206Pb 207Pb/206Pb Pb (ppm) AFA-003 Puerto Rico/FL 38. 2995 15.6399 18.6511 2.05352 0.838572 0.14 AFA-004 TN 38.4127 15.6477 18.8067 2.04244 0.832003 0.06 AFA-006 CA 38.3241 15.6381 18.6474 2.05520 0.838605 0.14 AFA-017 AZ 38.4275 15.6492 18.8189 2.04192 0.831561 0.09 AFA-021 AZ 38.3369 15.6418 18.6627 2.05420 0.838178 0.11 AFA-023 Guam 37.9804 15.5983 18.2690 2.07886 0.853778 0.04 AFA-025 MI 38.5135 15.6641 18.9965 2.02739 0.824583 0.40 AFA-031 CO 38.3722 15.6484 18.8110 2.03990 0.831875 0.43 AFA-032 Alberta/OR 38.2715 15.6369 18.6870 2.04802 0.836784 0.15 AFA-047 Philippines 37.3983 15.5568 17.6810 2.11503 0.879814 0.68 AFA-051 CO 38.3673 15.6436 18.7789 2.04311 0.833015 0.14 AFA-056 MN 38.3927 15.6595 18.7845 2.04391 0.833626 0.02 AFA-060 NE 38.6165 15.6749 19.0493 2.02726 0.822855 0.08 AFA-063 AK 38.2360 15.6316 18.6329 2.05207 0.838921 0.19 AFA-075 Ger/Belg/GA 38.0297 15.6013 18.2338 2.08576 0.855654 0.14 AFA-078 VT 38.3427 15.6374 18.6884 2.05170 0.836785 0.09 AFA-085 CT 38.2804 15.6291 18.5967 2.05846 0.840443 0.17 AFA-086 HI 38.2462 15.6274 18.5617 2.06049 0.841929 0.26 AFA-089 FL 38.3107 15.6324 18.6480 2.05440 0.838312 0.24 AFA-096 VA 38.3654 15.6432 18.7485 2.04639 0.834396 0.11 AFA-103 CO 38.2570 15.6279 18.6099 2.05566 0.839792 0.32 AFA-109 Lima 38.2572 15.6141 18.3808 2.08138 0.849489 0.50 AFA-111 CA 38.2731 15.6260 18.6255 2.05501 0.838940 0.11 AFA-116 CA 38.2845 15.6308 18.7206 2.04500 0.834949 0.95 AFA-133 AL 38.2865 15.6256 18.6444 2.05352 0.838097 0.05 AFA-134 MS 38.4202 15.6564 18.8606 2.03705 0.830099 0.05 AFA-143 Korea 38.1280 15. 6167 18.3654 2.07610 0.850340 0.35 AFA-146 ID 38.2489 15.6265 18.5676 2.05995 0.841478 0.06 AFA-148 Korea 38.1538 15. 6119 18.1632 2.10062 0.859540 0.60 AFA-163 TX 38.4059 15.6306 18.6732 2.05679 0.837086 0.12 AFA-164 GA 38.3386 15.6357 18.7481 2.04504 0.834003 0.09 AFA-173 CA 38.1825 15.6251 18.4710 2.06718 0.845922 0.14 AFA-174 KY 38.3703 15.6463 18.7275 2.04896 0.835468 0.20 AFA-176 FL 38.3703 15.6431 18.7920 2.04169 0.832413 0.09 AFA-184 Bulgaria 38.5500 15.6315 18.4734 2.08681 0.846187 2.60 AFA-220 Suriname 37.6305 15.5767 17.8462 2.10865 0.872829 0.92 were closely positioned with the CIL ratios associated with the Philippines. One outlying value is believed to be Laotian a nd quite evident when the plot of 208Pb/206Pb compared to 207Pb/206Pb is examined. It is possible this pe rson incorporated a lead signal from an object fashioned from foreign lead deposits, no t encountered by others in the East Asian

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128 37 37.2 37.4 37.6 37.8 38 38.2 38.4 38.6 38.8 39 16.51717.51818.51919.520 206Pb/204Pb208Pb/204Pb CIL USAFA Figure 5-11. Plot of 206Pb/204Pb compared to 208Pb/204Pb. 15.52 15.54 15.56 15.58 15.6 15.62 15.64 15.66 15.68 15.7 15.72 16.51717.51818.51919.520 206Pb/204Pb207Pb/204Pb CIL USAFA Figure 5-12. Plot of 206Pb/204Pb compared to 207Pb/204Pb. Korea Vietnam Suriname Korea Laos Solomon Islands Philippines Philippines

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129 0.78 0.8 0.82 0.84 0.86 0.88 0.9 0.92 0.94 1.91.9522.052.12.152.22.252.3 208Pb/206Pb207Pb/206Pb CIL USAFA Figure 5-13. Plot of 208Pb/206Pb compared to 207Pb/206Pb. group. This individual did not have very el evated bone lead levels though, thus it is difficult to speculate further why there is such a disparity in lead values for this one sample. Multiple runs of the GLM procedure were al so performed on each of the lead ratio means in the same fashion as the other isotop e ratios (Table F-3). Dividing the USAFA group into its US and foreign components produced the following results: 208Pb/204Pb Only the East Asian and U.S. group means demonstrated significant differences. 207Pb/204Pb 206Pb/204Pb 208Pb/206Pb 207Pb/206Pb Significant differences were found between the means of the East Asian samples and the U.S. samples and between the U.S. samples and the foreign samples. No statisti cal difference among the East Asian and foreign USAFA means was calculated. Dividing the USAFA group into its C ONUS and overseas components produced the same effect for all five lead isotop e ratios. Significant differences were found Laos Korea Korea

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130 between the means of the East Asian samp les and the CONUS samples and between the CONUS samples and the overseas samples. The East Asian and overseas means however, were statistically indistinguishable. For the table of separate group means by lead isotope, please see Appendix F. Semi-quantitative concentrations were also calculated for lead in a similar fashion to the strontium concentrati ons (see Appendix F for the raw data). While the strontium showed little variation among popul ations, the lead content of the CIL teeth appears to be an order of magnitude or two higher than the USAFA participants (Figure 5-14). Keeping in mind the large potential for error (30%-40%), the East Asian concentrations showed a very large range of values, from 0.3 ppm to 248 ppm, with two individuals showing lead contents above 43 ppm, one at 154.3 ppm and one at 248 ppm. It is difficult to say why the lead abundances in th ese two individuals are so high. They have obviously had considerable environmental interaction with lead substances, perhaps through lead-based glazes on ear thenware, lead pipes, or tin-soldering used in canned food items. Lead concentrations of approximately 30 ppm in bone correspond to a blood lead level of 10 g/dL (Smith & Flegal 1992). Th is blood level is considered the cutoff for lead toxicity, which often manifests itself in various developmental, skeletal, and neurological problems, especia lly in children (Smith & Flegal 1992). It is difficult to say how this figure directly equates to enam el values, although in a study of the lead concentration of multiple tissues, cortical bone abundances were found to be an average of 34 ppm greater than dentin for the same individuals (Bower et al. 2005).

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131 0.0 0.1 1.0 10.0 100.0 1000.0 Individual SamplesPb Concentration (ppm) Figure 5-14 Comparative hist ogram of CIL and USAFA sa mple Pb concentrations (semi-quantitative). Lead abundances as high as 340 ppm have b een recorded in the deciduous incisors of modern children residing near a lead-sme lting works in the United Kingdom (Delves et al. 1986). High lead levels were also found among 18th century Omaha Indian remains, where rib samples from 12 individuals demonstrated lead conc entrations near or above 200 ppm, 3 of which were clinically unprecedented measuring higher than 800 ppm (all children), with the highest at an incredible 2,567 ppm (Reinhard & Ghazi 1992). This lead contamination may have arisen from either the practice of molding lead musket balls by mouth or through the preburial pa inting of corpses w ith red, lead-based pigments, resulting in diagenetic changes to the bone lead levels (Reinhard & Ghazi 1992). The abundance of lead found in human sk eletal remains increased between the Medieval ages and the 19th century, from low prehistoric levels to values 10–100 times higher for countries within Europe, the Amer icas, and Asia (Jaworowski 1990). Overall, lead concentrations in hu man bone decreased in the 20th century to nearly prehistoric CIL USAFA

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132 levels. This contrasts with rising production and environmen tal emissions of lead-based products though, implying that the inhalation route of lead intake makes only a minor contribution to total lead intake by humans (Jaworowski 1990). The Air Force Academy values were, with one exception, all less than 1 ppm, with most in the range of 0.1 to 0.2 ppm. The mean lead concentration for the Academy cohort was 0.30 +0.46 ppm. In comparison, the lead content of enamel from Neolithic human teeth has been measur ed at approximately 0.31 + 0.04 ppm (Budd et al. 2000). The only USAFA value above 1 ppm was 2.6 ppm and belonged to a female who primarily resided in Bulgaria and Libya. According to Dr. George Kamenov, a native Bulgarian, the town of Nova Zagora where this person grew up is a major vegetable producing area (personal communication). Assumi ng the inconsistency of this value with the rest of the USAFA lead contents is not a result of error on the part of the semiquantitative estimate, it may be that the highe r lead concentration in this individual is related to higher vegetable consumption. Sh e did have the second most depleted carbon value of 222 USAFA samples run, at -12.69. It is obvious from the 13C measure that she had a very small C4 constituent to her diet. Higher vegetable intake may well have contributed to this relatively high value alt hough Aufderheide et al. do (1981) state, “The minute quantity of lead in plants, animals, and water does not normally exceed the capacity of human excretory mechanisms to prevent significant accumulation in the body.” Pollution effects, therefore must not be ruled out based on her residency in Eastern Europe and Libya. Regardless of why her lead concentration value is higher than the rest of the USAFA cohort, she is s till within the range of normal values.

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133 Table 5-14. Comparison of spiked lead concentr ation data (actual) with semi-quantitative data. (All values are in ppm.). Identifier Actual Semi-quant CIL-001 1.441.20 CIL-004 7.184.23 CIL-046 22.746.76 CIL-048 10.469.48 CIL-052 15.434.17 CIL-058 6.797.28 Six CIL samples, all thought to be Vietnamese, were successfully spiked for concentration analysis. All six va lues were above 1 ppm. As can be seen from Table 5-14, some of the semi-quantitative cal culations were fairly close (e.g. CIL-001 or CIL-058), while others were considerably off (e.g. CIL-046). Regardless of the numerical disparity, what is evident is that th e true lead concentration results demonstrate the semi-quantitative data is wi thin the correct range of valu es, albeit a little low from the table data. No actual CIL values showed a nything lower than 1.44 ppm. This seems to validate the finding from the semi-quantitative da ta, that there is a difference of at least an order of magnitude between the lead con centrations of the CIL teeth and the USAFAprovided teeth. This divergence of lead con centration values was quite unexpected and presents yet another possible avenue for distinction among Ea st Asians and Americans. Multi-element Approach Because the uptake of the four isotopes discussed differs based on cultural dietary preferences, geography, and geology, it only ma kes sense that as you increase the number of isotopes included in your analysis, you increase the discriminatory power. An example of this is the American from Ida ho who overlaps the East Asian carbon values. When the individual’s oxygen signature is examined in concert with his 13C value, it becomes clear as to which population he belongs.

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134 One of the goals of this study was to deve lop a linear discrimi nant function for stable isotopes that would quickly allow the CIL to assess the natal origins of a set of purported American service member remains ba sed on enamel isotope ratios. A linear discriminant function was created utilizing a ll of the CIL samples except the CIL outlier and the 222 USAFA participants who were rais ed for at least a portion of amelogenesis within the U.S. and its territo ries. Carbon and oxygen values were available for all of these individuals. In additi on, the strontium data and measurements of all five lead isotope ratios were incorporat ed into the equation for 35 of the CIL samples and 30 of the USAFA samples. The discriminant function was tested th rough resubstitution, whereby the same data set used to derive the discriminant functi on was run back through the formula (SAS 9.1). After resubstitution, 2 of 65 individuals were incorrectly classified as belonging to the wrong population. One CIL sample was incorr ectly identified as being American, and one USAFA sample was misidentified as East Asian. This equate d to a 97.14% correct classification rate for East Asians and a 96.67% correct classification rate for Americans, with an overall error rate fo r both populations of 3.08%. The misclassified CIL sample was CIL-052, an individual recovere d from Vietnam. This individual had the highest 13C value of the 35 CIL samples with values for all isotope ratios studied and the third highest value of the en tire compliment of 61 East Asian samples, excluding the extreme outlier. All of the heavy isotope ratios were very near the median values for the East Asian group, although all of the lead values tracked similarly to AFA-075.

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135 The only American misidentified was AFA-075, a self-reported Caucasian who spent the first 8 years (of 11 total) of third molar amelogenesis in Germany and Belgium. As previously mentioned, her carbon values ove rlapped the East Asian cluster. For every single lead value, this in dividual fell squarely among th e foreign USAFA samples and was the minimum American USAFA value for 206Pb/204Pb, the second lowest value behind Guam for 208Pb/204Pb and 207Pb/204Pb, and the maximum value for 208Pb/206Pb and 207Pb/206Pb. Each of this individual ’s lead values was well within the range of all of the CIL samples. Cross-validation was also performed to veri fy the discriminant function. This test is not as biased as resubstitution, since cla ssifies each observation using a discriminant function compiled from all of the other data, so in essence, each obs ervation is classified based on a slightly different equation. Af ter running all of the individuals from the original data set, two of the CIL sample s were misclassified as American and one USAFA sample was incorrectly classified as being of East Asian or igin. This led to correct classification rates of 94.29% for the East Asian samples and 96.97% for the American samples, with an overall error rate of 4.62%. All thr ee of the individuals already spoken to were misi dentified along with CIL-015. Th is individual was recovered from Laos. The 206Pb series for this person was significa ntly different than all other East Asian samples (Figures 5-11 throug h 5-13) with this individual’s 206Pb/204Pb, 208Pb/206Pb, and 207Pb/206Pb values all at the end of their resp ective spectrum for the East Asian group. In the case of each of these three isotopes, there is a much closer affinity of this individual with the American values compared to its East Asian cohort.

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136 This misclassification of CIL-015 and AFA-075 highlights a problem with this approach: of the eight isotope ratios used for the discrimi nant function, five are lead values and they are highly interrelated. Assu ming equal weight of all the variables, this gives undue influence to the lead ratios over the other more di screte isotopes. To combat this, the discriminant functi on was recalculated using only tw o of the five lead isotope ratios, 208Pb/204Pb and 207Pb/206Pb. These two were chosen because they represent all four stable lead isotopes. The isotopes of 206Pb and 207Pb reflect the signa ture of lead ore bodies, from which industrial lead is derived (Stille & Shields 1997). An added utility of this ratio is that it can be used as a ch eck for anthropogenic lead contamination. These isotopes differ from 204Pb and 208Pb, which reflect the signature of average crustal rock, and have a markedly different lead isot opic composition from ore bodies (Stille & Shields 1997). A new discriminant functi on was run utilizing only 208Pb/204Pb and 207Pb/206Pb, with only slightly better resu lts than the equation which incorporated all of the lead isotopes. The resubstitution procedure produc ed the exact same results as running the discriminant function with a ll five lead isotope ratios with an overall correct classification rate of 96.92%. The same two individuals, CIL-052 and AFA-075 were misclassified. This formula did vary how ever, under cross-valida tion. Here, CIL-015 was correctly classified, thus the results mi rrored those of the res ubstitution step. To reiterate then, utilizing only two isotope ratios instead of five resu lted in a 97.14% correct classification rate for East Asians and a 96.67% correct classification rate for Americans, with an overall error rate fo r both populations of 3.08%.

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137 One iteration of the discriminant functi on was run with only the Southeast Asian data (results from Korea, the Philippines, and Solomon Islands were excluded) and two lead isotopes. This changed the numbers sampled from each population to 30 each. After a resubstitution was performed, only AFA-075 was misclassified. Correct classification rates were 100% for Southeast Asians and 96.67% for Americans, with an overall error rate of 1.67%. Cross-validati on produced greater erro r rates as CIL-015 and AFA-075 were again misclassified, as well as CIL-052. Of the Southeast Asians sampled for all isotopes, CIL-052 r ecorded the most depleted 13C value (-12.20‰). All other isotope values were well within their respective ranges. With these three misclassifications, the error rates for crossvalidation were 6.67% for Southeast Asians, 3.33% for Americans, and 5% overall. While very promising, there are several po ints of consideration that must be addressed before this protocol is put into use. First, the sample sizes must be increased to improve the statistical robusticity of this equati on. As it currently stands, this formula is based only on the isotopic results of 66 individu als total. Weights need to be calculated for each of the isotope variables to understa nd what the impact of each variable is upon the equation. It would also be beneficial to create a quadratic discriminant function to enable use of the formula with missing data. The USAFA data has not been proven as an accurate proxy for Vietnam-era servicemen. Additional sampling needs to be conducted from the peers of those individuals we are seeking to identify. This is essential to identify what, if any, temporal effects ex ist between the USAFA donors and those still listed as missing in Southeast Asia. The ol dest individual sample d from the Air Force Academy was born in 1964, 3 year s after the conflict in Viet nam began (Reports 1985).

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138 Finally, a sex effect was noted for the 13C value (discussed in Chapter 6). As there are no female service members and only two civi lian females listed as missing in Southeast Asia it would be beneficial to include a co rrection factor in the equation to account for this observation.

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139 CHAPTER 6 VARIATION WITHIN USAFA SAMPLES The second aim of this study was to see if regional differences in natal isotopic signatures were discernable w ithin populations raised wi thin the U.S. Two-hundred twenty-two individuals fit this criterion (T able 6-1). The general linear model (GLM) procedure was run on the United States Air Force Academy (USAFA) subset of American-origin to determine if there were any effects evident based on the variables generated by the survey questions. In terms of this study, origin refers to locales during the period of amelogenesis of the third molar, which ranges from age 7 to age 18 (Fanning & Brown 1971). Tests of the diffe rences between means were run for the different responses for each question. The su rvey questions analyzed asked for date of birth, sex, race (open ended), tobacco use history, diet, and residency. Year of Birth Year of birth for the American partic ipants ranged from 1964–1987. This age distribution lacks the older ages found in the general military population, although individuals between 18 and 25 years-old do comprise roughly 60% of the military (Friedman et al. 1989). Of the 221 individuals who provided their birth year, all but seven were born in or after 1980. Those born during the 1980s are most indicative of the ages found within the cadet wing for the academic year 2005/2006. No significant differences in birth year were found with re spect to the carbon and oxygen mean values. It is highly possible though, that this is due to the large bias in the data, as 75% of all participants

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140 Table 6-1. USAFA-provided sampling demographics, American natal region only. Question Response Total Male Female Participants 222 173 49 Year of birth 1964 1 0 1 1968 1 1 0 1973 1 1 0 1975 1 1 0 1976 1 1 0 1979 2 1 1 1980 4 2 2 1981 5 4 1 1982 12 8 4 1983 45 38 7 1984 73 57 16 1985 47 35 12 1986 23 18 5 1987 5 5 0 No response 1 1 0 Race Native American 0 0 0 Caucasian 177 142 35 African American 10 8 2 Hispanic 12 10 2 Asian 11 7 4 Mixed 7 3 4 No response 5 3 2 Tobacco use Non-user 178 133 45 User 42 38 4 No response 2 2 0 Diet Meat eater 218 177 49 Vegetarian 1 0 1 Vegan 0 0 0 Mixed meat/ vegetarian 1 0 1 No response 2 1 1 were born between 1983 and 1985. Of the 30 i ndividuals sampled for the heavy isotopes, the year groups 1964, 1975, and 1982–1987 were repr esented. No significant differences for the means of strontium or any of the le ad stable isotope means were observed for these age cohorts.

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141 Sex Among the 173 males and 49 females sample d, a significant difference in mean carbon values was noted at a p-va lue of 0.0021. Males had a mean 13C value of -9.84‰ while females had a mean 13C of -10.21‰. No difference between the sexes was noted for oxygen. Sex differences in 13C have been noted in the li terature, but these are from ancient populations where it has been hypothe sized that the delta value incongruence was likely the result of different feeding regimes associated with social strata based on sex (Hedman et al. 2002). In laboratory studies it does not appear that there is any metabolic difference between the sexes in how they assi milate carbon isotope si gnatures into their tissues (Schwarcz and Shoeninger 1991). Fo r the case of the sex differences among the USAFA participants, it is likely that there is some difference in dietary intake among men and women, although all but tw o respondents indicated they grew up on a diet that included meat products. One female stated she was raised vegetarian, and one female indicated she switched to vege tarianism at the age of 12. The females have a more depleted mean 13C, which indicates less of a prevalence of C4-based foods in their diet. No female was found within the top 17% (most enriched) of the 13C values for the American group. This may be explained by the simple fact that while the women in this study do eat corn-fed meat, they eat proportiona lly less than their male counterparts, or there may be a much more complicated dynamic involved. No sex influences were noted for strontium, but among the lead isotopes, all but 208Pb/204Pb displayed significant differences between the means at =0.05 (the p-value for 208Pb/204Pb was 0.0523). This difference though may be more of an artifact of location, than do to an actual biological diffe rence. Of the 30 sampled for heavy isotope analyses, 6 were female. Of these, one was the individual who spent most of her

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142 childhood in Germany and Belgium and the other grew up in Guam. In the case of each lead isotope, these two individuals fell s quarely among the foreign USAFA samples and were the two minimum American USAFA values for 208Pb/204Pb, 207Pb/204Pb, and 206Pb/204Pb, and the maximum American values for 208Pb/206Pb and 207Pb/206Pb, by quite a margin. These two individuals undoubtedly sk ewed the female data because of their extreme values, as the remaining four female s’ lead ratios were fairly well distributed throughout the group. It is doubtful then that sex played a role in the heavy isotope values. Race Survey respondents were asked the openended question as to what race they considered themselves. From the partic ipants’ responses, six race classifications emerged, Native American, Caucasian, African American, Hispanic, Asian, and mixed race. For a breakout of USAF A individuals, see Table 5-1. Of the 217 individuals who answered this question, there was no marked difference between the mean 13C values for any of the racial groups. This finding wa s counter to a hypothesized difference among Asian Americans, perhaps indicating less of an adherence in younger individuals to traditional Asian diets. The greatest difference between any two groups was between Asians and Caucasians, but at a p-value= 0.4188 after a Tukey-Kramer adjustment, it is far from significant. A significant difference was found for the mean 18O values between Caucasians (-6.95‰) and those of mixed ancestry (-5.14‰). After a Tukey-Kramer adjustment for multiple comparisons was completed, a pvalue of 0.0316 was calcu lated between the two groups. The mixed ancestry group consis ted of seven individuals, four of which stated they were Asian and Caucasian, two who declared they were “White/Hispanic,”

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143 and one who answered “Korean/Mexican.” Al l of these individuals spent at least a portion of amelogenesis in a southern state, except for AFA-162 (“Korean/White”) who lived in Michigan, Maryland, and California. Within California, this individual spent 4 years in the San Francisco area. Of the ot her individuals, two lived in Texas, one in South Carolina, one in California and Florida, one in Texas/Michigan/Virginia, and one in Colorado/Panama/Hawaii/Texas/ Germany. The phenomenon of a much enriched 18O mean for those of mixed ancestry is proba bly a characteristic of this study and not indicative of the group as a whole across the United States. For the heavy isotopes, the racial brea kdown for samples run was as follows: 25 Caucasians, 4 Hispanics, and 1 Asian. No significant differences were found among the mean strontium values for the three racial categories at =0.05. Differences were found among all of the lead isotope ratios except for 208Pb/206Pb. In each instance the Asian differed from both the Caucasian and Hispanic groups. In the case of 208Pb/204Pb, the differences were at p-values < 0.01. No difference was noted between Caucasians and Hispanics. Because there was only one Asian sampled for lead, the differences seen are most likely locational in nature and not racially influenced. This individual was raised in Guam, where the volcanic, island geomorphology varies dramatically from the CONUS. Tobacco Use Two hundred twenty individuals responded to questions concerning their tobacco use history. Of these, 178 reported that th ey had never used tobacco products and 42 stated they had. Two individuals did not respond to this question. A significant difference among the means for 13C (-10.00‰) was noted between the tobacco users (-10.00‰) and non-users (-9.62‰), at a p-valu e=0.0033. No significant difference for

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144 oxygen was observed. Tobacco interactions have not been addressed in the literature for isotopes to date. In addition to asking whether participants had a history of tobacco use, the survey also queried those who answered yes as to what type of product was used and the frequency, in the form of an open-ended question. From this information, the following four categories were crafted for tobacco produc t use: no history of tobacco use; inhalant use (i.e., cigarettes and cigars ); smokeless (i.e., snuff and chew ); or a mixture of inhalant and smokeless products. The mean 13C value for product type was found to be significantly different between non-users and those who partook of smokeless tobacco, at a p-value of 0.0124 after Tukey-Kramer ad justment. The means equaled -10.00‰ for non-users and -9.41‰ for smokeless users. When queried as to the difference among means for 18O, SAS returned a p-value of 0.0553 that at least one mean differed, but after a Tukey-Kramer adjustment, the grea test statistical difference was between smokeless users and users of both inhalant s and smokeless tobacco, at a p-value of 0.0818, which does not meet the a priori criteria of =0.05. Tobacco use frequency was broken down into four categories as well: never used; used at least once a day (daily); used at least once a month but less than daily (occasional); used less than once a month (rarely). The only significant difference between means when tobacco use frequency was considered was between those who never used tobacco and those who stated they rarely used tobacco products for 13C at a p-val = 0.0042. The means were -10.00‰ for non-users and -8.87‰ for those who rarely partook of tobacco products. This does not intuitively make sense since one would think the rare-use group and never-used group would have the closest means. The simplest

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145 explanation is this observation may actually be an artifact of small sample size for the rarely-used group (N=5) compared to the non-user group (N=178). After examining all the data, it appear s the tobacco effect upon carbon is probably due to the type of product used, versus the frequency. Sixteen indi viduals responded they used smokeless tobacco and an addition six st ated they used both inhalant and smokeless forms. Twelve of the sixteen who used onl y smokeless tobacco report ed they use it daily, three occasionally, and one rarely. For thos e who used both forms of tobacco products, four said they use them daily and two responded they rarely used them. It is possible that smokeless tobacco has an effect upon tooth isot ope values because the product is in much closer proximity to the teeth for a longer pe riod of time compared to inhalant forms of tobacco. Additionally, when the substance mixe s with saliva, it is transported throughout the oral cavity and adheres more to the te eth than cigarette/cigar smoke. Tobacco products are known to stain teeth so there is obviously some interchange between enamel and the oral environment. Concerns have been raised concerning possi ble diagenetic change of surface tissues in teeth (Budd et al. 1998). While no such change has been noted for strontium (Hillson 1996), Budd et al. (1998) did observe a consistent a ppearance of highly lead-enriched surface tissues within the first 30 m of surf ace enamel. They did not believe this trait was a result of chemical exch ange in the oral environment in vivo though, as the presence of a surface lead peak was also found in an unerupted, modern, permanent premolar, and instead attributed it to biogenic processes during tissue formation. Heavy isotope readings did not appear to be influenced by tobacco use, since the means of the tobacco use categories did not sign ificantly vary for any of the strontium or

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146 lead isotope ratios. Of the 30 respondents us ed in this portion of the study, none reported using both inhalant and smoke less tobacco products. Diet All but four individuals of American-origin reported they were raised on a meatbased diet. One female indicated she was ra ised a vegetarian, one stated she switched from meat to vegetarianism at the age of 12 and the other two (one male, one female) did not provide a response to this question. Becau se of this pattern of answers, tests of differences of the means were not performed fo r diet. Strontium and lead analyses were not run on the vegetarian or individual who switched feeding regimes. The 13C value of the vegetarian was in the upper 40% (more en riched) of the American group, reflecting a larger C4 constituent to the diet that the major ity of the group, while the individual who switched feeding regimes was in the bottom 25% (more depleted). Both 18O values were near the mean for the American-reared cohort. Residency The sampled USAFA pool contained 222 indi viduals originating from 43 states within the United States plus the American territory of Guam. Of these 222, 43 persons lived in multiple locations with in the United States, while 19 lived in the U.S. and abroad (Table 6-2). Only the states of Delaware Kansas, Louisiana, Maine, New Hampshire, Nevada, and Rhode Island were not represented. Strontium Based on the successful applic ation of strontium data to questions of region of origin in human archaeological assemblages (e.g. berg et al. 1998, Beard & Johnson 2000, Montgomery et al. 2005) and in a few studies of c ontemporary individuals (berg

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147 Table 6-2. Locations during amelogenesi s represented by sampled USAFA teeth. Domestic Foreign Locale N Locale N Locale N Locale N AK 1 MA 4 PA 5 Korea 1 AL 2 MD 2 SC 4 Peru 1 AR 2 MI 5 SD 1 Philippines 1 AZ 3 MN 4 TN 5 Suriname 1 CA 13 MO 1 TX 19 Intl mixc 2 Locale N Locale N Locale N Locale N CO 10 MS 1 UT 2 CT 1 MT 4 VA 3 FL 6 NC 2 VT 2 GA 8 ND 1 WA 2 Guam 1 NE 3 WI 3 HI 1 NJ 5 WV 2 IA 2 NM 3 WY 1 ID 2 NY 3 Unknown 1 IL 4 OH 6 US mixa 43 IN 2 OK 2 US/Intlb mix 19 KY 1 OR 5 States Not Represented DE KS LA ME NH NV RI a1 resided primarily in Puerto Rico, 2 included Guam b1 resided primarily in Belgium/Germany, 1 primarily in Alberta, Canada c1 resided primarily in Bulgaria, 1 primarily in Korea et al. 1998, Beard & Johnson 2000, Juarez 2005), it was hoped that strontium might prove useful for distinguishing among the Am ericans in this study. When the foreignraised were removed from consideration, th e uniformity of American values was even more evident (Table 6-3). While it has been estimated that the variation of 87Sr values for the CONUS ranges from +5 to +390 (Figur e 6-1) due to the differences in age of basement rocks (Beard & Johnson 2000), the Am erican samples for this study (including Alaska, Hawaii, Puerto Rico, and Guam) onl y extended from +49 to +80. The average value for all Americans sampled was 0.709265 ( 87Sr = +68). The mean

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148 Table 6-3. Strontium isotope values for Am erican USAFA samples, in ascending order. Identifier Location 87Sr/86Sr 87Sr Identifier Location 87Sr/86Sr 87Sr AFA-173 CA 0.707969 +49 AFA-134 MS 0.709264 +68 AFA-086 HI 0.708247 +53 AFA-056 MN 0.709333 +69 AFA-003 Puerto Rico/FL 0.708348 +55 AFA-021 AZ 0.709355 +69 AFA-111 CA 0.708464 +56 AFA-116 CA 0.709388 +69 AFA-063 AK 0.708616 +58 AFA-133 AL 0.709439 +70 AFA-032 Alberta/OR 0.708770 +61 AFA-146 ID 0.709499 +71 AFA-004 TN 0.708783 +61 AFA-096 VA 0.709574 +72 AFA-075 Ger/Belg/GA 0.708820 +61 AFA-006 CA 0.709597 +72 AFA-174 KY 0.708919 +63 AFA-060 NE 0.709994 +78 AFA-176 FL 0.708938 +63 AFA-085 CT 0.710012 +78 AFA-163 TX 0.708971 +63 AFA-017 AZ 0.710052 +79 AFA-023 Guam 0.708984 +64 AFA-078 VT 0.710055 +79 AFA-089 FL 0.709012 +64 AFA-031 CO 0.710130 +80 AFA-025 MI 0.709149 +66 AFA-051 CO 0.710463 +85 AFA-164 GA 0.709188 +67 AFA-103 CO 0.710606 +87 87Sr/86Sr ratio is alarmingly similar to the value for sea water, at 0.709165 ( 87Sr = +66) (Stille & Shields 1997), indicati ng this group as a whole is approaching the global mean for strontium or the unlikely possibility that al l have diets primarily derived from the sea. This finding seems to reinforce the orig inal fear of the potential for erroneous conclusions due to true 87Sr values being washed out by the effects of the global economy. As stated in Chapter 1, archaeological research does not us ually concern itself with such matters because food tended to be locally grown and c onsumed. After the Industrial Revolution and the es tablishment of global trade networks, food in the U.S. was very rarely grown in the localities were people lived. Younger, contemporary Americans may present a more amalgamated strontium signature because their food,

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149 Figure 6-1. Strontium isotope compos ition of the U.S. showing inferred 87Sr values, as calculated by ag e variations in basement rocks. Image reproduced from Bear d and Johnson (2000), with permission.

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150 especially produce and some meats, comes in creasingly from other states or foreign nations. Local water may provide some diet ary strontium reflective of the region in which they live, but the overall effects of this world food network dietary “noise” seem quite apparent from this study and may prove difficult to overcome in strontium analyses of contemporary populations. It would be useful to have the data from Vietnam War era individuals to see what the temporal prevalence of damped true loca l strontium signals are due to global dietary homogenization. It is assumed that older individuals would better reflect their natal strontium ratios, since U.S. food importati on was not as wide-spread in the 1940s-1960s as compared to the subjects in this study, w ho largely did not begin third molar formation until the late 1980s and beyond. By further exam ining the variation of strontium values for the same regions during different eras, we can make a better e ducated opinion as to the viability of strontium analyses both spatially and temporally. Lead No clear trend emerged among the Americans concerning their lead isotope values. As was stated in Chapter 5, national or region al borders, by and large, do not conform to the delineations of geological formations. Th e result is that one state may have several very diverse values for a single lead isotope ratio that are all equally valid. Perhaps if the sample number had been increased for the he avy isotopes, a trend would have emerged or at least a better idea of why a lack of one exists. Only 30 Americans were tested, hailing from 19 states, Puerto Rico, Guam, and th e two individuals w ho spent a significant portion of their childhood in Alberta, Canada, and the Germany/Belgium region. Of these samples, there were multiple individuals tested from four states: Arizona (2); California (4); Colorado (3); and Florida (2). In hindsight, it would have been better to

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151 test people who had lived in the same location, but since th e teeth came in over a period of 8 months, most of these relationships ha d not yet been established when the heavy isotope testing was being performed. These individuals were also compared agai nst significant lead ore deposits to rule out the lead isotope signat ures being a reflection of anthropogenic contamination. Because 206Pb and 207Pb represent the signa ture found in lead ore bodies, the ratio of 207Pb/206Pb was used. Table 6-4 lists the 207Pb/206Pb ratio for nine of the major lead mining deposits and/or district s in the United States, while Table 6-5 lists the American 207Pb/206Pb values. As can be seen, only one of the USAFA values falls near any of the lead ore values compiled by Sangster et al. (2000 ). None of the individuals who lived in the same state as a major lead mining operation appear to have incorporated the mine’s lead isotope ratios in to their tissues. The individual from Michigan has a similar 207Pb/206Pb value to the Austinville/Ivanhoe deposit in Virginia. A ll of the other lead isotope ratios for this person, with th e exception of 208Pb/204Pb, are also within the resp ective standard errors of the lead isotope ratios for the mine. At an estimated lead concen tration rate of 0.4 ppm, the person is well within the normal range, and would not have suffered from lead toxicity. It may be a coin cidence or there may have been something in the home fashioned from lead from the Virginia depos it that influenced the individual’s isotope signature, such as lead-based paint in an older home. Regionality In order to reduce the number of locales within the United States to a more manageable number, nine regions (Table 6-6 and Figure 6-2) were identified based on the 18O least squares means for location effect for the USAFA samples of American

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152 Table 6-4. Mean 207Pb/206Pb values for major U.S. lead ore deposits. Deposit/District 207Pb/206Pb Upper Mississippi Valley 0.71124 Tri-State (OK/KS/MO) 0.72706 Southeast Missouri 0.74683 Central Tennessee District 0.80308 Eastern Tennessee District 0.80632 Austinville/Ivanhoe (VA) 0.82461 Red Dog (AK) 0.84710 Shafter (TX) 0.86790 Balmat (NY) 0.91929 Data compiled by Sangster et al. (2000) Table 6-5. 207Pb/206Pb values Americans reared in th e United States, in ascending order. Location 207Pb/206Pb Location 207Pb/206Pb NE 0.822855 TX 0.837086 MI 0.824583 AL 0.838097 MS 0.830099 AZ 0.838178 AZ 0.831561 FL 0.838312 CO 0.831875 Puerto Rico/FL0.838572 TN 0.832003 CA 0.838605 FL 0.832413 AK 0.838921 CO 0.833015 CA 0.838940 MN 0.833626 CO 0.839792 GA 0.834003 CT 0.840443 VA 0.834396 ID 0.841478 CA 0.834949 HI 0.841929 KY 0.835468 CA 0.845922 Alberta/OR 0.836784 Guam 0.853778 VT 0.836785 Ger/Belg/GA 0.855654 origin (N=222). After a Tukey-Kramer adju stment for multiple comparisons, the mean 18O values of location pairs were examined and those exhibiting a p-value of 1.00 were considered for consolidation. All members w ithin a particular region share a p-value for the least squares mean for location effect of 1.00. Additionally, all st ates within a region are a contiguous, with the excep tion of region 9, which includes the islands of Hawaii and Guam. Regions are not wholly inclusive, in that some states were eligible

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153 for membership in more than one region. For instance, Arkansas would have fit with any region from 4–9. In such instances of ambiguous group membership, states were arranged in the most latitudinally contiguous fashion. Those individuals from mixed U.S. locales were included with region 7, and those whose natal regions in cluded international locales were included in region 8. Table 6-6. Region membership based on 18O values. Regions States/Locations (1) AK (2) ID/MT/ND (3) CO/UT/WY (4) AZ/CA/NM/OR/WA (5) AR/OK/TX (6) AL/FL/GA/MS (7) KY/MD/NC/SC/TN/VA/WV/US mix (8) CT/IA/IL/IN/MA/MI/MN/MO/NE/N J/NY/OH/PA/SD/VT/WI/US-int'l mix (9) Guam/HI Figure 6-2. Region map based on 18O values. 1 9 7 8 6 3 5 2 4 + Guam

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154 When the differences between the mean 13C values for region were compared, it was discovered that region 6 (southeast) diffe red significantly from regions 2 and 4, with respective p-values of 0.0208 and 0.0120. Regions 3 and 4 did not differ from one another. The means for these three regions were as follows: region 2 = -10.54‰; region 4 = -10.24‰; and region 6 = -9.42‰. The results indicate individual s in the southeast have a larger C4 component to their diets than thos e who reside in Idaho/Montana/North Dakota and on the west coast/southwest and may reflect regional food preferences (i.e., corn-based products such as grits [C4] versus potatoes [C3]). No other significant differences were found for carbon. It is intere sting to note that region 8 has a mean group 13C value of -9.93‰, which is only +0.01‰ from the American mean. As is to be expected, most of the 18O values were signifi cantly different between regions, since the regions were created based on the 18O values for each state/location. Exceptions were expected, because as previous ly stated, there was some overlap between groups as well. Each region’s mean 18O value overlapped with at least one other region (Table 6-7) at =0.05. Region 9 (Hawaii & Guam) s howed the greatest homogeneity with the other regions, overlapping with five of them. Regions 7 and 8 showed mean differences at a p-value of 0.0311. All othe rs region pairs had p-values below 0.004, 21 of which were <0.0001. All nine regions were represented among the samples run for strontium and lead, although region membership was small since the total sample size of 30 was spread across the nine regions. After a TukeyKramer adjustment, two region pairs did demonstrate significant difference for their 87Sr/86Sr means. Region 3 (Colorado/Utah

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155 Table 6-7. Region-pair co mparison for difference in 18O means. /Wyoming) varied from region 7 (central east) with a p-value of 0.0370, and region 3 varied from the islands of Hawaii and Guam (re gion 9). This is heartening news, because any difference in regional values indicates that strontium signatures are not totally washed out by the global food trade. None of the region pairs exhibited significant differences among any of the stable lead isot ope mean. This does not mean they do not exist. One must keep in mind that the samp le size was small (three regions only had one representative) and th e fact that the region break-out was based on oxygen values. It could very well be that the lead regions within the Unites States differ, thus leading to the current lead signature results. These discoveries justify c ontinued examination of, at a minimum, strontium values to see if trends mentioned above hold true with a larger pool of samples. An effort was made to create a lineardiscriminant function for regional affinity among the American samples. Using the 30 sa mples that had been run for all data and the two lead isotopes of 208Pb/206Pb and 207Pb/206Pb, rather than the full complement of five lead isotopes, the results proved bette r than anticipated. After resubstitution, the error rate for misclassificati on of region was 26.67%. All of the individuals from regions 1, 2, 3, 5, and 6 were correctly classified. Upon cross-validation how ever, the resultant Region Pair P-value 1 & 2 0.7105 1 & 3 0.0835 2 & 3 0.2286 4 & 9 0.1158 5 & 6 0.9908 5 & 9 0.9999 6 & 9 0.9929 7 & 9 0.9961 8 & 9 0.7855

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156 error rate increased dramatically to 66.67%. The validation process will always misclassify an individual who comprises th eir own group, because validation removes the observation under consideration from the pool from which the discriminant function is drawn. In the case of this study, regions 1, 2, and 5 only had one person each, and region 9 had two. Hopefully, if the data set can be expanded so as to include approximately 10 values from each state, then regional groupings can be better drawn with more discriminating power. This analysis also highlights the f act that if the object is to maximize the effectiveness of the discrimina nt function, then perhaps the criteria of contiguous borders should be abandoned and Hawaii and Guam incl uded in one of the other groups as with the U.S. mix or U.S./international groups. Relationship Between 18O Values and Latitude As stated elsewhere, a rough latitudinal cline appears to have emerged for the USAFA 18O values, with the higher latitudes sh owing more depletion and the lower latitudes more enrichment. This phenome non has been noted elsewhere (MacFadden et al. 1999a, Kendell & Coplen 2001). In fact, the American 18O values essentially mirror Kendell and Coplen’s (2001) observation that U.S. river waters are generally most depleted in Alaska, Montana, and North Dakot a (less than -20‰), and most enriched in Florida and Texas (greater than 0‰). Oxygen values are frac tionated in human tissue, so the river water values do not directly compar e to the enamel values, but the two regions with the most depleted 18O values were region 1 (Alaska) and region 2 (Idaho/Montana/North Dakota). Additionall y, the values from Florida and Texas residents were consistently the highest among the USAFA samples. The river water

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157 values for Hawaii and Puerto Rico were high (Kendell & Coplen 2001), but not above most Florida and Texas values. The same was true of the USAFA 18O values. To assess the effect of latitude upon the 18O values for this study, the grid coordinates for 143 individuals of American origin who resided in one location for the duration of third molar amelogenesis were examin ed. As can be seen, in general, as one progresses from north to south, the mean 18O values increase (Fi gure 6-3 and Table 6-8), except for the more equatorial islands. Variatio ns in this latitude gradient appear to arise primarily from differences in altitude be tween locations found along the same line of latitude. In many instances, those latitudes w ith the greatest standard deviation among individuals sampled pass through areas with marked differences in altitude. This is well reflected in Table 6-8, where more often than not, the more mountai nous states occupy -14.00 -12.00 -10.00 -8.00 -6.00 -4.00 -2.00 10 15 20 25 30 35 40 45 50 55 60 65 Figure 6-3. Plot of latitude compared to 18O values. Error bars equal 1 std dev. Latitude ( oN ) 18 O (‰)

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158 Table 6-8. Summary statistics for American USAFA 18O values based on latitude. All values are in ‰. Min Max Lat (o) N Mean Std Dev Value State Value State 61 N 1 -12.57-------------48 N 2 -9.300.56-9.70MT-8.91 MN 47 N 2 -8.600.12-8.69WA-8.51 WA 46 N 3 -10.690.51-11.20MT-10.18 ND 45 N 6 -8.311.72-11.60MT-6.93 MN 44 N 5 -7.490.88-9.03OR-6.87 VT 43 N 7 -7.171.04-9.22NY-5.82 NY 42 N 11 -7.461.63-11.46ID-6.28 IL 41 N 6 -6.510.50-7.17PA-5.67 NJ 40 N 16 -7.431.25-10.30UT-6.07 NJ 39 N 11 -8.171.80-10.21CO-5.85 IN 38 N 10 -7.401.59-9.79CO-5.55 MD 37 N 4 -6.660.70-7.57CA-5.88 KY 36 N 5 -5.800.75-6.53TN-4.75 OK 35 N 7 -6.561.14-8.22NM-5.45 NC 34 N 8 -6.491.17-8.84CA-5.41 AL 33 N 14 -6.491.45-8.58CA-4.73 TX 32 N 6 -4.980.98-6.83NM-4.11 TX 31 N 1 -5.12-----------TX 30 N 4 -5.650.54-6.15TX-4.98 TX 29 N 7 -5.450.53-6.41TX-4.76 TX 28 N 2 -4.481.02-5.20FL-3.75 FL 26 N 3 -4.440.27-4.64FL-4.13 FL 21 N 1 -5.52-----------HI 13 N 1 -5.98-----------Guam Latitudinal information drawn from Rand McNa lly Atlas (1998) and Google Earth (2006) Table fashioned after MacFadden et al. (1999a) minimum 18O positions for a specific latitude, while lower elevations make up the maximum values. A correlation function was performed to ex amine the relationship between latitude and the 18O of human enamel. Based on 142 values input into this procedure (Guam was omitted because of an eastern longitude ), latitude was shown to have a weak negative correlation with 18O, at r2 = -0.41. This data was in line with Kendall and Coplen (2001) who found a strong negative correlation (r2 = -0.79) for the eastern U.S.

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159 and a weak relation in the west (r2 = -0.35). This information begs the question how do altitude and annual preci pitation correlate to 18O values in comparison to latitude. Longitude for the same data set was found to be negatively correlated as well, at r2 = -0.2642. Longitudinal impacts upon human is otopes, are not frequently mentioned in the literature. One exception is Millard et al. (2004), who declared of oxygen isotopes, “Considering the scale of the variation, it is evident that it is potentially possible to pinpoint a person's place of origin with a usef ul resolution, particularly with respect to east-west differences.” The most reasonable ex planation within the United States is that the major mountain ranges run north to south, with the highe r elevation Rockies to the west of the older, more weathered Appalachia ns. It has already been established that altitude has a definite imp act upon oxygen isotope ratios, and the fact that the higher altitudes are in the west w ill affect the appearance of longitudinal influence upon group 18O values. This longitudinally-influenced 18O trend has also been observed for Great Britain, although the values tend to generally decrease as one goes from east to west (Budd et al. 2004), whereas the c line is reversed in the USAFA American values, which generally increased from east to west. Duplicate Residences Within this study, there were nine cities in which more than one individual resided during the entire period of amelogenesis. This presented a unique opportunity to examine how closely the oxygen values matched (Table 6-9 and Figure 6-4). It is assumed that people from the same city should have sim ilar oxygen values since they are primarily influenced by climate and geography. If the machine precision of the PRISM for 18O was 0.14‰, then any difference between values from the same locality greater than

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160 0.14‰ shows a variation in values that cannot be attributable to th e mass spectrometer. The difference beyond this value is assumed to be attributable to some underlying Table 6-9. 18O values corresponding to cities in wh ich multiple participants resided. (All values in ‰.) Identifier 18O City State Std Dev AFA-017 -7.83Phoenix AZ AFA-225 -7.14Phoenix AZ 0.49 AFA-116 -8.84Los Angeles CA AFA-250 -6.73Los Angeles CA 1.49 AFA-223 -8.58Laguna Niguel CA AFA-006 -8.27Laguna Niguel CA 0.22 AFA-029 -5.75Houston TX AFA-181 -5.44Houston TX AFA-163 -5.36Houston TX AFA-172 -4.76Houston TX 0.41 AFA-031 -9.52Denver CO AFA-065 -6.79Denver CO 1.93 AFA-194 -9.79Colorado Springs CO AFA-241 -9.13Colorado Springs CO AFA-270 -9.06Colorado Springs CO AFA-265 -8.25Colorado Springs CO 0.63 AFA-093 -7.13Cincinnati OH AFA-235 -6.28Cincinnati OH 0.61 AFA-051 -10.21Arvada CO AFA-024 -10.16Arvada CO 0.04 AFA-112 -8.22Albuquerque NM AFA-013 -8.00Albuquerque NM 0.16

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161 -11.00 -10.00 -9.00 -8.00 -7.00 -6.00 -5.00 -4.00Ph o enix L o s An g el e s L a g u na Nig u el Ho u st o n De n ver Colora d o Spring s C i n ci nnat i Arvad a Al bu querq u e Figure 6-4. Range of 18O values produced from this study for specific cities. biological or cultural factor independent of residence. The likelihood of differences arising due to physiological reasons is minimized because only third molars were used, ages of individuals were similar, and all were presumed to be of good health, a requirement of military service. The two residents from Arvada, CO, had surprisingly close values with difference in 18O of only 0.05‰. The two individuals from Denver, CO, on the other hand had the largest diffe rence of 2.73‰. The Denver value of -6.79‰ seems a bit enriched for someone belonging to a mountain state and could be attributable to differential oxygen fractionation compared to the general populace, however unlikely, or the individual may have been reliant on bottled water that was not from a local source. Comparison to the Literature While sparse, some studies of contemporary individuals or fairly recent historic remains are available to which a comparison of the USAFA data can be made. One such study was Beard and Johnson’s (2000) atte mpt to identify the remains of three commingled individuals from the Vietnam War. Background information on the three City 18O (‰)

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162 indicated one was from north-c entral California, one Detroi t, and the other spent their childhood in Vermont and Massachusetts. Th e individual they identified from northcentral California matched surprisingly well with the USAFA donor from Mt. Shasta, California (also in the north-central portion of the stat e). The strontium compositions calculated by Beard and Johnson (2000) based on bedrock geology led the researchers to conclude that these i ndividuals should have 87= +30 to +60 if they were from this locale. The Vietnam veteran has a mean third molar value (one tooth, three samples) of 0.70850 ( 87=+57) while the USAFA cadet measured 0.70846 ( 87=+56). This could indicate one of several things: either the effects of strontium homogenization from global food importation into the United States is not as prevalent as origina lly thought; the effects were being felt as early as the 1950s-1960s when Vietnam veterans were undergoing third molar amelogenesis; or this specific person ate a high percentage of locally grown food. Beard and Johnson (2000) where unable to distinguish among the other two individuals in their collection of remains. One presented strontium values of 0.71180 ( 87=+104 for a third molar and a mean of 0.71153 ( 87=+100) for another. A third molar from the last individual had a mean of 0.71062 ( 87=+87). One of these persons was from Detroit, Michigan. In the present study there was participant from Rochester, Michigan, some 27 miles from Detroit (Google Earth 2005) Unfortunately, the strontium value for the USAFA molar measured 0.70915 ( 87=+66), far from either of the veteran values, and outside the calculated strontiu m isotope composition range of 87= +90 to +200 for that area. Moreover, the USAFA value is even further off when compared to deer antler from Michigan analyzed by Beard and Johnson (2000), mean value 0.71373 ( 87=+131). There

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163 also was a USAFA donor from Vermont who measured 0.71006 ( 87=+79). This is closest to the unknown veteran with a mean value of 0.71062 ( 87=+87). One unaccounted for individual resi ded in both Vermont and Mass achusetts, but it is difficult to say what the influence of the second lo cale would be on the overall strontium ratio, thus it is inappropriate at pr esent to conclude that the me an value represents someone from Vermont. Strontium analyses were not run on any USAFA samples from those whose natal state was Massachusetts. Carlson (1996) performed an examination of the lead isotope ratios of eight unidentified skeletons from a 19th century fur trade cemetery in Alberta, Canada. One of the individuals analyzed was a male Caucasian believed to have been between the ages of 23–25 when he died. USAFA-032 is a male Cau casian who lived in Alberta until the age of 14 and was 22 at the time his third molars were extracted. While the bone of the fur trader was sampled versus a tooth for the Academy personnel, because lead does not fractionate, the values should be comparable. Based on the values presented in Table 6-10, it can be seen that only the 207Pb/204Pb lead ratio overlaps in these two individuals. All the other lead values show quite a leve l of divergence. Of course, the bone lead signature of the fur trader represents a more recent biogeochemical signal than the enamel of the USAFA sample, because of its turnover rate. There is no documentation to prove the fur trader was a native to the area or even permanently resided in Alberta at the time of his death. The USAFA individual di d move to Oregon at the age of 14, and hence his isotope values could represen t and amalgam of the two residences. Additionally, the older sample is much more susceptible to diagenetic contamination because of the porosity of bone contributing to exchange with the interment environment.

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164 Table 6-10 Comparison of Albe rta fur trader lead values to USAFA donor from Alberta Time Period Sample 208Pb/204Pb 207Pb/204Pb 206Pb/204Pb 208Pb/206Pb 207Pb/206Pb 2005 AFA-032 38.271515.6369 18.68702.04802 0.836784 1835-1856 Fur Trader 38.4515.62 18.442.085 0.847 *Carlson (1996)

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165 CHAPTER 7 SUMMARY/CONCLUSION This study was initiated with the hope of assisting forensic investigators in identifying remains of unknown or uncertain provenance, with the explicit goal of contributing to the Joint POW/MIA Acc ounting Command-Central Identification Laboratory’s mission of identifying those sti ll unaccounted for from previous conflicts, and specifically, the Vietnam War. The goa l was not only to dis tinguish isotopically between Southeast Asian and American dental remains, but to pinpoint, at least to a regional level, where in the United States an individual was reared. Unfortunately, high impact crashes and the destructive forces of modern weaponry frequently reduce what was once an intact body to a handful of remains, rendering most conventional identification met hods inadequate. Matters ma y be further complicated by the advance of time, unknown whereabouts of a body, and/or commingling of multiple military, terrorist, and/or civilian remain s. While DNA mapping technologies are now routinely employed in identification operations unless there are antemortem or familial profiles to compare them against, such procedures are of limited utility. Furthermore, remains are often too frag mentary or damaged to perform DNA extraction. Nowhere is this better exemplified than by the difficulties encountered in the identification efforts for those who perished during the 11 Septembe r 2001 terrorist attack on the World Trade Center. While nearly 20,000 individual samples were recovered from the area in and around the Twin Towers, the physical destruction was so great, that only 1,592 individuals out of a presumed 2,749 w ho died have been positively identified

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166 (Messer 2006). Likewise, similar difficu lties are encountered by anthropologists attempting to match a name to the remains of those service personnel from previous conflicts who still remain missing. For this project, the stable isotopes of carbon, oxygen, strontium, and lead were examined. Combined, they account for cultu ral dietary practices, climate and geography of natal areas, and the underlying geology of where an individual was reared. Enamel from the teeth of 61 individuals believed to be of East Asian origin, spanning the time period from World War II to the Vietnam Wa r was collected from the Joint POW/MIA Accounting Command-Central Identificati on Laboratory’s (JPA C-CIL) “Mongoloid” hold collection. The associated isotope values of these samples were compared to those of the enamel from the extrac ted third molars of 228 recent patients of the Unites States Air Force Academy (USAFA), Department of Oral and Maxillofacial Surgery. Since the USAFA participants were all living subjects, they were able to complete surveys detailing physiological, behavioral and residential information that affect isotope values. The least squares means for all isotope values examined exhibited significant differences between the CIL and USAFA c ohorts based on a conservative multivariate analysis of variance. This appears to demonstrate that all isotopes considered are potentially useful in distinguishing between these two populations. The carbon isotopes, reflecting dietary practices, were the most disc riminatory of the four examined. The Air Force Academy values were more enriched indicating a heavier C4 component to the diet, likely due to considerable corn consump tion. The East Asian values were more indicative of a C3 diet, undoubtedly related to their dietary reliance upon rice. When the extreme CIL outlier was excluded from co mparison, only six USAFA values overlapped

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167 with the main cluster of USAFA values. Of these overlapping values, all but one corresponded to individuals reared prim arily outside of the United States. The East Asian oxygen values clustered relatively tightly, wh ile the American values had a much greater range, extending a pproximately 2‰ on either side of the East Asian samples. The wide latitudinal breadth of the United States is likely the primary influence for this effect. For all the strontiu m and lead isotopes, the range of East Asian values encompassed the American values. A linear discriminant function was creat ed using all eight isotope ratios ( 13C, 18O, 87Sr/86Sr, 208Pb/204Pb, 207Pb/204Pb, 206Pb/204Pb, 208Pb/206Pb, and 207Pb/206Pb), that correctly classified individuals, through resubstitution an d cross-validation, as belonging to one of these two groups by 95% or better. A second version of the discriminant function was computed utilizing only 208Pb/204Pb and 207Pb/206Pb for the lead component of the equation instead of all five lead isotopes. This changed improved the overall accuracy rate of correct classification of an individua l as East Asian or American to nearly 97% after both resubstitution and cr oss-validation. A third disc riminant function was run attempting to distinguish individuals from S outheast Asia from the United States. While the same individuals were misclassified for the second and third discriminant functions calculated, because of smaller sample size in this instance, the third iteration produced a correct classification rate of 95%. Among those USAFA donors who resided for at least a portion of the period of amelogenesis of the third molar within the United States (henceforth referred to as Americans), significant differen ces were found for the mean values of several of the survey variables. The sexes differed as to their carbon ratios with females displaying

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168 more enriched values than males, likely due to differing diet preferences and not biological factors. Significant differences were also noted for 13C means among those who have never used tobacco products and thos e who partook of smokeless tobacco. It is possible that because this produc t mixes with saliva and is in close proximity to the teeth within the oral cavity that some form of chem ical exchange with the enamel is occurring in vivo This is the first time this phenome non has been noted however, thus there is much speculation as to the mechanisms behind the observation. As was feared the American strontium va lues displayed a distinct trend toward homogenization, with the mean value for 87Sr/86Sr varying only slightly from that of seawater. This indicates that the widespr ead importation of foodstuffs into the United States has had a dramatic effect upon populat ion strontium ratios. While this may dampen the utility of this isotope for geol ocational purposes within the United States, it still may prove useful when examining geogr aphically diverse populations and may serve as a temporal indicator when populations from varied periods are compared. In order to identify natal origin among Amer icans, nine regions were created within the United States based on 18O values. Good discrimination was noted between the mountain states and the southern states. A discriminant func tion analysis proved disappointing though, and at this point, additional sampling from most states is needed to improve the statistical robusticity of the model. Interrelated to this, a definite latitudinal cline was observed, with 18O values becoming more depleted as one moves north. If forced to choose between the light and heavy isotopes, a combined carbon and oxygen analysis provides the “biggest bang fo r the buck,” both in terms of money and time, when compared to strontium and lead analysis. One pass through the mass

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169 spectrometer provides information pertai ning to cultural di etary practices ( 13C) and geographic location ( 18O). Materials required are less numerous and less costly and from start to finish, chemical processing can be done a few days sooner, with much less manpower. When at all possible, however; it is highly recommended that an investigator employ as many stable isotopi c elements as possible. One cannot use a single traditional geoloca tional isotope such as oxygen, strontium, or lead singly to prove conclusively than an individual is from a given area. In making attributions it can only be said that the person in question gr ew up in one particular area or from some other regions which are geoc hemically similar (Brill & Wampler 1965). If the comparative sample is sound though, a lone isotope value does permit one to make a conclusive negative judgment. With a multi-element approach however, the odds are dramatically increased that region of origin can be pinpointed, especially within a closed population. The two greatest weaknesses of this study are the lack of American Vietnam-era samples and the requirement for greater numbers of samples from each state/territory within the United States. While the data generated from the USAFA samples is overwhelmingly positive, this sample set has yet to be proved as an appropriate proxy for Vietnam-era servicemen who still remain un accounted for. Food consumption patterns have changed measurably within the United St ates over the past 40 years (Kantor 1998). Bottled water and prepared beverages are now standard fare. A global economy guarantees the exchange of foodstuffs worldwide and lead-based products are still found in high prevalence (Sangs ter 2000). The utility of stable isotope analyses is predicated upon the widely held assumption that you are what you eat. Thus the isotopic ratios

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170 calculated through mass spectrometr y must bear direct relation ship to the food and drink ingested and imbibed by the individual over the course of their lifetim e. We, as members of the human species though, especially in t oday’s global climate, do not necessarily eat from which we live. The present study has barely scratched the surface of the possible avenues to explore with stable isotope analyses in the medico-legal realm. Some future areas it would be interesting to explore include how the lead isotope signatures and content in enamel have changed over the course of the la st 100 years in this c ountry, especially after lead-based gasoline and paints were phased out. How do dental restorat ions affect stable isotope readings? Does soaki ng teeth in jet fuel or burning them, as might happen in an aircraft crash, impact dent al isotopic signatures? How do altitude and annual precipitation correlate to 18O values in comparison to latitude and longitude? One thing is certain; individuals of a much greater range of ages must be included in isotope studies if stable isotopes are going to be used fo r general geolocational purposes. Hopefully, with the continued assistance of the Veterans Affairs Dental Clinic, an adequate sample set can be acquired. This study is novel in that it is the first of its kind to co mpile a reference sample of isotopic values associated with known natal regi ons to be utilized in forensic work. More importantly, the information gleaned from th is study will be applied in support of the JPAC’s mission to achieve the fullest possibl e accounting of all Americans missing as a result of our nation’s past conflicts. Th e databases compiled here and the analyses performed will boost discriminatory measures for identification, especially in instances where DNA has degraded and is unavailable or there is no reference sample to compare a

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171 profile against. This will prove especially us eful in cases of commingled remains. When compared to DNA processing, especially m itochondrial DNA, isotope analyses are quicker, less expensive, and much less vulnerable to contamination. The results of this study will hopefull y have wide-reaching effects across the medico-legal spectrum. This body of research will serve as the foundation for a database of modern, human, geolocatio nal isotope values that wi ll assist not only in the identification of fallen servicemen and wome n, but in the identific ation of victims of mass fatality incidents, undocumented aliens who perish attempting entry into the U.S., and local skeletal “Jane and John Doe” cases. This database will be available to the public free of charge and hopefully grow th rough future personal efforts and the contributions of other researchers.

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APPENDIX A REPLICATED VETERANS AFFAIRS BINDER

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173 Stable Isotopes Research Master Documents

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174 Table of Contents Protocol Approval Letters 1) UF IRB-01 2) VA SCI 3) VA R&D Committee 4) USAFA IRB Dental Staff Instructions CPRS/VISTA Instructions Subject Identifier Key HIPPA/Informed Consent (VA Research Consent Form) master Survey master Completed Example Survey master Background Information master Completed Patient HIPPA/I nformed Consent forms CD

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181 DEPARTMENT OF THE AIR FORCE HEADQUARTERS UNITED STATES AIR FORCE ACADEMY USAF ACADEMY COLORADO MEMORANDUM FOR MAJ OUELLETTE 22 July 2005 FROM: HQ USAFA/XPX SUBJECT: Protocol FAC2005026H Approved 1. The HQ USAFA Institutional Review Boar d considered your protocol FAC2005026, Isotopic Determination of Region of Origin in Modern Peoples: Applications for Identification of U.S. War-dead from the Vi etnam Conflict at its 21 July 2005 meeting. The study was approved as exempt from IRB oversight in accordance with 32 CFR 219.101, paragraph (b)(2). The board agreed that suffici ent safeguards were in place to protect research participants and deemed th is study can be exempt; you will not need to use the ICD or HIPPA Forms. Please place th e following statement at the bottom of your recruitment material: 'Approved: HQ US AFA IRB FAC2005026H.' This will inform potential subjects that your re search has been reviewed a nd approved. Please note that the USAFA Authorized Institutional Offi cial, HQ USAFA/DS and the Biomedical Research and Compliance Office of the Surgeon General’s Office, AFMSA/SGRC review all USAFA IRB actions and may ame nd this decision or identify additional requirements. 2. The protocol will be considered closed, but will be retained in XPX for 5 years then sent to permanent storage. As the princi pal investigator on th e study, the Biomedical Research and Compliance Office requires that y ou retain your data, reports, etc. for 3 years following completion of the study. 3. If the conditions under which you have b een granted exempt status change, you must notify the IRB Chair or IRB Administrator immediately. We will advise you on whether additional IRB review is required 4. If you have any questions or if I can be of further assistance, please don't hesitate to contact me at 333-3091 or the IRB Chair, Dr. Kate Carson at 333-2597. KATHLEEN A. O’DONNELL, PhD HQ USAFA IRB Administrator

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182 JPAC Isotope Study Dental Staff Instructions Thank you for your assistance with this worthw hile project. The purpose of this study is to determine if stable isotopes serve as geographic markers for regions of childhood residency. Patients, to include cad ets, active-duty, retire es, and dependents, already identif ied by the 10 DS for tooth extraction will be asked to participate in a brief survey and donate their extrac ted teeth for analysis. The i nvestigators will be examining the mineral elements in human tooth enam el. Initial efforts will focus on carbon, oxygen, strontium, and lead stable isotope ratios. No DNA analysis will be performed. The survey information will be used to help determine if geographic regions of the United States have specific isotopic signatures that b ecome incorporated into dental tissues. This study will also assess if these isotopic signatures vary throug h time. When compared to isotopic signatures developed for geographic areas of Southeast Asia, it is hoped the information will assist in identifying the origin of unknown dental remains unilaterally turned over to, or recovered by, the Jo int POW/MIA Accounting Command (JPAC) Central Identification Laboratory. Additiona lly, this information may be applied to identification efforts of fallen servicemen a nd women in conflicts outside of Southeast Asia and in the identification of victims of ma ss disasters such as airliner crashes and the events of 11 September 2001. A total of 100-200 participants will be surveyed from this facility. A further 200-300 patients will be sampled following the same protocol from the 10th Dental Squadron, U.S. Air Force Academy. Participant involvement is limited to tooth donation and completion of the JPAC Cent ral Identification Labo ratory survey. If you would like more information about this project, see the Isotope Study Information document provided for participants’ information. Please familiarize yourself all the necessary paperwork asso ciated with this study and with these instructions. If you have any questions please contact Dr Jack Meyer or Dr Ray Berringer, at 379-4040, or Maj Laura Regan 392-6772. Instructions Please query all patients sc heduled for dental extractions of permanent teeth as to their willingness to particip ate in this study. We need as large and broad of a sample population as possible. This is best done as soon as the patients are notified their teeth will be pulled. This will give them time to consider participating prior to enrollment in the study. o Minors, prisoners, UF students, and t hose with diminished mental capacity will not be included in this study. o Please emphasize to the patient th at nonparticipation or voluntary withdrawal from the investigation ca nnot, and will not, be the basis for any retribution brought against the subject.

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183 If the patient would like additional info rmation, please provide them with a copy of the study information question and answer material provided with this package. At patient intake/inprocessing, completion of two forms is required of all subjects: 1. VA Research Consent Form (sign two copies) This is a combined Informed Consent and Health Insurance Portability & Privacy Act (HIPAA) form specific to this study and separate from any forms that may need to be administered for routine care. The dental staff member must sign last page of the form as the “Person Obtaining Consent and Authorization” (1st blank) Volunteer/participant needs to read form, fill in their name in both locations on the front page and sign the back page in front of a witness as the “Person Consenting and Authorizing” (2nd blank) Allow the volunteer as much time as needed to read the form. Witness signs last page of form (3rd and last blank) verifying volunteer’s signature Each individual needs to sign two original forms. After all three individuals have signed, the volunteer/participant must be given one of the copies of the form prior to participation in the study. The second hard c opy needs to be filed in the study master document binder. 2. JPAC Survey with map Volunteer/participant needs to read form and answer all questions including drawing the numbers associ ated with where they lived as children on the map. (question #7) Once completed, please go over the in formation with the patient to ensure it is all accurate. Read the questions and answers to the patient affirming the answers provided are correct. Check the map for completion. An example of a completed surv ey is provided for guidance to staff and patients. Please make it available to patients should they desire it. Retain all forms. A binder will be provided for storage of the original VA Research Consent Forms and the JPAC Is otope Study Subject Identifier Key. The survey form should be maintained in th e patient’s record until the date of extraction. Just prior to extraction, a ssign each individual a unique st udy identifier utilizing the JPAC Isotope Study S ubject Identifier Key. o It is critical that no numbers are repeated so completely fill in the log as numbers are assigned. The first person assigned to the study should have number VA-001, the second person volunteering for the study should be VA-002, and so on. o Prior to assigning a number, check ques tion #8 on the survey. If a subject has not had a dental extraction perfor med by your facility within the last year (i.e., answered “No”) then assign them the next number in the log. If they answered “Yes,” you need to go back in their record and cross-check

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184 it with the log to find out what thei r original number was and give them the same number, except add on a “b” suffix. So if someone was assigned identifier VA-123 for their first ex traction, their sec ond visit would be VA-123b. o All teeth extracted on the same day will have the same identifier number. The number is associated with the individual, not each tooth Write the sample identifier number on the member’s survey on the appropriate blank in the gray “For Dental Staff Use Only” box. If multiple teeth are being pulled, collect as many teeth as possible from each individual. Write the tooth number (position in the ar cade) for all teeth donated and the date of extraction on the appropriate blank on th e member’s survey in the gray “For Dental Staff Use Only” box. Write the sample identifier and tooth numb er on the storage vial with a Sharpie or other permanent marker. Only place one tooth into each vial. Write the sample identifier on the plasti c pouch with a Sharpie or other permanent marker. Place all of the tooth vials in the plastic pouch, seal the pouch, and staple the pouch to the volunteer/participant surve y. All vials, the pouch, and the survey should have the same sample identifier. Dr Meyer will identify a container for stor age of the associated teeth and surveys. The surveys and associated teeth will be picked up by Major Laura Regan. If you need to contact her for some reason, her information is as follows: Laura Regan C.A. Pound Human Identification Laboratory University of Florida Bldg 114, Radio Road Gainesville FL 32611-2545 Phone: 392-6772 Fax: 392-2071 This study is scheduled to end 1 Aug 2006. At the completion of the study, Major Re gan will pick up all forms and arrange for final disposition.

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185 Isotope Study CPRS/VISTA Instructions All research participation within the VA medica l system must me documented in the subject’s electronic medical record (CPRS). This action complies with federally mandated standards regarding research documentation. The electronic document eliminates the need to file the Informed Consent form in the subject’s paper me dical record. The template must be added to CPRS on the date the subject signs the Informed Consent document. Since subject participation is limited to a single office visit, the research fl ag must be removed from the system within one week of dental extraction(s). To Enter Patient in Study: Open CPRS Select the patient’s name Click on the “Notes” tab on the bottom toolbar On the left sidebar, click on “New Notes” or under the top “Action” tab, click on “New Progress Note” In the Location for Current Activity window, type in in “ GHIS ” (for GHistorical) in the visit location box and check the small right-hand box for historical visit Click “OK” In the title box, type in “ Dental Research-W (T) ” Click “OK” The IRB number is 474-05 Click “OK” On the left sidebar, click on “Templates” Under the “My Templates” file, select “Dental Isotope Study” (you may need to click on the “+” sign to reveal) Sign the note per normal CPRS procedures The patient now has a clinical warning research alert To Remove Patient from Study/Remove Research Alert: Log into VISTA At the Select Dental/Control Point Official Menu Option prompt, type in “ ^TIU ” and press enter When it asks, Would you like to resume editing now? type in “ No ” and press enter At the Progress Notes User Menu prompt, Type in “ 1 ” for “Select progress notes/discharge summary” and press enter At the Progress Notes User Menu Option prompt, type in “ 2 ” to review progress notes by patient and press enter Type in the patients name (last name, first) and select There may be a pause at this point while the system performs a means test If the patient was entered in the study today, type “ T ” and press enter, otherwise type in “T-(number of days ago patient was entered in study)” Select the number of the record with th e “Dental Research-W (T)” warning title At the Select Action prompt, type “ CT ” and press enter You should receive a Dental Research-W (T) prompt; type in “ COMPLETED ” (all in caps) and press enter Exit VISTA If you go back into CPRS, the template shoul d now read “Dental Research Completed”

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186 JPAC Isotope Study Subject Identifier Key Extraction Date Attending Dentist Tooth Position Study Identifier Example 7 July 2005 Dr Berringer 1,16,17,32 VA-000 1 VA-001 2 VA-002 3 VA-003 4 VA-004 5 VA-005 6 VA-006 7 VA-007 8 VA-008 9 VA-009 10 VA-010 11 VA-011 12 VA-012 13 VA-013 14 VA-014 15 VA-015 16 VA-016 17 VA-017 18 VA-018 19 VA-019 20 VA-020 21 VA-021 22 VA-022 23 VA-023 24 VA-024 25 VA-025

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APPENDIX B CENTRAL IDENTIFICATION LABORATORY SAMPLING

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204Table B-1. Central Identifica tion Laboratory sampling data. Identifier Sample Origin Tooth Drill/ Loose Chip Wt Powder Wt Comments CIL-001 01A Vietnam 16 0.0670 02A Vietnam 32 0.1230 CIL-002 01A Vietnam 16 0.1057 02A Vietnam 17 0.0892 CIL-003 01A Vietnam 17 D 0.0991 Carie bucco-medial cusp/chip same side 02A Vietnam 32 D 0.1181 Chipped while drilling CIL-004 01A Vietnam 1 0.0980 Very small 02A Vietnam 15 0.1137 CIL-005 01A Vietnam 1 0.1250 02A Vietnam 32 D 0.1298 Partially impacted CIL-006 01A Vietnam 16 0.0730 Some wear 02A Vietnam 32 0.0891 Carie/mod calculus / worn 2 dentine mesial 5mm wide CIL-007 01A Vietnam 17 0.1482 Mild calculus 02A Vietnam 32 0.1331 Moderate calculus CIL-008 01A Vietnam 32 D 0.0961 Sm side and center caries 02A Vietnam 31 D 0.0800 Minor calculus/caries center & lingual CIL-009 01A Solomon Isl. 18 0.1143 No 3rds/buccal carie 02A Solomon Isl. 31 0.1113 No 3rds/central & small side caries CIL-010 01A Vietnam 17 0.1158 Roots immature 02A Vietnam 32 D 0.1101 Roots immature/impacted/root abscess CIL-011 01A Vietnam 32 D 0.0120 0.1592 Rich red dirt/ 3 chips frm drilling in sep vial 02A Vietnam 31 D 0.0381 0.0774 Rich red dirt/mod wear/2 lrg chips frm drilling CIL-012 01A Vietnam 17 0.1632 Center carie

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205Table B-1. Continued. Identifier Sample Origin Tooth Drill/ Loose Chip Wt Powder Wt Comments 02A Vietnam 32 0.1427 CIL-013 01A Laos ~32 L 0.1262 Appears MNI=2 based on wear/articulation CIL-014 01A Vietnam 17 D 0.0687 0.1038 Chipped drilling out/3 chips 02A Vietnam 18 0.0723 Mild-mod wear CIL-015 01A Laos 17 0.1067 02A Laos 32 0.1212 CIL-016 01A Vietnam 15 0.0307 0.0829 Glued in/nicked dentine but didn't use pwdr/3 chips 02A Vietnam 13 D 0.0550 Glued into alveoli CIL-017 01A Vietnam ~17 L 0.1896 Roots incomplete/ #1 impacted 02A Vietnam ~32 L 0.1700 CIL-018 01A Vietnam 31 D 0.0509 0.1700 02A Vietnam 30 0.0097 0.0499 Heavy brown stain/req heavy cleaning CIL-019 01A Vietnam 1 (2) L 0.0284 0.0508 Enamel chalky 02A Vietnam 16 (17) L 0.1099 0.0777 Enamel chalky/roots encased red wax CIL-020 01A Vietnam 17 (18) L 0.1970 Ambig place in jaw due to alveolar resorb/min wear CIL-021 01A Laos 1 0.1220 Minor wear/min chippng--did not keep 02A Laos 32 0.0927 Enamel chalky/heavy stain/cleaning CIL-022 01A Vietnam 18 0.1096 Rich red dirt/heavy stain/cleaning CIL-023 01A Vietnam 3 D 0.0843 Heavy wear/dentin exposed/heavy calculus 02A Vietnam 14 D 0.0338 0.0217 Heavy wear/dentin exposed/heavy calculus CIL-024 01A Vietnam 15 0.1043 Teeth black after cleaning/drilled stain off 02A Vietnam 14 D 0.0463 Same/chalky brown enamel undr black CIL-025 01A Vietnam 17 0.01485

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206Table B-1. Continued. Identifier Sample Origin Tooth Drill/ Loose Chip Wt Powder Wt Comments 02A Vietnam 31 D 0.1782 CIL-026 01A Korea 18 0.0089 0.0362 No 3rds/hea vy wear/occlusal surface dentin only 02A Korea 31 0.0503 Same but no chips/#6 bit CIL-027 01A Vietnam 19 0.0954 Buccal carie/3 small occlus caries CIL-028 01A Korea 2 D 0.2577 0.0489 Glued in/flaked apart on drilling 02A Korea 30 0.2431 ----------Large chips prior to drilling CIL-029 01A Korea 17 0.0877 Center carie 02A Korea 18 0.0924 Center carie CIL-030 01A Vietnam 2 L 0.0121 0.0879 Mild wear CIL-031 01A Vietnam 18 0.0986 Crude black center fillng/mod wear 02A Vietnam 19 D 0.0686 0.0890 Mod wear/center & side caries CIL-032 01A Cambodia 18 0.0258 0.0681 Heavy brown stain 02A Cambodia 19 D 0.0733 0.0636 Glued in CIL-033 01A Cambodia 17 0.0323 0.0801 Brown stain 02A Cambodia 19 0.0649 0.0127 Heavy wear & chippng/little to work with CIL-034 01A Cambodia 18 0.0425 0.0568 No 3rds/heavy wear/thin wall left 2 work with 02A Cambodia 20 0.0526 No 3rds/heavy wear CIL-035 01A Vietnam 15 L 0.1164 No 3rds/2 center caries 02A Vietnam 31 L 0.1044 No 3rds/side carie CIL-036 01A Vietnam 19 L 0.0963 0.0357 Mod wear/black occlusal surf/mult chips/looks rough CIL-037 01A Cambodia 16 0.1392 Maybe dent mixd 02A Cambodia 15 0.1375 CIL-038 01A Vietnam 18 0.1038 0.0277 Heavy wear

PAGE 224

207Table B-1. Continued. .Identifier Sample Origin Tooth Drill/ Loose Chip Wt Powder Wt Comments CIL-039 01A Philippines 16 0.0408 0.1287 02A Philippines 15 0.1166 Buccal carie CIL-040 01A Solomon Isl. 18 0.1897 CIL-041 01A Vietnam 32 D 0.1470 Occlusal surface brown 02A Vietnam 31 D 0.0987 Occlusal surface brown CIL-042 01A Vietnam 17 0.1164 Large center carie 02A Vietnam 31 0.1356 CIL-043 01A Vietnam 1 0.1750 Nasals more rounded 02A Vietnam 16 0.1895 4 roots CIL-044 01A Vietnam 32 (w) 0.0352 0.1069 Heavy cleanp/buccal & center caries/red mesial stain 02A Vietnam 31 0.0713 Very large lingual chips CIL-045 01A Vietnam 18 (w) 0.3215 0.0312 No 3rds/t ooth fract upon drill/may be contaminated 02A Vietnam 31 0.1145 No 3rds/mult fracturess in other teeth CIL-046 01A Vietnam 17 (w) D 0.0078 0.1232 Chip mes/ling cusp 02A Vietnam 31 0.1187 CIL-047 01A Vietnam 18 (w) 0.1403 02A Vietnam 19 D 0.0615 0.1414 Glued & red wax/mild wear CIL-048 01A Vietnam 32 (w) 0.1246 Brown stain/smallish size 02A Vietnam 30 0.1210 0.0625 Light brown stain/heavy wear CIL-049 01A Vietnam 31 (w) 0.1333 02A Vietnam 30 0.1225 Mod wear CIL-050 01A Vietnam 17 (w) 0.1714 Allignment of mandible 1/2s iffy

PAGE 225

208Table B-1. Continued. Identifier Sample Origin Tooth Drill/ Loose Chip Wt Powder Wt Comments 02A Vietnam 18 0.1630 CIL-051 01A Vietnam 32 (r) 0.0641 Distal surf gone/enamel flaked 02A Vietnam 30 0.0367 0.0664 Heavy wear CIL-052 01A Vietnam 30 (r) 0.0356 0.1241 No 3rd/lrg mesial, sm distal & lingual caries CIL-053 01A Vietnam 17(L) 0.0805 Large buccal chip 02A Vietnam 19 0.1348 Min wear CIL-054 01A Vietnam 17(L) 0.1344 Red wax/distal carie 02A Vietnam 19 D 0.0736 0.0703 Heavy wear CIL-055 01A Vietnam 18(17) 0.1451 Red wax 02A Vietnam 19(w) L 0.0307 0.0747 Red wax/distal chip/heavy wear CIL-056 01A Vietnam 32(r) 0.1087 Red wax 02A Vietnam 31 0.1116 Mod-heavy wear/red wax CIL-057 01A Vietnam 31(w) D 0.1852 0.1232 Large distal/lingual chip/red wax 02A Vietnam 19 D 0.0800 Heavy wear CIL-058 01A Vietnam 18(L) 0.1026 Large lingual chip/glued 02A Vietnam 19 D 0.1571 Glued/tough get out/heavy clean CIL-059 01A Vietnam 32(?) D 0.1746 Allignment of mandible 1/2s iffy 02A Vietnam 31 D 0.1199 chose lrg chip over ? Tooth/#6 CIL-060 01A Vietnam 17(w) 0.1679 Center/buccal carie 02A Vietnam 18 0.1218 CIL-061 01A Vietnam 32(w) D 0.0789 1/2 crown missing

PAGE 226

209 Figure B-1. Sample CIL chain of custody form.

PAGE 227

210 APPENDIX C UNITED STATES AIR FORCE ACADEMY SURVEY RESULTS USAFA Survey Code Key Field CodeDefinition Dates day/month/year Sex1 Male 2 Female Tobacco Product Code0 Non-user 1 Inhalent 2 Smokeless 3 Both 1 & 2 Diet1 Meat 2 Vegetarian 3 Vegan 4 Changed regimes

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211Table C-1. United States Air Force Academy survey data. Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # AFA-001 1984 1 Cauc 0 . 1 1984 1991 Coer D'Alene ID 1 1991 2002 Seaside OR 16 2002 2005 Colorado Springs CO 32 AFA-002 1986 2 Cauc 0 . 1 1986 1996 Chicago IL 1 1996 1998 Iro Peru 16 1998 2003 Santa Ana Costa Rica 17 2003 2004 Leysin Switzerland 32 AFA-003 1985 1 Hisp 0 . 1 1985 1997 Guaynabo Puerto Rico 1 1997 2000 Tampa FL 16 2000 2002 Newport News VA 17 32 AFA-004 1982 1 White 0 . 1 1982 1986 Clarksville TN 1 1986 1986 Cleveland TN 16 1986 2002 Clarksville TN 17 2002 2003 Marion AL 32 2003 2005 Clarksville TN AFA-005 1986 2 White 0 . 1 1986 1988 West Islip NY 13 1988 2005 Syracuse NY 16 AFA-006 1986 1 White 0 . 1 1986 2004 Laguna Niguel CA 1 2004 2005 Colorado Springs CO 16 32 AFA-007 1981 2 Black 0 . 1 1981 1991 Chicago IL 1 1991 1997 Colorado Springs CO 16 1997 1999 Las Vegas NV 17 32 AFA-008 1980 1 White . . 1 1980 1986 San Antonio TX 16 1986 2001 El Paso TX 17 2001 2005 Colorado Springs CO 32 AFA-009 1984 2 Cauc/Asian 0 . 1 1984 2002 Greenville SC 1 16 AFA-010 1985 1 Cauc 3 2001 2005 1 pack/week 1 1985 1990 Camarillo CA 1

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Table C-1. Continued. 212 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 2002 2005 1 can/week 1990 1992 Guam 16 1992 1998 Youngsville NC 17 1998 2003 Omaha NE 32 AFA-011 1986 1 White 2 2000 2005 3-5 times/day 1 1986 1986 Wichita Falls TX 1 1986 1991 Davie FL 16 1992 2004 Manila AR 17 2004 2005 Colorado Springs CO 32 AFA-012 1985 2 White 0 . 1 1985 1995 Hyde Park VT 1 1995 2001 Preston ID 16 2001 2003 Layton VT 32 2003 2004 Wichita Falls TX 2005 2005 Colorado Springs CO AFA-013 1985 1 Mex 0 . 1 1985 2003 Albuquerque NM 1 16 17 32 AFA-014 1984 1 Cauc 2 1995 2005 1 can/week 1 1984 2002 Georgetown TX 1 2002 2005 Colorado Springs CO 32 AFA-015 1 Cauc 0 . 1 . Omaha NE 1 . Aurora CO 16 . Colorado Springs CO 17 32 AFA-016 1985 1 Cauc 1 2002 2005 occasionally 1 1985 1986 Ft Walton Beach FL 1 1986 1988 Lutz FL 16 1988 1990 Virginia Beach VA 17 1990 1991 Birmingham AL 32 1991 1993 Marshall TX 51 1993 2001 Schell City MO 66 2001 2002 Ft Walton Beach FL 2002 2005 Colorado Springs CO AFA-017 1984 1 Hisp 1 . 1/day 1 1984 2002 Phoenix AZ 1 2002 2005 Colorado Springs CO 16

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Table C-1. Continued. 213 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 32 AFA-018 1984 1 Cauc 0 . 1 1984 1993 Austin TX 1 1993 2002 Acworth GA 16 AFA-019 1981 1 White 1 1998 2005 1/2 pack/day 1 1981 1983 Littleton MA 17 1983 1999 Everett MA AFA-020 1984 2 White 0 . 1 1984 1985 Vail CO 1 1985 1992 Alexandria VA 16 1992 2002 San Antonio TX 2002 2005 Colorado Springs CO AFA-021 1983 1 White 2 2002 2005 1 can/week 1 1983 2002 Goodyear? AZ 1 2002 2005 Colorado Springs CO 16 AFA-022 1984 2 Asian/White 0 . 1 1984 1995 Oceanside CA 1 1995 2000 Jacksonville FL 16 17 32 AFA-023 1985 2 Pac Isl 0 . 1 1985 2003 Agat Guam 1 2003 2004 San Antonio TX 16 2004 2005 Colorado Springs CO 17 32 AFA-024 1985 2 3 2004 2004 3 pinches/day 1 1985 2003 Arvada CO 1 2003 2003 5/day 2003 2005 Colorado Springs CO 16 17 32 AFA-025 1964 2 Cauc 0 . 1 1964 1988 Rochester MI 17 AFA-026 1983 1 Cauc 0 . 1 1983 1984 Miami FL 1 1984 1989 Andrews AFB MD 16 1989 1994 Danville PA 17 1994 1995 Charlotte NC 32 1995 2002 San Antonio TX 2002 2005 Colorado Springs CO AFA-027 1982 1 White 0 . 1 . UT 1 . AZ 16

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Table C-1. Continued. 214 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # . UT 17 . CA 32 . UT . NM . TX . CO . Russia AFA-028 1982 1 Cauc 0 . 1 1982 2000 Katy TX 1 16 17 32 AFA-029 1985 1 White 0 . 1 1985 2003 Houston TX 1 2003 2005 Colorado Springs CO 16 17 32 AFA-030 1984 1 Filipino 0 . 1 1984 1985 Oakland CA 1 1985 2002 Vallejo CA 16 2002 2005 Colorado Springs CO AFA-031 1982 1 Hispanic 0 . 1 1982 2001 Denver CO 1 2001 2005 Colorado Springs CO 16 17 32 AFA-032 1983 1 White 1 2004 2005 1 pack/week 1 1983 1997 Sprucegrove? Alberta, Canada 1 1997 2005 Roseburg OR 16 32 AFA-033 1984 1 White 0 . 1 1984 1999 Boston MA 1 1999 2002 Andover MA 16 2002 2005 Colorado Springs CO 32 AFA-034 1984 1 White 0 . 1 1984 2003 Seattle WA 1 2003 2005 Colorado Springs CO 16 17 32

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Table C-1. Continued. 215 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # AFA-035 1985 1 Asian 0 . 1 1985 1990 Flushing NY 1 1990 1993 NJ 16 1993 1996 Flushing NY 17 1996 2004 Old Bridge NJ 32 2004 2005 Colorado Springs CO AFA-036 1984 1 Cauc 0 . 1 1984 1985 Weymouth MA 1 1985 1990 Charlotte NC 16 1990 2002 Tryon NC 17 32 AFA-037 1983 1 Cauc 0 . 1 1983 1987 Nashville TN 1 1987 1990 Whitehouse TN 16 1990 2001 Portland TN AFA-038 1985 1 Mexican 0 . 1 1985 1991 La Mirada CA 1 1991 1995 Hobbs NM 17 1995 1999 Alamogordo NM 32 1999 2003 El Paso TX AFA-039 1982 1 White 1 2004 2005 twice a week 1 1982 1983 Salt Lake City Utah 1 1983 1985 Santa Maria CA 16 1985 1989 Abilene TX 17 1989 2002 Lubbock TX 32 2002 2005 Colorado Springs CO AFA-040 1983 1 White 0 . 1 1983 1991 Greenville SC 1 1991 2002 Fort Mill SC 32 AFA-041 1986 1 Afr Am 0 . 1 1986 1988 Phoenix AZ 1 1988 1994 UNK Germany 16 1994 1994 Fort Walton FL 17 1994 1997 Sheppard AFB TX 32 1997 2000 Alamogordo NM 2000 2002 Keflivik Iceland 2002 2005 Del Rio TX 2005 2005 USAFA CO AFA-042 1985 1 Afr Am 0 . 1 1985 1987 Barksdale AFB LA 1

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Table C-1. Continued. 216 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1987 1989 Castle AFB CA 17 1989 1990 Winnipeg Canada 32 1990 1993 Plattsburg NY 1993 1996 Pickerington OH 1996 2003 Warner Robins GA 2003 2005 Colorado Springs CO AFA-043 1983 1 Black 0 . 1 1983 1989 Colorado Springs CO 1 1989 1996 Norcross GA 16 1996 2001 Anstell GA 17 32 AFA-044 1984 1 Cauc 0 . 1 1984 1989 Monte Vista CO 1 1989 1990 Lidge Field MN 16 1990 2003 Thief River Falls MN 17 2003 2005 USAF Academy CO 32 AFA-045 1985 1 American 0 . 1 1985 1991 International Falls MN 1 1991 1995 Duluth MN 16 1995 1999 Carlsbad NM 17 1999 2003 Naples FL 32 AFA-046 1986 1 Korean 0 . 1 1986 1990 Niles IL 1 1990 2004 Mundelein IL 16 2004 2005 Colorado Springs CO 17 32 AFA-047 1982 1 Asian 1 1999 1999 1/week 1 1982 1999 Taytay, Rizal Phillipines 1 1999 2003 UP Los Banos Phillipines 5 2003 2004 PMA Baynio City Phillipines 12 2004 2005 Colorado Springs CO 16 32 AFA-048 1986 1 White 0 . 1 1986 2004 New Braunfels TX 1 2004 2005 Colorado Springs CO 16 17 32 AFA-049 1984 1 Cauc 0 . 1 1984 1986 Zanesville OH 1

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Table C-1. Continued. 217 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1986 2003 Dover OH 16 2003 2005 Colorado Springs CO 17 32 AFA-050 1984 1 White 0 . 1 1984 1988 Spangdahlem AFB Germany 17 1988 1990 Las Vegas NV 32 1990 1994 Hahn AB Germany 1994 1997 Tucson AZ 1997 2003 Goldsboro NC 2003 2005 Colorado Springs CO AFA-051 1984 1 White 0 . 1 1984 2002 Arvada CO 17 32 AFA-052 1983 1 Filipino 0 . 1 1983 1987 Laspias, Manilla Phillipines 1 1987 2001 Anaheim CA 16 2001 2005 Colorado Springs CO 17 AFA-053 1983 1 Cauc 2 2000 2005 2 cans/wk 1 1983 1998 Tampa FL 1 1998 2001 Faribault MN 16 AFA-054 1986 1 Cauc 0 . 1 1986 1989 Enid OK 1 1989 1992 Rapid City SD 16 1992 1995 Colorado Springs CO 17 1995 1996 Rapid City SD 32 1996 1997 Monterey CA 1997 2000 Buenos Aires Argentina 2000 2001 West Springfield WV 2001 2004 Athens Greece 2004 2005 Colorado Springs CO AFA-055 1985 1 Cauc 0 . 1 1985 1985 Oakland CA 1 1985 1988 Honolulu HI 16 1989 1992 NavCams Guam 17 1992 1994 Lexington Park MD 32 1994 2004 Sumner WA 2004 2005 Colorado Springs CO AFA-056 1985 1 White 0 . 1 1985 2003 Chaska MN 17

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Table C-1. Continued. 218 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 32 AFA-057 1985 1 White 0 . 1 1985 1988 San Mateo CA 17 1988 1995 Fremont CA 32 1995 2003 Tracy CA AFA-058 1984 1 White 2 2002 2005 1 can/wk 1 1984 1985 State College PA 1 1985 2002 Centerville VA 2002 2005 Colorado Springs CO AFA-059 1985 1 White 0 . 1 1985 2003 West Milwaukee WI 1 2003 2005 Colorado Springs CO 16 17 32 AFA-060 1985 1 Cauc 0 . 1 1985 2003 Kearney NE 1 16 17 32 AFA-061 1985 2 Other 0 . 1 1985 1987 San Mateo CA 16 1987 1989 Gathersburg MD 17 1989 2005 San Mateo CA 32 AFA-062 1986 1 Cauc 1 2003 2005 2 pack/day 1 1986 1998 Allen TX 16 1998 2004 Bonham TX 17 2004 2005 Wichita Falls TX 32 2005 2005 St Louis MO 2005 2005 Colorado Springs CO AFA-063 1984 1 White 0 . 1 1984 1985 Riverside CA 1 1985 2002 Anchorage AK 16 17 32 AFA-064 1982 1 Cauc 0 . 1 1982 1983 Boston MA 1 1983 1985 Albequerque NM 16 1985 1990 Dayton OH 17 1990 1993 Redlands CA 32 1993 2002 Dayton OH

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Table C-1. Continued. 219 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 2002 2005 Colorado Springs CO AFA-065 1984 2 White 0 . 1 1984 1988 Lakewood CA 1 1988 2002 Denver CO 16 17 32 AFA-066 1985 1 Cauc 0 . 1 1985 1996 Bedwyn IL 1 1996 2003 Aurora IL 16 2003 2005 Colorado Springs CO 17 32 AFA-067 1983 1 Hispanic 1 . 1/week 1 1983 1987 NYC NY 1 1987 1988 Paris France 16 1988 1992 NYC NY 17 1992 1993 Hong Kong China/UK 1993 2002 NYC NY AFA-068 1984 1 Other 0 . 1 1984 1986 Scott AFB IL 1 1986 1987 ??? MS 16 1987 1989 Hickam AFB HI 17 1989 1992 Misawa AB Japan 32 1992 1996 Woodbridge VA 1996 1998 Howard AB Panama 1998 2005 Colorado Springs CO AFA-069 1984 2 Cauc 0 . 4 1984 1987 Uppder Saddle River NJ 1 1987 1990 Ridgewood NJ 16 1990 1991 Paramus NJ 17 1991 2004 Allendale NJ 32 2004 2005 Paramus NJ AFA-070 1984 1 Indian 0 . 1 1984 1991 Bronx NY 1 1991 2002 Coconut Creek FL 16 2002 2005 Colorado Springs CO AFA-071 1985 1 Nat Hawaiian 2 2004 2005 3 per day 1 1985 1989 Japan 1 1989 1992 San Pedro CA 16 1992 1996 San Antonio TX 17

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Table C-1. Continued. 220 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1996 2003 Bremerton WA 32 2003 2005 Colorado Springs CO AFA-072 1985 1 Cauc/Korean 0 . 1 1985 1986 Fulda Germany 1 1986 1988 Radcliffe KY 16 1988 1992 ?? TX 17 1992 1994 ?? MI 32 1994 1998 Colonial Heights VA 1998 2003 Midlothian VA AFA-073 1985 2 Cauc 0 . 1 1985 2003 Athens TX 1 2003 2005 Colorado Springs CO 16 17 32 AFA-074 1982 2 White 0 . 1 1982 1983 Valdosta GA 16 1983 1984 San Antonio TX 17 1984 2000 Cabot AR AFA-075 1983 2 Cauc 0 . 1983 1993 Berlin Germany 1 1993 1995 Geilenkirchen Germany 16 1995 1997 Mons Belgium 17 1997 2002 Warner Robins AFB GA 32 2002 2005 Colorado Springs CO AFA-076 1985 1 White 0 . 1 1985 1993 Redlands CA 1 1993 2004 Victorville CA AFA076B 16 32 AFA-077 1984 1 White 0 . 1 1984 1994 Arlington TX 1 1994 2000 Burleson TX 16 2000 2002 Dover NH 17 2002 2003 Colorado Springs CO 32 AFA-078 1985 1 White 0 . 1 1985 2003 Burlington VT 1 2003 2004 Kansas City MO 16 2004 2005 Colorado Springs CO 17

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Table C-1. Continued. 221 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 32 AFA-079 1984 1 Cauc 0 . 1 1984 2000 Kalispell MT 17 2000 2002 Spokane WA 2002 2003 Roswell NM 2003 2005 Colorado Springs CO AFA-080 1985 2 White 1 2000 2005 1/2 pack/day 1 1985 2003 Port Clinton OH 1 2003 2005 Colorado Springs CO 16 17 32 AFA-081 1983 2 Cauc 0 . 1 1983 1985 Tumwater WA 1 1985 1987 Provo UT 16 1987 1991 Tri cities WA 17 1991 1994 Missoula MT 32 1994 2001 Lebanon OR AFA-082 1987 1 Cauc 0 . 1 1987 2005 Boone IA 1 16 17 32 AFA-083 1983 2 Cauc 0 . 1 1983 2001 Jacksonville TX 1 2001 2001 Taichung Taiwan 16 2002 2005 Colorado Springs CO 17 32 AFA-084 1983 1 Hispanic 2 2004 2005 once a day 1 1983 1995 San Antonio TX 1 1995 1998 Irvine CA 16 1998 1998 Peoria AZ 17 1998 2002 San Antonio TX 32 2002 2005 Colorado Springs CO AFA-085 1987 1 Cauc 0 . 1 1987 2005 Torrington CT 1 2005 2005 Colorado Springs CO 16 17 32 AFA-086 1982 1 Cauc 0 . 1 1982 2001 Honolulu HI 1

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Table C-1. Continued. 222 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 2001 2005 Colorado Springs CO 16 17 32 AFA-087 1984 2 White 0 . 1 1984 1985 Atlanta GA 1 1985 2003 Newnan GA 16 17 32 AFA-088 1983 1 Cauc 1 2002 2005 1 every 2-3 mos 1 1983 1985 Wooster OH 1 1985 1986 South Bend IN 16 1986 2002 Wooster OH 17 2002 2005 Burbank CA 32 AFA-089 1984 1 Cauc 2 2004 2005 3 x per wk 1 1984 1986 Columbus OH 1 1986 2003 Orlando FL 16 2003 2003 Crestline CA 17 2004 2005 Colorado Springs CO 32 AFA-090 1985 2 White 0 . 1 1985 1988 Cary NC 1 1988 2003 Methuen MA 16 2003 2005 Colorado Springs CO 17 32 AFA-091 1985 1 White 0 . 1 1985 1990 Victoria TX 1 1990 1991 Albuquerque NM 16 1991 2004 Ft Davis TX 17 2004 2005 Colorado Springs CO 32 AFA-092 1986 1 White 0 . 1 1986 1992 Jacksonville NC 1 1992 2004 Elon NC 16 2004 2005 Colorado Springs CO 17 32 AFA-093 1985 1 White 0 . 1 1985 2003 Cincinnati OH 1 6 17 32

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Table C-1. Continued. 223 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # AFA-094 1984 1 White 0 . 1 1984 2003 Summerville SC 1 2003 2005 Colorado Springs CO 16 17 32 AFA-095 1973 1 Cauc 0 . 1 1973 1994 Scranton PA 1 16 17 32 AFA-096 1984 1 White 0 . 1 1984 2002 Bland VA 1 16 17 32 AFA-097 1986 1 Cauc 0 . 1 1986 2004 College Station TX 1 2004 2005 Colorado Springs CO 16 17 32 AFA-098 1985 2 Cauc 3 2004 2005 each day 1 1985 1986 Honolulu HI 1 2005 2005 each day for 1 mo 1986 1988 Platte City MO 5 1988 2004 Corning IA 12 2004 2005 Colorado Springs CO 16 17 20 29 32 AFA-099 1983 1 White 0 . 1 1983 1987 Evergreen CO 1 1987 2002 Littleton CO 16 32 AFA-100 1984 1 White 0 . 1 1984 1986 Valdosta GA 1 1986 1989 Apple Valley CA 16 1989 1992 Naples Italy 17 1992 1994 Ellensburg WA 32 1994 1995 Baker City OR

PAGE 241

Table C-1. Continued. 224 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1995 1996 Camdenton MO 1996 1996 McNeal AZ 1996 2000 Manila Phillipines 2001 2002 Ellensburg WA 2002 2003 Manila Phillipines 2003 2005 Colorado Springs CO AFA-101 1984 1 White 0 . 1 1984 1986 Springfield MA 17 1986 1989 Chicopee MA 32 1989 2002 Springfield MA AFA-102 1985 1 White 2 2004 2005 1 can/mo 1 1985 2003 Sparta NJ 1 16 17 32 AFA-103 1983 1 Whte 0 . 1 1983 2002 Golden CO 1 2002 2005 Colorado Springs CO 16 17 32 AFA-104 1983 1 White 0 . 1 1983 1986 Naples Italy 1 1986 1989 Maine 16 1990 1998 Monrovia MD 17 1999 2001 Kailua HI 32 2001 2005 Colorado Springs CO AFA-105 1983 1 White 0 . 1 1983 2001 Short Hills NJ 1 16 17 32 AFA-106 1984 1 Cauc 0 . 1 1984 1985 Norfolk VA 1 1985 1988 Charleston SC 16 1988 1992 Poway CA 17 1993 1993 Chesapeake VA 32 1994 2002 Longmeadow MA 1996 1998 (summers only) Guam

PAGE 242

Table C-1. Continued. 225 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1998 2000 (same) Oceanside CA 2000 2002 (same) Rocky Mount NC AFA-107 1968 1 Wh 3 1984 1996 1/2 pack/day 1 1968 1971 Lebanon OR 1 1999 2005 1/2 can/day 1971 1986 Albany OR 16 17 32 AFA-108 1987 1 Black 0 . 1 1987 1989 Watertown NY 1 1989 1993 Tacoma WA 16 1994 2000 Beaverton OR 17 2000 2005 Hillsboro OR AFA-109 1986 2 Hispanic 0 . 1 1986 2004 Lima Peru 1 2004 2005 Colorado Springs CO 16 17 32 AFA-110 1984 1 White 1 2002 2004 3-4 per yr 1 1984 1984 Newport News VA 1 1985 1987 Sumter SC 16 1987 2003 Southlake TX 17 2003 2005 Colorado Springs CO 32 AFA-111 1984 1 white 0 . 1 1984 2002 Old Shasta CA 1 17 32 AFA-112 1985 1 Cauc 0 . 1 1985 1986 McAllen TX 1 1986 1990 Tulsa OK 16 1990 2003 Albuquerque NM 17 32 AFA-113 1986 1 Black 0 . 1 1986 1998 Albany GA 1 1998 1999 Camilla GA 16 1999 2004 Albany GA 17 2004 2005 Leesburg GA 32 AFA-114 1983 1 White 3 2004 2005 rarely 1 1983 1985 Ann Arbor MI 1 2004 2005 rarely 1985 1986 Los Angeles CA 16 1986 1993 Dallas TX 32

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Table C-1. Continued. 226 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1993 2002 Atlanta GA AFA-115 1985 1 White 0 . 1 1985 2003 Franklin TN 1 16 17 32 AFA-116 1975 1 Caucasian 2 1995 2005 1 can/week 1 1975 1994 Los Angeles CA 1 17 AFA-117 1985 2 Caucasian 0 . 1 1985 2004 Kent WA 1 16 17 32 AFA-118 1984 1 White 0 . 1 1984 1984 Loveland CO 17 1984 1989 Suanee GA 32 1989 1990 Lawrenceville GA 1990 2002 Loganville GA 2002 2005 USAFA CO AFA-119 1979 1 White Cauc 1 1996 2005 1/2 pack 1 1979 1984 Barre Vermont 1 1984 1986 Greenfield Mass 16 1986 1999 Newport Vermont 17 1999 2002 Minot North Dakota 32 2002 2005 Colorado Springs CO AFA-120 1980 2 African-Amer 0 . 1 1980 1988 Linden Guyana 17 1988 2004 Brooklyn NY 32 2004 2005 Colorado Springs CO AFA-121 1986 1 White 0 . 1 1986 1991 Payson UT 1 1991 1993 Missoula MT 16 1993 2004 Florence MT 17 AFA-122 1985 1 White 0 . 1 1985 1987 AL/LA 1 1987 1990 Liverpool NY 16 1990 1996 North Syracuse NY 17 1996 2003 Havelock NC 32 2003 2005 Colorado Springs CO

PAGE 244

Table C-1. Continued. 227 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # AFA-123 1981 1 Cauc 0 . 1 1981 1982 Bryson City NC 1 1982 1984 ?? AL 16 1984 1986 Dothan AL 17 1986 1988 Greenville MS 32 1988 1991 Marysville WA 1991 1994 Arlington WA 1994 1995 Dothan AL 1995 1998 Homestead FL 1998 2000 Manheim PA AFA-124 1984 1 White 1 2000 2005 1 pack per day 1 1984 1985 Greensboro NC 1 1985 1990 Trenton MO 16 1990 2003 Lincoln NE 17 2003 2004 TX 32 2004 2005 Colorado Springs CO AFA-125 1981 1 Caucasian 0 . 1 1981 2003 Oregon City OR 1 16 17 32 AFA-126 1985 1 Hispanic 0 . 1 1985 1997 Los Angeles CA 1 1997 2003 Benicia CA 16 2003 2005 Colorado Springs CO 17 32 AFA-127 1984 2 White 0 . 1 1984 1991 San Jose CA 1 1991 1996 Austin TX 16 1996 1997 San Jose CA 17 1997 2002 Pleasanton CA 32 2002 2005 Colorado Springs CO AFA-128 1985 1 White 0 . 1 1985 1988 March AFB CA 1 1988 1991 Mather AFB CA 16 1991 1997 New Baden IL 17 1997 2003 Altus AFB OK 32

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Table C-1. Continued. 228 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # AFA-129 1986 1 White 1 2003 2005 1-3 per month 1 1986 1987 Charlotte NC 1 1987 1995 Coral Springs FL 16 1995 1996 West Palm Beach FL 17 1996 2004 Wantage NJ 32 AFA-130 1982 2 Cauc 0 . 1 1982 1983 Ft Myers FL 1 1983 1987 Winchester VA 16 1987 1991 Cape Coral FL 17 1991 1993 Bealeton VA 32 1993 1994 Slanesville WV 1995 2000 Inwood WV AFA-131 1985 1 White 0 . 1 1985 2000 Esko MN 1 2000 2004 Clovis CA 16 2004 2005 USAF Academy CO 17 32 AFA-132 1984 1 White 0 . 1 1984 1986 Yuma AZ 1 1986 1989 AL 16 1989 1992 Panama City Panama 17 1992 1995 Ramstein Germany 32 1995 2005 Colorado Springs CO AFA-133 1986 2 White 0 . 1 1986 2004 Scottsboro AL 1 2004 2005 Colorado Springs CO 16 17 32 AFA-134 1985 1 White 0 . 1 1985 2005 Southaven MS 1 16 17 32 AFA-135 1984 1 White 2 2003 2005 1can/week 1984 1988 Orlando FL 1 1988 1995 Grand Polanic TX 16 1995 1997 Louisville KY 17 1997 2003 Arlington TX 32

PAGE 246

Table C-1. Continued. 229 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 2003 2005 USAF Academy CO AFA-136 1983 1 Caucasian 0 . 1 1983 2001 Jerome ID 1 16 17 32 AFA-137 1984 1 White 0 . 1 1984 1985 Gaithersburg MD 1 1985 1987 Shenandoah VA 16 1987 1992 Belfast ME 17 1992 2003 Newark DE 32 2003 2005 USAF Academy CO AFA-138 1984 1 Asian 0 . 1 1984 2002 Gastonia NC 1 16 17 32 AFA-139 1982 2 Korean/Mexican 1 2003 2004 1pack/week 1 1982 1984 Colorado Springs CO 1 1984 1988 Los Angeles CA 16 1988 1991 Colorado Springs CO 1991 1993 Panama City Panama 1993 1996 Waniawa HI 1996 1998 El Paso TX 1998 1999 Vilseck Germany 1999 2001 Heidelburg Germany 2001 2005 Colorado Springs CO AFA-140 1986 1 White 1 2003 2004 1/2 per month 1 1986 1988 Bitburg AB Germany 1 1988 1991 Luke AFB AZ 16 1991 1995 Eielson AFB Alaska 17 1995 1998 Hickam AFB HI 32 1998 2003 Lake Mary FL AFA-141 1983 2 Asian 0 . 1 1983 1992 Seoul/Incheon Korea 1 1992 1995 West Point NY 16 1995 1998 Heidelburg Germany 17

PAGE 247

Table C-1. Continued. 230 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1998 1999 London England 32 1999 2002 Seoul Korea 2002 2005 Colorado Springs CO AFA-142 1983 1 Caucasian 0 . 1 1983 1985 Austin TX 1 1985 1988 Goppingen Germany 16 1988 1991 Madrid Spain 32 1991 1994 Landstuhl Germany 1994 2001 Enid OK AFA-143 1983 1 Filipino-Amer 0 . 1 1983 1983 Ohahron Saudi Arabia 1 1983 1999 Seoul Korea 16 17 32 AFA-144 1983 2 Mexican-Amer 0 . 1 1983 1984 San Diego CA 1 1984 1985 Yuma AZ 16 1985 1987 Landsdale PA 17 1987 1991 Cherry Point NC 32 1991 2000 Yuma AZ 2000 2005 Colorado Springs CO AFA-145 1986 1 Caucasian 0 . 1 1986 1986 Buffalo WY 1 1986 1991 Lake Charles LA 1991 1993 Elida OH 1993 1997 Cheyenne WY 1997 1999 Glenrock WY 1999 2004 Cheyenne WY AFA-146 1983 1 White 0 . 1 1983 2001 Burley ID 17 32 AFA-147 1983 1 Caucasian 2 2001 2005 2/day 1 1983 1988 Oklahoma City OK 1 1988 2002 Tullahoma TN 16 2002 2005 USAF Academy CO 17 AFA-148 1982 1 Asian 1 . 4 pieces/day 1 1982 2001 Yeosu Korea 1 16 17

PAGE 248

Table C-1. Continued. 231 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 32 AFA-149 1983 2 Caucasian 0 . 1 1983 2002 Chaffee MO 1 2002 2005 USAFA CO 16 17 32 AFA-150 1985 1 White 0 . 1 1985 1987 Loring AFB Maine 1 1987 1988 Montgomery Alabama 16 1988 1991 Osan AFB/Soeul Korea 17 1991 1995 Grand Forks ND 32 1995 1999 Newport News VA 1999 2002 Jakarta Indonesia 2002 2004 Newport News VA 2004 2005 USAF Academy CO AFA-151 1983 1 Hispanic 0 . 1 1983 1985 Farmers Branch TX 1 1985 1986 Youngstown OH 17 1986 1988 Aromoore OK 32 1988 2002 Farmers Branch TX 2002 2005 USAF Academy CO AFA-152 1984 2 Asian 0 . 1 1984 1986 Yokota AFB Japan 1 1986 1989 Holoman AFB NM 16 1989 1993 Yokota AFB Japan 1993 1998 Ramstein AFB Germany 1998 2002 Brookings SD 2002 2005 USAF Academy CO AFA-153 1985 1 Whit 0 . 1 1985 2004 Pittsburg PA 1 2004 2005 USAF Academy CO 16 17 32 AFA-154 1 Caucasian 0 . 1 . Schaumburg IL 1 . Peachtree City GA 16 . USAF Academy CO 17 32

PAGE 249

Table C-1. Continued. 232 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # AFA-155 1981 1 White 0 . 1 1981 1985 Salt Lake City Utah 1 1985 1999 Allen TX 16 1999 2001 USAFA CO 17 2001 2003 Siberia Russia 32 2003 2005 USAFA CO AFA-156 1985 1 White-Hispanic 0 . 1 1985 1987 Ft. Worth TX 17 1987 2004 Cleburne TX AFA-157 1985 2 White/NonHispanic 0 . 1 1985 2003 Norfolk NE 1 2003 2005 Colorado Springs CO 16 17 32 AFA-158 1987 1 White 0 . 1 1988 1990 Monterey CA 1 1990 2000 Columbia MD 16 2000 2005 Hanover MD 17 32 AFA-159 1986 2 White 0 . 1 1986 1988 San Diego CA 1 1988 1991 Monterrey CA 16 1991 1992 Newport RI 17 1992 1994 Goose Creek SC 32 1994 1997 Puyallup WA 1997 2003 Germantown MD 2003 2005 USAFA CO AFA-160 1986 1 Caucasian 0 . 1 1986 1988 Ronkonkoma NY 1 1988 2004 Boca Raton FL 16 2004 2005 McKinney TX 17 32 AFA-161 1983 1 White . . 1 1983 1988 Cheboygan MI 1 1988 1989 Las Vegas NV 16 1989 2002 Cheboygan MI 17 2002 2002 Keesler AFB MS 32 2002 2004 Spangdahlem AB Germany

PAGE 250

Table C-1. Continued. 233 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 2004 2005 Colorado Springs CO AFA-162 1987 1 Korean/White 0 . 1 1987 Bethesda MD 1 . Seattle WA 16 1993 Key West FL 17 1993 1996 Sault St. Marie MI 32 1996 1997 Waldorf MD 1997 1999 Concord CA 1999 2001 Alameda CA 2001 2002 Waldorf MD 2002 2005 Indian Head MD 2005 2005 USAF Academy CO AFA-163 1984 2 Hispanic 0 . 1 1984 2002 Houston TX 1 2002 2005 Colorado Springs CO 16 AFA-164 1984 1 White 2 2004 2005 1 can/4 days 1 1984 2002 Washington GA 1 2002 2005 Colorado Springs CO 16 17 AFA-165 1983 1 White 0 . 1 1983 1993 Mt. Gilead OH 1 1993 1996 Kissimmee FL 16 1996 2002 Mt. Gilead OH 17 32 AFA-166 1984 1 Caucasian 1 2004 2005 1/4 pack per day 1 1984 2003 Bismarck ND 1 16 17 32 AFA-167 1984 1 White 0 . 1 1984 1987 Santa Fe TX 1 1987 2003 Alvin TX 16 2003 2005 USAFA CO 17 32 AFA-168 1984 1 Black 0 . 1 1984 2005 Monroeville PA 1 16 17

PAGE 251

Table C-1. Continued. 234 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 32 AFA-169 1983 1 White 0 . 1 1983 1998 Grundy Center IA 1 1998 2001 Dodge Center MN 16 17 32 AFA-170 1985 1 Caucasian 0 . 1 1985 1990 Indinapolis IN 1 1990 1992 Madison WI 16 1992 2003 Watertown WI 17 2003 2005 Colorado Springs CO 32 AFA-171 1985 1 African-Amer 0 . 1 1985 2003 Vauxhall NJ 1 16 17 32 AFA-172 1983 1 White 0 . 1 1983 2001 Houston TX 1 2001 2002 Lexington MO 16 2002 2005 USAF Academy CO 17 32 AFA-173 1984 2 Caucasian 0 . 1 1984 2002 Vacaville CA 1 16 AFA-174 1984 1 White 0 . 1 1984 2003 Elizabethtown KY 1 2003 2005 USAFA CO 16 17 32 AFA-175 1986 1 White 1 2004 2005 2-3/week 1 1986 1995 Denver CO 17 1995 1997 Atlanta GA 32 1997 2000 Lake Oswego OR 2000 2005 Leesburg VA AFA-176 1984 1 White/Caucasian 0 . 1 1984 2003 Tallahassee FL 1 2003 2005 Colorado Springs CO 16 32 AFA-177 1984 1 0 . 1 1984 Ceiba Puerto Rico 1 1991 Peachtree City GA 16

PAGE 252

Table C-1. Continued. 235 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1991 1994 Wiesbaden Germany 1994 2003 Fairfax VA 2003 2005 Colorado Springs CO AFA-178 1984 1 Caucasian 0 . 1 1984 1985 Dyersburg TN 16 1985 1987 Dotham AL 17 1987 1994 Niceville FL 1994 1997 Incirlik AB Turkey 1997 2002 Fairborn OH 2002 2003 Tallahassee FL 2003 2005 Colorado Springs CO AFA-179 1984 1 White 0 . 1 1984 1986 Lancaster OH 1 1986 2003 Columbus OH 16 17 32 AFA-180 1985 2 White 0 . 1 1985 1992 Miami FL 1 1992 2002 Coral Springs FL 16 2002 2005 USAF Academy CO 17 32 AFA-181 1984 2 White/Hispanic 0 . 1 1984 2003 Houston TX 1 16 17 32 AFA-182 1984 1 Caucasian 0 . 1 1984 2002 East Stroudsburg PA 1 2002 2005 USAF Academy CO 16 17 32 AFA-183 1980 2 White 0 . 1 1980 1996 La Quinta CA 1 1996 1997 Palm Desert CA 3 1997 2001 Ft. Carson CO 16 2001 2002 La Quinta CA 17 2002 2005 USAF Academy CO 32 AFA-184 1982 2 Caucasian 0 . 1 1982 1983 Veliko Tarnovo Bulgaria 1

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Table C-1. Continued. 236 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1983 1995 Nova Zagora Bulgaria 16 1995 1997 Tripoli Libya 17 1997 2000 Nova Zagora Bulgaria 32 AFA-185 1984 1 White 0 . 1 1984 2002 Corcoran CA 16 2002 2006 USAFA CO 17 AFA-186 1984 1 Caucasian 0 . 1 1984 2002 Inola OK 1 16 17 32 AFA-187 1983 1 Caucasian 3 2002 2006 1/week 1 1983 2002 Indianapolis IN 1 2005 2006 2/week 2002 2006 Colorado Springs CO 16 17 32 AFA-188 1983 1 Caucasian 0 . 1 1983 1989 Grand Rapids MI 1 1990 2002 Spring Lake MI 16 2002 2005 USAFA CO 32 AFA-189 1986 1 Caucasian 2 2003 2006 1/week 1 1986 1995 Belmont CA 17 1995 1997 High Springs FL 32 1997 2004 Sandy UT 2004 2006 USAFA CO AFA-190 1985 1 White 0 . 1 1985 1990 Kansas City KA 1 1990 1996 North Bend WA 16 1996 2003 Mercer Island WA 17 32 AFA-191 1981 1 Caucasian 0 . 1 1981 1986 Syracuse NY 1 1986 1994 Houghton LA 16 1994 2000 Manhawkin NJ 17 2000 2001 Dixon CA 32 2001 2005 USAFA CO AFA-192 1984 1 Caucasian 0 . 1 1984 2002 Portland OR 16 17 AFA-193 1984 1 White 0 . 1 1984 1990 Fresno CA 16

PAGE 254

Table C-1. Continued. 237 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1990 2002 Modesto CA AFA-194 1983 1 Hispanic 3 1999 Present 2/week 1 1983 1984 Ft. Stuart GA 1 1999 Present 2/week 1984 2006 Colorado Springs CO 16 AFA-195 1984 1 Caucasian 0 . 1 1984 2002 Hackettstown NJ 1 2002 2005 USAFA CO 16 AFA-196 1980 1 W 2 1999 2004 daily 1 1980 1999 Bonaire GA 17 AFA-197 1983 1 Caucasian 0 . 1 1983 2002 St. Johns MI 1 16 AFA-198 1983 1 Caucasian 0 . 1 1983 2001 Brooklyn Park MN 1 16 17 32 AFA-199 1983 1 Caucasian 0 . 1 1983 2004 Dallas TX 1 2004 2006 USAFA CO 17 AFA-200 1983 1 Caucasian 0 . 1 1983 1984 Houston TX 1 1984 2002 Carlbad NM 16 17 32 AFA-201 1983 1 Caucasian 0 . 1 1983 2002 New Castle IN 1 2002 2005 USAFA CO 16 17 32 AFA-202 1984 1 Asian 0 . 1 1984 1996 Chicago IL 16 1996 2002 Mundelein IL AFA-203 1984 2 White 0 . 1 1984 2003 Burnsville MN 1 16 17 32 AFA-204 1983 1 Caucasian 0 . 1 1983 2002 Buffalo Grove IL 1 16 AFA-205 1983 1 Caucasian 2 2002 2005 4/week 1 1983 2002 Atlanta GA 1 2002 2006 Colorado Springs CO 16

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Table C-1. Continued. 238 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 17 32 AFA-206 1983 1 White 0 . 1 1983 1992 Portland TX 1 1992 1997 Eagan MN 16 1998 2001 Portland TX 32 2001 2006 USAFA CO AFA-207 AFA-208 1984 1 Caucasian 0 . 1 1984 1989 Arvada CO 1 1989 2002 Broomfield CO 16 2002 2006 USAFA CO 17 32 AFA-209 1985 1 Caucasian 0 . 1 1985 1986 Norfolk VA 16 1986 1989 LasVegas NV 17 1989 1992 Bitburg Germany 1992 1993 Clifton VA 1993 2006 Colorado Springs CO AFA-210 1984 1 White 0 . 1 1984 1984 Red Bank NJ 1 1984 1986 Ft. Leavenworth KS 16 1986 1989 Ft. Bliss TX 17 1989 1992 Kitzogen Germany 32 1992 1997 Ft. Hood TX 1997 2000 Ft. Riley KS 2000 2002 Ft. Hood TX AFA-211 1983 1 Caucasian 0 . 1 1983 1986 Houston TX 1 1986 2002 Park City UT 17 2002 2006 Colorado Springs CO 32 AFA-212 1983 2 White 0 . 1 1983 2002 Butte MT 1 16 17 32 AFA-213 1984 1 Caucasian 0 . 1 1984 1985 Ft. Stewart GA 1 1985 1988 Ft. Lee VA 16

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Table C-1. Continued. 239 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1988 1990 Ft. Leavenworth KS 32 1990 1991 Heidelberg Germany 1991 1993 Reichenbach Germany 1993 1997 Carbondale IL 1997 2002 Davidson NC 2002 2003 Colorado Springs CO AFA-214 1986 1 Pacific Island 0 . 1 1986 1994 Vallejo CA 1 1994 2000 Puna HI 16 2000 2004 Kapolei HI 17 32 AFA-215 1984 2 Multiracial 0 . 1 1984 1993 Rock Island IL 1 1993 2002 Moline IL 16 2002 2003 Cedar Rapids IA 2002 2003 Urbana IA AFA-216 1985 2 White 0 . 1 1985 1986 West Point NY 17 1986 1989 Alamagordo NM 32 1989 1990 Brighton MI 1990 1995 Fairfax VA 1995 2005 Manassas VA AFA-217 1984 1 White 0 . 1 1984 1986 Munich Germany 1 1986 2002 Knoxville TN 16 2002 2006 Colorado Springs CO 17 32 AFA-218 1984 1 White 0 . 1 1984 2002 Hicksville NY 1 16 17 32 AFA-219 1984 1 White 0 . 1 1984 2003 Bay City TX 16 2003 2006 USAFA CO 32 AFA-220 1983 1 Mixed 0 . 1 1983 2004 Paramaribo Suriname 1 5 12

PAGE 257

Table C-1. Continued. 240 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 16 17 32 AFA-221 1984 1 White 1 2005 2005 2/week 1 1984 2003 Nitro WV 1 2003 2006 USAFA CO 16 17 32 AFA-222 1986 1 White 0 . 1 1986 2004 Tulsa OK 1 2004 2006 USAFA CO 16 AFA-223 1984 2 White 0 . 1 1984 2003 Laguna Niguel CA 1 16 17 32 AFA-224 1982 1 Caucasian 0 . 1 1982 2000 Boulder CO 1 2000 2001 Roswell NM 16 2001 2006 Colorado Springs CO 17 32 AFA-225 1983 1 Caucasian 2 2002 2005 not often 1 1983 1986 Lansing MI 1 1986 2001 Phoenix AZ 17 AFA-226 1983 1 White 0 . 1 1983 1989 Vancouver WA 1 1989 2001 Billings MT 16 17 32 AFA-227 1984 1 White 0 . 1 1984 1984 Knoxville TN 1 1984 1990 Lawrenceville GA 16 1990 2002 Spartanburg SC 17 32 AFA-228 1984 1 Caucasian 0 . 1 1984 2003 Wheeling WV 1 16 17 32 AFA-229 1985 1 Caucasian 1 2004 2005 1pk/month 1 1986 1986 Oakland CA 1

PAGE 258

Table C-1. Continued. 241 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1986 1988 Memphis TN 16 1988 1990 ??? WA 17 1990 1993 Lubbock TX 32 1993 1995 Corpus Christi TX 1995 1996 Pensacola FL 1996 1999 Crofton MD 1999 2002 . AFA-230 1985 1 White 0 . 1 1985 2000 Montgomery IL 1 2000 2003 Yorkville IL 16 17 32 AFA-231 1984 1 White 0 . 1 1984 2002 Grand Haven MI 1 2002 2006 Colorado Springs CO 16 17 32 AFA-232 1985 1 White 0 . 1 1985 1987 Ventura CA 1 1987 1990 Long Beach CA 16 1990 1998 Tehachupi CA 17 1998 2003 Mission Viejo CA 32 2003 2005 USAFA CO AFA-233 1984 1 Caucasian 0 . 1 1984 1992 Limestone ME 17 1992 1998 Plattsburgh NY 32 1998 2002 Niagra Falls NY AFA-234 1984 2 Caucasian 0 . 1 1984 1985 Enid OK 1 1985 1986 Washington DC 16 1987 1991 Plattsburgh NY 17 1991 1994 San Antonio TX 32 1994 1997 Lubbock TX 1997 2003 San Antonio TX AFA-235 1986 2 White 0 . 1 1986 2004 Cincinnati OH 1 2004 2006 Colorado Springs CO 16 17

PAGE 259

Table C-1. Continued. 242 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 21 28 32 AFA-236 1983 1 Caucasian 0 . 1 1983 1984 Del Rio TX 1 1984 1986 Arlington VA 16 1986 1987 Plattsburgh NY 1987 1991 York ME 1991 1994 Vienna VA 1994 1998 Dallas TX 1998 2002 Virginia Beach VA 2002 2006 Colorado Springs CO AFA-237 1985 2 Caucasian 0 . 1 1985 1990 Colome SD 17 1990 1992 Utica NE 32 1992 1995 Colome SD 1995 2004 Gregory SD AFA-238 1983 1 Caucasian 0 . 1 1983 1989 Eglin AFB FL 1 1989 1992 Missawa AFB Japan 16 1992 1998 Holloman AFB AZ 17 1998 2000 Tucson AZ 32 2000 2001 Hickam AFB HI 2001 2006 USAFA CO AFA-239 1985 1 White 0 . 1 1985 1988 White Haven PA 16 1988 1989 Natchez MI 17 1989 1991 Durham NC 32 1991 1998 Cape Cod MA 1998 2002 Corpus Christi TX 2002 2005 Colorado Springs CO AFA-240 1976 1 Caucasian 0 . 1 1976 1990 Denton TX 1 1990 1995 Pueblo CO 16 1995 2003 Denton TX 17 32 AFA-241 1984 1 White 0 . 1 1984 1985 Wright-Pat AFB OH 1

PAGE 260

Table C-1. Continued. 243 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1985 1988 Colorado Springs CO 16 1988 1990 Chanute AFB IL 17 1990 2002 Colorado Springs CO 32 AFA-242 1983 1 Caucasian 0 . 1 1983 1985 Corona del Mar CA 1 1985 2002 St Helena CA 16 2002 2006 Colorado Springs CO 17 32 AFA-243 1983 1 Caucasian 0 . 1 1983 1985 Hope MI 1 1985 1986 Grand Blanc MI 16 1986 1990 Hope MI 17 1990 1993 Franklin TN 32 1993 2002 Murfreesboro TN 2002 2006 USAFA CO AFA-244 1984 1 White 0 . 1 1984 1989 Salinas CA 1 1989 2003 King City CA 16 17 32 AFA-245 1985 1 Hispanic 0 . 1 1985 1985 El Centro CA 1 1985 1992 Kearny AZ 6 1992 2002 Albuquerque NM 16 2002 2003 Carlsbad NM 17 32 AFA-246 1984 2 White 0 . 1 1984 2003 Kalispell MT 1 2003 2006 USAFA CO 16 17 32 AFA-247 1984 1 White 0 . 1 1984 1984 Port Chester NY 1 1984 1990 Yuba City CA 16 1990 1995 Oakland OR 17 1995 2002 Roseburg OR 32 2002 2006 Colorado Springs CO AFA-248 1982 1 Caucasian 1 2001 2006 5 cigs/week 1 1982 1985 CA 1

PAGE 261

Table C-1. Continued. 244 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1985 1987 ND 16 1987 1988 MI 17 1988 1990 AZ 32 1990 2001 CO 2001 2002 AL 2002 2006 CO AFA-249 1983 1 Caucasian 0 . 1 1983 1984 Fayetteville NC 1 1984 1985 Fort Jachuka AZ 16 1985 1988 Lenoir NC 17 1988 1994 Steamboat Springs CO 32 1994 1997 Wytheville VA 1997 2002 Marion VA AFA-250 1984 1 White 0 . 1 1984 2002 Los Angeles CA 1 16 17 32 AFA-251 1984 2 Caucasian 0 . 1 1984 1985 Lewisville TX 1 1985 1990 Morganton NC 16 1990 2002 Hoover AL 17 2002 2002 USAFA CO 32 AFA-252 1986 1 Caucasian 0 . 1 1986 1987 Monteray CA 1 1987 1990 Woods Bridge VA 16 1990 2004 Orem UT 17 2001 2005 USAFA CO 32 AFA-253 1984 1 Caucasian 0 . 1 1984 1985 Williams AFB AZ 1 1985 1987 McChord AFB WA 16 1987 1988 Cannon AFB NM 32 1988 1995 Lakenheath AB England 1995 1999 Seymour Johnston AFB NC AFA-254 1984 1 Asian 0 . 1 1984 1988 Durham NC 1 1988 1991 State College PA 16 1991 2002 Fredericksburg VA 17

PAGE 262

Table C-1. Continued. 245 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 32 AFA-255 1983 1 Caucasian 1 2003 2006 2/year 1 1983 1984 Stuttgart Germany 1 1984 1996 Manhattan KS 17 1996 1998 Seward NE 1998 2002 Independence KS AFA-256 1983 1 Black 0 . 1 1983 1984 Holloman AFB NM 1 1984 1991 McGuire AFB NJ 16 1991 1995 Eielson AFB AK 17 1995 2002 Baltimore MD 32 AFA-257 1984 1 Caucasian 0 . 1 1984 2002 Montoursville PA 1 2002 2006 Colorado Springs CO 16 17 32 AFA-258 1983 1 White 0 . 1 1983 1987 George AFB CA 1 1987 1992 Homestead AFB FL 16 1992 1993 Gunter AFB AL 17 1993 1995 Langley AFB VA 32 1995 1999 Mountain Home AFB ID 1999 2000 Paris France 2000 2002 Naples Italy 2002 2006 USAFA CO AFA-259 1983 2 Hispanic 0 . 1 1983 2002 San Diego CA 1 2002 2006 Colorado Springs CO 16 17 32 AFA-260 1982 1 Caucasian 0 . 1 1982 1984 Drexell Hill PA 1 1984 1987 Satellite Beach FL 16 1987 1991 Greenville PA 17 1991 2001 King of Prussia PA 32 2001 2006 USAFA CO AFA-261 1984 1 White 0 . 1 1984 1987 San Diego CA 1 1987 1990 Suore Point Phillipines 16

PAGE 263

Table C-1. Continued. 246 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1990 1995 Meridian MS 17 1995 2002 Memphis TN 32 2002 2006 Colorado Springs CO AFA-262 1986 1 Caucasian 0 . 1 1986 1986 Colorado Springs CO 1 1986 1999 Grand Rapids MI 16 1999 2003 Metamora IL 17 2003 2006 Colorado Springs CO 32 AFA-263 1984 2 Indian 0 . 2 1984 1992 Nairobi Kenya 17 1992 2002 Old Bridge NJ 32 2002 2006 Colorado Springs CO AFA-264 1984 1 White 0 . 1 1984 1990 Cincinnati OH 1 1990 2006 Barron WI 16 32 AFA-265 1979 2 White 0 . 1 1980 1998 Colorado Springs CO 1 16 17 32 AFA-266 1984 2 White 0 . 1 1984 1985 Harrogate England 17 1986 1988 Sacramento CA 32 1988 2001 Cameron Park CA AFA-267 1982 2 White 0 . 1 1982 1983 Charleston SC 1 1983 1990 Monrovia MD 16 1990 2000 . 32 AFA-268 1984 1 White 0 . 1 1984 1984 Dayton OH 1 1985 1987 Ithaca NY 16 1987 1990 Omaha NE 17 1990 1993 Deddington United Kingdom 32 1993 1995 San Antonio TX 1995 1999 Yokota Japan 2000 2002 Dayton OH AFA-269 AFA-270 1984 1 White 0 . 1 1984 1985 Aurora CO 1

PAGE 264

Table C-1. Continued. 247 Tobacco Use Residency Subject Identifier YOB Sex Race Prod Code Used From Used Until Frequency Diet From To City State/ Country Tooth # 1985 2005 Colorado Springs CO 16 AFA-271 1984 1 Anglo Sax/White 0 . 1 1984 1985 Goldsboro NC 1 1985 1988 George AFB CA 32 1988 1988 Sevel Germany 1988 1990 Flavione Belgium 1990 1992 Spangdelhm AFB Germany 1992 1996 Smithfield VA 1996 1999 Niborg Denmark 1999 2000 Montgomery AL 2000 2002 Belton TX AFA-272 1982 1 Black 1 2001 2006 2 packs/year 1 1982 2001 Macon GA 1 2001 2006 USAFA CO 16 17 32 AFA-273 1984 1 Caucasian 0 . 1 1984 2002 Easton MD 1 16 AFA-274 1983 1 White 0 . 1 1983 1987 Elmhurst IL 1 1987 2002 Inverness FL 16 2002 2006 Colorado Springs CO 17 32 AFA-275 1984 1 African American 0 . 1 . VA 1 . New Brunswick NJ 16 . NJ 17 . Brooklyn NY 32 . Hamilton (Trenton) NJ AFA-276 1985 1 White 0 . 1 1985 2003 Rochester NY 1 16 17 32

PAGE 265

248 APPENDIX D EXAMPLE PRISM LOAD SHEET Figure D-1. Example PRISM load sheet

PAGE 266

249 APPENDIX E COLUMN CHEMISTRY VESSEL AND IMPLEMENT CLEANING INSTRUCTIONS DISHWASHING Everyone who uses the beakers and vials in the clean lab is expected to help clean them. 1. Remove labels. If some glue or residue is left behind, use acetone to remove it. 2. Rinse out the beakers in house DI water (f rom the grey tap). Use a gloved finger to wipe the inside of the beaker and remove any stuck-on material. 3. Put the beakers in a soap and water bat h. There are large containers under the sink for this purpose. The soap and wate r solution is made of house DI water and few drops of Versaclean soap. 4. Soak at least overnight in the soap and water. 5. NON-TEFLON: Remove from the soap and water bath, rinse with 2X H2O and let dry on a Kimwipe by the sink. 6. TEFLON: Drain the soap and water solu tion from the container. Add house DI water, shake and rain it off. Repeat the rinse step until soap is gone. 7. Put the beakers in the 50% (7M) HNO3 bath. (Note: the HNO3 bath should always be left in the fume hood!) 8. Let sit overnight or longer. 9. Add a little 2X H2O to clean Tupperware container. 10. Remove beakers from the bath using tongs Place them in the clean Tupperware. 11. Add enough 2X H2O to cover the beakers, swish it around, and dump it out. 12. Repeat Step 11 two more times 13. Put the beakers in the 50% HCl bath (This bath contains half concentrated trace metal grade HCl and half 4X H2O). 14. Add a little 2X H2O to the clean Tupperware container. 15. Remove beakers from the bath using tongs Place them in the clean Tupperware. 16. Add enough 2X H2O to cover the beakers, swish it around, and dump it out. 17. Repeat Step 16 two more times. 18. Put the beakers in the 2X H2O bath. 19. Let sit overnight or longer. 20. Drain off the H2O 21. Fill the container with 2X H2O, swish it around and dump it out. 22. Repeat Step 21 twice. 23. Put the beaker and caps in a laminar flow hood, on a lint-free wiper. Cover loosely with parafilm and allow to dry. 24. Do not let the beakers sit for days and da ys “drying”, as they will pick up dust. 25. When they are dry, put the caps on and put them away in the drawer in Bay 2.

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250 PIPETTE TIPS 1. Place purchased pipette tips, tip down in a Teflon cup. 2. Pre-leach Sr & Pb from the plastic pipette tips by covering the tips, inside and out, with 6N HCl. 3. Shake out any air bubbles and fill to below the cup rim. 4. Place the covered cup in the fume hood overnight, on a labeled napkin. 5. Rinse tips twice in 4X H2O and leave soaking overnight in a third wash of 4X H2O. 6. Rinse tips twice in 4X H2O and shake out any excess water. 7. Put the cup of tips on the top shelf of the laminar flow hood to dry. Remove the lid, and place it askew on the cup leav ing as much of the tips exposed to the passing air as you can. 8. Once dry, recap the cup and place it in the drawer of clean pipette tips.

PAGE 268

251 APPENDIX F MISCELLANEOUS HEAVY ISOTOP E RESULTS WORKSHEETS Strontium Raw, uncorrected concentrati ons (in ng) of Sr were de rived from mass spectrometer readings. Corrected concentration equation: Sr (ppm) = (init dilution/dilu tion used)(column dissolution/vol loaded)(raw ng) (powder wt*1000) ex. CIL-001 = (3/2)(200/100)(1372) = 81.20 ppm (0.0507*1000)

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252 Table F-1. Semi-quantitative Sr concentration calculation matrix. powder initial dilutionColumn weight dilutionused dissolved loadedSr Sr (g) (mL) (mL) (L) (L) (ng) (ppm) CIL-001 0.0507 322001001372 81 CIL-003 0.0867 32200100593 21 CIL-004 0.0564 32200100555 30 CIL-005 0.0984 322001002722 83 CIL-007 0.1168 322001004773 123 CIL-009 0.0829 323501001638 104 CIL-011 0.1278 32200100— — CIL-012 0.1318 322001001420 32 CIL-013 0.0948 32350100204 11 CIL-015 0.0753 323501001131 79 CIL-020 0.1656 322001003580 65 CIL-021 0.0906 323501001535 89 CIL-025 0.1171 322001003300 85 CIL-026 0.0189 323501001755 488 CIL-028 0.0175 32350100819 246 CIL-029 0.0563 323501001560 145 CIL-032 0.0367 323501001560 223 CIL-033 0.0487 323501001120 121 CIL-034 0.0254 323501001365 282 CIL-037 0.1061 322001001772 50 CIL-039 0.0973 323501003300 178 CIL-040 0.1583 322001001852 35 CIL-042 0.1042 322001005444 157 CIL-043 0.1581 322001002722 52 CIL-044 0.0755 32200100332 13 CIL-046 0.1089 322001002600 72 CIL-047 0.1403 322001004171 89 CIL-048 0.0427 322001001600 112 CIL-049 0.1019 322001001155 34 CIL-050 0.1400 322001006333 136 CIL-052 0.1241 322001001914 46 CIL-054 0.1030 32200100930 27 CIL-055 0.1137 322001001400 37 CIL-058 0.0640 322001001444 68 CIL-059 0.1432 323501002730 100 CIL-060 0.1365 322001003333 73

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253 Table F-1. Continued. powder initial dilutionColumn weight dilutionused dissolved loadedSr Sr (g) (mL) (mL) (L) (L) (ng) (ppm) AFA-003 0.1007 323501001949 102 AFA-004 0.1011 323501001999 104 AFA-006 0.1252 32200100940 23 AFA-017 0.1244 32200100270 7 AFA-021 0.1328 322001001432 32 AFA-023 0.1436 323501001016 37 AFA-025 0.1429 32350100776 29 AFA-031 0.1267 322001001016 24 AFA-032 0.1046 32350100707 35 AFA-047 0.1106 32350100742 35 AFA-051 0.1129 32200100785 21 AFA-056 0.1039 32200100587 17 AFA-060 0.1030 32350100942 48 AFA-063 0.0650 32350100977 79 AFA-075 0.1111 323501001094 52 AFA-078 0.1000 323501001691 89 AFA-085 0.1060 32200100559 16 AFA-086 0.1120 322001001316 35 AFA-089 0.1090 32200100740 20 AFA-096 0.1090 32350100667 32 AFA-103 0.1020 32200100390 11 AFA-109 0.1020 32350100993 51 AFA-111 0.1070 32200100243 7 AFA-116 0.1212 322001001298 32 AFA-133 0.0977 32200100404 12 AFA-134 0.1327 32350100554 22 AFA-143 0.1343 32350100533 21 AFA-146 0.0988 32200100559 17 AFA-148 0.1063 323501001287 64 AFA-163 0.0912 32350100726 42 AFA-164 0.1152 32200100137 4 AFA-173 0.0887 32200100252 9 AFA-174 0.0945 32200100587 19 AFA-176 0.1578 32200100705 13 AFA-184 0.0683 32350100746 57 AFA-220 0.0949 323501001211 67

PAGE 271

254 Lead Raw, uncorrected concentrati ons (in ng) of Pb were de rived from mass spectrometer readings. Corrected concentration equation: Pb (ppm) = (init dilution/dilu tion used)(column dissolution/vol loaded)(raw ng) (powder wt*1000) ex. CIL-001 = (3/2)(300/200)(27) = 1.20 ppm (0.0507*1000)

PAGE 272

255 Table F-2. Semi-quantitative Sr concentration calculation matrix. powder initial dilution Column weight dilution used dissolved loaded Pb Pb (g) (mL) (mL) (L) (L) (ng) (ppm) CIL-001 0.0507 32300 20027 1.2 CIL-003 0.0867 32600 200211 11.0 CIL-004 0.0564 32300 200106 4.2 CIL-005 0.0984 32300 200290 6.6 CIL-007 0.1168 32300 200240 4.6 CIL-009 0.0829 32600 20070 3.8 CIL-011 0.1278 32300 20060 1.1 CIL-012 0.1318 32300 200250 4.3 CIL-013 0.0948 32600 200550 26.1 CIL-015 0.0753 32600 200120 7.2 CIL-020 0.1656 32300 20028 0.4 CIL-021 0.0906 32600 200310 15.4 CIL-025 0.1171 32300 20040 0.8 CIL-026 0.0189 32600 200125 29.8 CIL-028 0.0175 32600 200600 154.3 CIL-029 0.0563 32600 200190 15.2 CIL-032 0.0367 32600 200350 42.9 CIL-033 0.0487 32600 20042 3.9 CIL-034 0.0254 32600 2001400 248 CIL-037 0.1061 32300 20070 1.5 CIL-039 0.0973 32600 200350 16.2 CIL-040 0.1583 32300 20048 0.7 CIL-042 0.1042 32300 20080 1.7 CIL-043 0.1581 32300 20020 0.3 CIL-044 0.0755 32600 200159 9.5 CIL-046 0.1089 32300 200327 6.8 CIL-047 0.1403 32600 200101 3.2 CIL-048 0.0427 32300 200180 9.5 CIL-049 0.1019 32600 200326 14.4 CIL-050 0.1400 32300 200960 15.4 CIL-052 0.1241 32300 200230 4.2 CIL-054 0.1030 32600 200120 5.2 CIL-055 0.1137 32600 200507 20.1 CIL-058 0.0640 32300 200207 7.3 CIL-059 0.1432 32600 200150 4.7 CIL-060 0.1365 32300 200105 1.7

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256 Table F-2. Continued. powder initial dilution Column weight dilution used dissolved loaded Pb Pb (g) (mL) (mL) (L) (L) (ng) (ppm) AFA-003 0.1007 32400 2004.6 0.14 AFA-004 0.1011 32400 2002.0 0.06 AFA-006 0.1252 32600 2004.0 0.14 AFA-017 0.1244 32600 2002.6 0.09 AFA-021 0.1328 32600 2003.3 0.11 AFA-023 0.1436 32400 2001.9 0.04 AFA-025 0.1429 32400 20019.1 0.40 AFA-031 0.1267 32600 20012.2 0.43 AFA-032 0.1046 32400 2005.3 0.15 AFA-047 0.1106 32400 20025.2 0.68 AFA-051 0.1129 32600 2003.6 0.14 AFA-056 0.1039 31*400 2000.3 0.02 AFA-060 0.1030 32400 2002.8 0.08 AFA-063 0.0650 32400 2004.2 0.19 AFA-075 0.1111 32400 2005.2 0.14 AFA-078 0.1000 32400 2003.1 0.09 AFA-085 0.1060 32600 2004.1 0.17 AFA-086 0.1120 32600 2006.5 0.26 AFA-089 0.1090 32600 2005.9 0.24 AFA-096 0.1090 32400 2003.9 0.11 AFA-103 0.1020 32600 2007.3 0.32 AFA-109 0.1020 32400 20017.0 0.50 AFA-111 0.1070 32600 2002.6 0.11 AFA-116 0.1212 32600 20025.5 0.95 AFA-133 0.0977 32600 2001.1 0.05 AFA-134 0.1327 32400 2002.1 0.05 AFA-143 0.1343 32400 20015.8 0.35 AFA-146 0.0988 32600 2001.4 0.06 AFA-148 0.1063 32400 20021.4 0.60 AFA-163 0.0912 32400 2003.6 0.12 AFA-164 0.1152 32600 2002.4 0.09 AFA-173 0.0887 32600 2002.8 0.14 AFA-174 0.0945 32600 2004.3 0.20 AFA-176 0.1578 32600 2003.0 0.09 AFA-184 0.0683 32400 20059.1 2.60 AFA-220 0.0949 32400 20029.0 0.92 *Note: AFA-056 had to be rerun through column, so back-up dilution used.

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257 Table F-3. Comparison of the means for multip le runs of the GLM procedure for Pb. (CIL outlier excluded) Group Variable N Mean Std Dev Min Max East Asia 208Pb/204Pb 3538.074157 0.35506937.17610 38.88370 207Pb/204Pb 3515.603740 0.04009515.52880 15.69580 206Pb/204Pb 3518.090809 0.43583216.99170 19.62050 208Pb/206Pb 352.105517 0.0404001.95801 2.25445 207Pb/204Pb 350.862969 0.0187260.79998 0.91475 USAFA 208Pb/204Pb 3638.268931 0.22569837.39830 38.61650 207Pb/204Pb 3615.631292 0.02256515.55680 15.67490 206Pb/204Pb 3618.595094 0.28082217.68100 19.04930 208Pb/206Pb 362.058318 0.0209012.02726 2.11503 207Pb/204Pb 360.840787 0.0117960.82286 0.87981 USAFA (US only) 208Pb/204Pb 3038.318790 0.12183337.98040 38.61650 207Pb/204Pb 3015.637293 0.01589515.59830 15.67490 206Pb/204Pb 3018.683780 0.16894718.23380 19.04930 208Pb/206Pb 302.051028 0.0124522.02726 2.08576 207Pb/204Pb 300.837004 0.0068060.82286 0.85565 USAFA (foreign) 208Pb/204Pb 638.019633 0.42542937.39830 38.55000 207Pb/204Pb 615.601283 0.02832315.55680 15.63150 206Pb/204Pb 618.151667 0.32145517.68100 18.47340 208Pb/206Pb 62.094765 0.0156752.07610 2.11503 207Pb/204Pb 60.859700 0.0137950.84619 0.87981 USAFA (CONUS) 208Pb/204Pb 2738.337078 0.10648138.02970 38.61650 207Pb/204Pb 2715.639315 0.01463115.60130 15.67490 206Pb/204Pb 2718.705548 0.15509618.23380 19.04930 208Pb/206Pb 272.049609 0.0117292.02726 2.08576 207Pb/204Pb 270.836129 0.0062430.82286 0.85565 USAFA (overseas) 208Pb/204Pb 938.064489 0.35116437.39830 38.55000 207Pb/204Pb 915.607222 0.02574815.55680 15.63160 206Pb/204Pb 918.263733 0.31959317.68100 18.63290 208Pb/206Pb 92.084446 0.0209782.05207 2.11503 207Pb/204Pb 90.854759 0.0137580.83892 0.87981

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278 BIOGRAPHICAL SKETCH Laura A. Regan received her Bachelor of Science in biology from the United States Air Force Academy. She received a Master of Science in management from Troy State University and a Master of Science in z oology from Colorado State University. She received her Ph.D. in anthropology, specializi ng in osteology, skeletal biology, skeletal biochemistry, and forensic identification a nd trauma analysis. In 2003 she was awarded the Captain William F. Goodner Award for Teach ing Excellence from the Department of Biology, United States Air Force Academy. She is currently a major in the United States Air Force. She is a lifetime member of th e United States Air For ce Academy Association of Graduates; Beta, Beta, Beta Biological Honor Society; and American Society of Mammalogists. She is a student affiliate of the American Association of Physical Anthropologists and the American Academy of Forensic Sciences.


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Title: Isotopic Determination of Region of Origin in Modern Peoples: Applications for Identifying U.S. War-Dead from the Vietnam Conflict
Physical Description: Mixed Material
Copyright Date: 2008

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ISOTOPIC DETERMINATION OF REGION OF ORIGIN IN MODERN PEOPLES:
APPLICATIONS FOR IDENTIFICATION OF U.S. WAR-DEAD FROM THE
VIETNAM CONFLICT













By

LAURA A. REGAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006


































Copyright 2006

by

Laura A. Regan































To Ronald D. Reed, Ph.D., Brigadier General, USAF
11 November 1948 20 April 2005

It is with bittersweet appreciation that I thank an incredible mentor for taking a risk and
agreeing to this absolutely insane adventure; for the opportunities he provided and his
unwavering confidence in me. It is with great regret that I cannot share my success in
this endeavor with him. I was fortunate to know him and I dedicate this work to his
memory.















ACKNOWLEDGMENTS

I could not have met all the crazy deadlines of this breakneck-paced program

without the assistance of a great many people. First, I wish to extend my sincere

appreciation to the members of my doctoral committee, Drs. Anthony B. Falsetti (Chair),

David Daegling, Thomas Holland, Connie Mulligan, and David Steadman. Not one of

you ever indicated you had any reservations about my ability to complete this program in

a blistering 3 years. Your faith in me fueled me on when I was doubting myself.

Boss, I have never encountered a professor who is so nurturing of his students, yet

has no qualms about telling us when we're being knuckleheads. I cannot express my

gratitude for all you have taught me, your unwavering friendship, your constant

confidence in me, and your personal support throughout this program. You've opened

countless doors for me. I will never be able to repay you for all of your kindness,

generosity, and all of the laughs. You take good care of your "kids." Please take good

care of yourself as well. No setting yourself on fire any more.

Dr. Daegling opened my mind and challenged me to think critically on a whole

new plane. The academic rigor of his courses was both mildly overwhelming and

incredibly fulfilling. Receiving an "A" in his course was truly something to covet. A

great thank you goes out to Dr. Thomas Holland, Joint POW/MIA Accounting

Command-Central Identification Laboratory Scientific Director, for supporting me and

this project, allowing me to intern with him for three incredible months, and putting me

on that week-long C-17 ride to Vietnam. I have partaken in some once in a lifetime









experiences through your generosity, and those memories I will always cherish. Please

don't forget about me. I'll be looking for ajob in about 8 years.

Dr. Mulligan broke down my internal block when it came to understanding genetics

and taught me a great deal about attention to detail and organization. She held my feet to

the fire and made me not only address but fully understand the flaws in my work, vastly

improving the quality of my scientific work. The journey to enlightenment could sure be

frustrating though. Dr. Steadman was a constant source of enthusiasm and energy. His

positive attitude kept me going and allowed me to overcome a long seeded loathing of

avian fauna that arose during my days in undergraduate Vertebrate Zoology. Birds are

cool!

I owe Dr. Andy Tyrell a great deal of gratitude as well, for getting me se up at CIL

and through his continued guidance and assistance. To Col. (ret) Thomas and Col. Merle

Sprague, thank you for allowing me to mooch off of you for 3 months. You opened your

hearts and home to me. I am truly grateful and a better person for knowing you. I would

also like to pass along my appreciation to LTC Mark Gleisner, for showing me the basics

of drilling teeth and along with the rest of the CIL dental guys, answering my many,

many questions.

I owe a great deal to Col. Nancy Perry and Maj. Albert Ouellette, 10th Dental

Squadron, U.S. Air Force Academy for agreeing to assist me with this project and

especially to Albert, who provided over 1000 freshly extracted third molars to me during

the course of this study (are you sure you guys do not have a quota?). Thanks also go out

to Drs. Jack Meyer and Ray Berringer from the North Florida/South Georgia Veterans

Health System, Veterans Affairs Dental Clinic..









I am indebted to Dr. Bruce MacFadden, Florida Museum of Natural History, who

really exposed me to the possibilities of isotope studies and in whose class this project all

took shape. He graciously allowed me the use of his laboratory to prepare samples, took

keen interest in my progress, and always greeted me with a smile, no matter what the

circumstances. I also learned a great deal about the basics of isotope work from Dr.

Joann Labs Hochstein and am grateful for her tutelage as I was starting out.

I would like to thank Dr. John Krigbaum for planting the seed of awareness of

stable isotope studies and bailing me out during a great time of need. Your genuine

concern for your students is well known. I would like to acknowledge the contributions

of George Kamenov, who showed me the ropes of heavy isotopes and gave me great

insight into their power and Dr. Jason Curtis, who worked around my crazy schedule,

even when his was just as bad, and always had time to answer my questions, even when

he was out of the country.

To the "Frogs," I cannot wait to rejoin your ranks. A special thank you goes out to

Col (ret) James Kent. Sir, you have been there for the course of my journey in academia.

I thank you for your patience, guidance, and gentle pushes in the right direction. Look-I

did not change my major once this degree program!

I would like to thank my family and friends for all of their love and support over

the years. You mean the world to me. Anna, you are the most selfless friend anyone

could ever be blessed with. I don't know what I would have done without you but I do

know I can never repay your kindness nor the countless times you bailed me out of a

difficult situation. Greg, you were a constant sounding board and helped me through

some very difficult times. Hang in there my friend. There is light at the end of the









tunnel, and no, it isn't a train. Shanna and Erin, I can't tell you how my stress melted

away when I was in your company-and thanks to some apple juice-laced wine. I'll miss

our girls' dinners more than you will ever know. Thanks to Laurel and her technical

wizardry and extraordinary and often utilized dog sitting skills. I also wouldn't have

been able to launch this project if it hadn't been for the assistance of Alicia during the 3

months I was away. I can't tell you how much your help eased my mind. I owe much

appreciation to Carlos for teaching me the basics of tooth identification and to Miss

Shiela for assisting me with numerous mind-numbing tasks. Have a great Air Force day!

I'd also like to thank the rest of the Pound Lab rats and lab rats by-proxy: Dr. Mike

Warren, Shuala, Joe, Trey, Paul, Ron, Nicolette, Kathy, Debbie, Pat, Melissa, Megan, and

Jennifer; and my friends Chad, Laurie, and Erin. You added immeasurable levity to my

life during a period of extreme stress and thoroughly deprogrammed me. I couldn't ask

for a better cohort to be associated with. It's going to be tough going back to the real

world.

I also owe a huge debt of gratitude to two undergraduate assistants, Ursula Zipperer

and Ana del Alamo, who spent countless hours helping me with the most mundane of

tasks. Lastly, I must express my heartfelt appreciation to Calvin and Hobbes. I would

not have survived this program, especially the first year, without you guys, but it would

have been nice if you had not eaten the door ..twice.

To all the men and women who have gone before me in service to our nation and to

those who currently serve, I salute you. I am proud to be among your company. It has

been an incredible honor to complete this project with the hopes of reuniting families

with their long, lost, loved ones. Until they are home ...









My tuition was provided by the United States Air Force. This research was funded

in part by the Joint POW/MIA Accounting Command-Central Identification Laboratory,

the C.A. Pound Human Identification Laboratory, a William R. Maples Scholarship, and

my savings account.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F T A B L E S ........ .......................................................... ..................... xii

LIST OF FIGURES ......... ....... .................... .. ....... ........... xiv

ABSTRACT ........ .............. ............. ........ ..................... xvi

CHAPTER

1 ST A B L E ISO T O PE S ............ .............................................................. ......... .. ....... 1

Stu dy Isotopes ............................................... 6
C arb on ............................................................. . 6
O x y g en ....................................................... 7
Strontiu m ................................................................... 8
L e a d ........................................................................................................1 0
Fractionation ................... ...... ......................................... ...............11
Frequently Sampled Human Tissues .............................. ...............13
B o n e ............................................ ...... ............................................................ 1 4
T e e th ............................................ ....... ........................................1 5
H air ........... ........ ..... .............................. .......... 17
F ingernails and T oenails ........................................................ 18
S k in ............................................ ................................................1 9
Com plications .................................. .......................... .... .... ........ 20
Diagenesis ................................ ......... 20
Anthropogenic Contamination ................................ ............... 27
G global E conom y ..................................... ....................................................... ...... 28

2 APPLICATIONS OF STABLE ISOTOPE ANALYSES ........................................30

T ra c in g S tu d ie s ...................................................................................................... 3 0
F ractionation Studies .............................................................32
Z o ology an d E ecology ............................................................................................. 3 3
A rch aeology .................................3.............................5
D iet A ssessm ent .............................................................35
Introduction of m aize ......................................................... ..... .......... 35
W meaning practices ......................................................... 37










R region of O rigin .................. ........................................................39
M material Culture.............. .. ................ ................ .. 41
Forensic Investigations ............. .... ............. ................................... 42

3 HUMAN FORENSIC IDENTIFICATION......................................................50

M military Identification M measures ........................................ .......................... 54
P resent Study ................................................................... 58

4 M ATERIALS AND M ETHODS ........................................ ......................... 66

D en tal P ro to c o ls .................................................................................................... 6 7
S am pling ............................................................................................................... 73
Central Identification Laboratory ................................................. .........74
United States Air Force Academy and Veterans Affairs................ .............. ....77
Carbon and Oxygen Sample Preparation......................................... ............... 80
Central Identification Laboratory Samples ............................... ............... 80
United States Air Force Academy Samples ................................................83
Strontium and Lead Sample Preparation ............ .........................................84
Statistical A analyses .......................................................... ... ...... ..... 90

5 ANALYTICAL COMPARISON OF EAST ASIAN AND AMERICAN
S A M P L E S ................................................................9 3

L eight Isotop es ....................................................... 93
C a rb o n ........................................................................................................... 9 3
O x y g e n .............................................................................................................. 1 0 8
Acetic Acid Test ...... .................. .......... ........114
Heavy Isotopes .......... .. .. ................ .......... 118
Strontium ............... ......... .......................118
Lead .............. ............................................ ..... ..... ......... 125
M ulti-elem ent A approach ..................................................................................... 133

6 VARIATION WITHIN USAFA SAMPLES ................. ................. ...........139

Y e a r o f B irth ....................................................................................................... 1 3 9
S e x .........................................................................1 4 1
R a c e .................................................................................................................... 1 4 2
T tobacco U se ................................................................ 143
D ie t ........................................................................ 1 4 6
R e sid e n c y ............................................................................................................ 1 4 6
S tro n tiu m ....................................................... 14 6
Lead ....................................... 150
Regionality ......................................... ......................................... 151
Relationship Between 6180 Values and Latitude ...................................... 156
Duplicate Residences ................................. ........................... ........... 159
Comparison to the Literature ................................. ...............................161



x









7 SU M M A R Y /C ON CLU SION ........................................................ .....................165

APPENDIX

A REPLICATED VETERANS AFFAIRS BINDER................... ........................ 172

B CENTRAL IDENTIFICATION LABORATORY SAMPLING.............................203

C UNITED STATES AIR FORCE ACADEMY SURVEY RESULTS ....................210

D EXAM PLE PRISM LOAD SHEET.......................... .................... ............... 248

E COLUMN CHEMISTRY VESSEL AND IMPLEMENT CLEANING
IN STRU CTION S ............................................ .. .. .... ......... ......... 249

F MISCELLANEOUS HEAVY ISOTOPE RESULTS WORKSHEETS ..................251

L IST O F R E FE R E N C E S ..................................................................... ..... .................258

B IO G R A PH IC A L SK E T C H ........................................ ............................................278
















LIST OF TABLES


Table page

1-1 Stable isotope standard materials and calibrants............... ..... ................. 5

1-2 Mean age of completion of permanent crown mineralization..............................16

2-1 Mean and standard deviations for selected groups of immigrant teeth (enamel).....49

3-1 Form s of forensic identification. ............................... ............................ ................ 54

3-2 Numbers of unaccounted for U.S. prisoners of war and/or those missing in
a ctio n ............................................................................ 5 9

3-3 United States casualties in Southeast Asia by race. ............................................60

3-4 United States military listed as unaccounted for in Southeast Asia by race. ...........60

4-1 Crown formation/tooth eruption.............. ............................... 74

4-2 Isotope sam pling m atrix. ............................................... ................................ 78

5-1 Summary statistics and general linear model results of all isotopes examined for
CIL samples compared to USAFA samples (CIL outlier excluded). All values
are in % o ............................................................................. .9 4

5-2 Carbon and oxygen isotope results. All values are in %o. ....................................95

5-3 Central Identification Laboratory outlier run data. ..............................................102

5-4 613C value comparison. Twelve most enriched CIL samples and 12 most
depleted USAFA samples (CIL outlier excluded). All values measured in %0.....103

5-5 Summary statistics and general linear model results of all isotopes examined for
American and foreign USAFA comparison (CIL outlier excluded). All values
are in %o. ................... ..... ........... ................... ................. 105

5-6 Summary statistics and general linear model results of all isotopes examined for
CONUS and overseas USAFA comparison (CIL outlier excluded). All values
are in %o. .......................... ........... ......................... ................ 105

5-7 East Asian 6180 values, in ascending order. .................................. .................109









5-8 Partial list of USAFA 6180 values, in ascending order (30 most depleted and 30
m ost enriched) ................................................................. ... ......... 110

5-9 Results of acetic acid test, with intertooth comparison when available...............16

5-10 Strontium isotope values for CIL and USAFA samples, in ascending order.........119

5-11 Comparison of the means for multiple runs of the GLM procedure for 87Sr/86Sr.
(CIL outlier excluded.) .......................................... ............ ................. 124

5-12 Lead isotope results for East Asia. ............................................... ............... 126

5-13 Lead isotope results for USAFA. .........................................................................127

5-14 Comparison of spiked lead concentration data (actual) with semi-quantitative
data. (All values are in ppm .). .................................. ................................ 133

6-1 USAFA-provided sampling demographics, American natal region only..............140

6-2 Locations during amelogenesis represented by sampled USAFA teeth. .............147

6-3 Strontium isotope values for American USAFA samples, in ascending order......148

6-4 Mean 207Pb/206Pb values for major U.S. lead ore deposits................................ 152

6-5 207Pb/206Pb values Americans reared in the United States, in ascending order......152

6-6 Region membership based on 6180 values.............. ..................... ........ ....... 153

6-7 Region-pair comparison for difference in 6180 means. .......................................155

6-8 Summary statistics for American USAFA 6180 values based on latitude. All
values are in %o. ........... ..... .................................................. .................. 158

6-9 6180 values corresponding to cities in which multiple participants resided. (All
values in %o.) .......................... ........... .............. 160

6-10 Comparison of Alberta fur trader lead values to USAFA donor from Alberta......164

B-l Central Identification Laboratory sampling data. .............................................204

C-l United States Air Force Academy survey data. ................................................211

F-l Semi-quantitative Sr concentration calculation matrix .......................................252

F-2 Semi-quantitative Sr concentration calculation matrix.......................................255

F-3 Comparison of the means for multiple runs of the GLM procedure for Pb. (CIL
outlier excluded)........ ...... .......................................... ......... 257














LIST OF FIGURES


Figure page

4-1 Joint POW/MIA Accounting Command survey. ............................................. 70

4-2 Pre-drilling photo of CIL-033 #19 with data card. Note: the accession number is
purposely, partially obscured. ............................................................................76

4-3 Pre-drilling photo of AFA-093 #32...................................... ..................... 80

4-4 Loaded tray for PRISM mass spectrometer analysis. ............. .............. 82

5-1 Carbon and oxygen isotope results with overlapping value overlay......................99

5-2 Carbon and oxygen isotope results for American and foreign USAFA
com prison ...................................................... ................. 104

5-3 Carbon and oxygen isotope results for CONUS and overseas USAFA
com prison ...................................................... ................. 106

5-4 Latitudinal dispersion of major natal regions featured in this study. East Asia is
on the right. Information drawn from Rand McNally Atlas (1998)......................111

5-5 Weighted Annual 6180 for Asia. Map reproduced from IAEA (2001) ..............112

5-6 Weighted Annual 6180 for North America. Map reproduced from IAEA (2001). 112

5-7 Plot of strontium values compared to 208Pb/204Pb. .......................................... 121

5-8 Plot of 87Sr/86Sr compared to 206Pb/204Pb ........... ............................. ............. 122

5-9 Box and whisker plot of 87Sr/86Sr values. ................................................... 123

5-10 Comparative histogram of CIL and USAFA sample Sr concentrations (semi-
quantitative).................................... ................................ .......... 125

5-11 Plot of 206Pb/204Pb compared to 208Pb/204Pb. ............................... ............... 128

5-12 Plot of 206Pb/204Pb compared to 207Pb/204Pb. .......... ........................................ 128

5-13 Plot of 208Pb/206Pb compared to 207Pb/206Pb. .......... ........................................ 129









5-14 Comparative histogram of CIL and USAFA sample Pb concentrations (semi-
quantitative).................................... ................................ ..........131

6-1 Strontium isotope composition of the U.S. showing inferred s87Sr values, as
calculated by age variations in basement rocks. Image reproduced from Beard
and Johnson (2000), with permission ............... ........................... .............. 149

6-2 Region m ap based on 6180 values..................................... ......................... 153

6-3 Plot of latitude compared to 6180 values. Error bars equal 1 std dev .................157

6-4 Range of 6180 values produced from this study for specific cities ......................161

D -1 Exam ple PRISM load sheet......................................................... ............... 248















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ISOTOPIC DETERMINATION OF REGION OF ORIGIN IN MODERN PEOPLES:
APPLICATIONS FOR IDENTIFICATION OF U.S. WAR-DEAD FROM THE
VIETNAM CONFLICT

By

Laura A. Regan

August 2006

Chair: Anthony B. Falsetti
Major Department: Anthropology

This study is novel in that it is the first of its kind to compile a reference sample of

isotopic values associated with known natal regions to be utilized in forensic work.

Stable isotopes of carbon, oxygen, strontium, and lead were examined to determine if

natal origins could be assessed isotopically between Southeast Asian and American

dental remains as well as regionally within the United States. Teeth believed to be of

East Asian origin were compared to the extracted third molars of recent American dental

patients. Living subjects completed surveys detailing physiological, behavioral, and

residential information that affect isotope values. The least squares means for all isotope

values examined exhibited significant differences between the East Asian and American

cohorts. Based on this information, a discriminant function was created that correctly

classified individuals, through resubstitution and cross-validation, as belonging to one of

these two groups by 95% or better. The sexes differed significantly as to their carbon

ratios with females displaying more enriched values than males. Significant differences









were also noted for 613C means among those who have never used tobacco products and

those who partook of smokeless tobacco. American strontium values displayed a distinct

trend toward homogenization, with the mean value for 87Sr/86Sr varying only slightly

from that of seawater. In order to identify natal origin among Americans, nine regions

were created within the United States based on 6180 values. Good discrimination was

noted between the mountain states and the southern states. A discriminant function

analysis proved disappointing though, and additional sampling from most states is needed

to improve the statistical robusticity of the model. The results of this study will have

wide-reaching effects across the medico-legal spectrum. This body of research will serve

as the foundation for a database of modern, human, geolocational isotope values that will

assist not only in the identification of fallen servicemen and women, but in the

identification of victims of mass fatality incidents, undocumented aliens who perish

attempting entry into the U.S., and local skeletal "Jane and John Doe" cases.














CHAPTER 1
STABLE ISOTOPES

Ascertaining the national origin of unidentified human remains is problematic,

especially with the passage of time. Often, the number of identifiable bony elements is

so few, fragmentary and/or degraded by the chemical properties of the soil, that

estimating biological profiles and DNA analyses cannot effectively be performed. This

challenge is particularly acute for the Joint POW/MIA Accounting Command's Central

Identification Laboratory (JPAC-CIL). The identification of unknown remains believed

to be missing U.S. service personnel is frequently hampered by high levels of degradation

and fragmentation as a result of circumstances of loss and subsequent taphonomic

regimes. If the geo-political region of origin for a set of remains could be established, it

would facilitate the construction of identification shortlists, especially from large, open-

ended decedent populations. This, in turn, would provide a highly effective means of

excluding possible candidates for identification, notably for human remains whose

provenience is either unknown or suspect. One potential tool in determining

geolocational origins of skeletal material is that of stable isotope analyses. Developed

primarily in the geochemical community (Fogel et al. 1997), stable isotope work has

revolutionized the anthropological realm, beginning with pioneering, archaeological,

dietary studies in the late 1970s (DeNiro & Epstein 1978a and 1978b, van der Merwe &

Vogel 1978).

Isotopes of a particular element are atoms whose nuclei contain the same number of

protons but differ in their number of neutrons (Hoefs 2004). It is the number of protons









in the atom that determines what the element is as well as how many electrons the atom

has (Herz & Garrison 1998). An atom at rest has a neutral charge; therefore, the normal

state for an atom is to have the same number of protons within the nucleus as electrons

outside of the nucleus. As stated previously, isotopes vary because of the differing

number of neutrons within the nucleus. This neutron variation, will in turn, affect the

atomic masses of different isotopes of the same element because the atomic mass is a

measure of the sum of the number of protons and neutrons (Hoefs 2004).

For example, carbon has an atomic number of "6," meaning an atom of carbon

contains 6 protons within the nucleus. Even though the number of protons is constant

within a carbon atom, it can take on three isotopic forms: 12C, 13C, 14C. A carbon atom

with a mass of 12 (denoted 12C) has 6 protons and 6 neutrons, one less neutron than a

carbon atom with a mass of 13 (13C) and two fewer neutrons than 14C.

Since chemical reactions are largely determined by the ionic or atomic electron

configuration, the varying isotopes of an individual element will have the same chemical

properties (Schwarcz & Schoeninger 1991). Different isotopes of a single element will

have different kinetic and thermodynamic properties when they undergo chemical

reactions though, because of differences in reaction rates and heat capacity influenced by

their different atomic masses (Urey 1947). So, while isotopes of a like element will react

the same chemically, they will react at different rates, due to their different atomic masses

and sizes. Different metabolic and chemical processes therefore change the ratios

between the isotopes in a characteristic manner (van der Merwe 1982). It is also noted

that as atomic weight increases, the differences in thermodynamic properties between

isotopes generally decrease (Urey 1947). In other words, light isotopes such as those of









hydrogen, carbon, and oxygen will have a much greater variation in their thermodynamic

and kinetic characteristics than heavier isotopes such as strontium and lead.

Stable isotopes are not radioactive (Hoefs 2004), thus they do not spontaneously

change into another atom or another isotope of the same element (Herz & Garrison

1998). Revisiting the carbon example, when considering the three isotopic forms of

carbon (12C, 13C, 14C), the former two are stable isotopes, while the latter is radioactive

(van der Merwe 1982), and commonly utilized for archaeological dating purposes.

Stable isotopes may also be characterized as radiogenic or nonradiogenic. A

particular isotope is classified as radiogenic if it is the product of the decay of a "long-

lived" radioactive isotope (Schwarcz & Schoeninger 1991). Strontium ( Sr) and lead

(206Pb, 207Pb, 208Pb) are the primary radiogenic isotopes used in nutritional ecology

studies. 87Sr forms from the radioactive decay of rubidium (87Rb) while 206Pb and 207Pb

arise from the decay of uranium (238U in the case of 206Pb and 235U for 207Pb) and 208Pb

results from the decay of thorium (232Th) (Herz & Garrison 1998). These radiogenic

isotopes vary considerably in abundance with respect to their associated non radiogenic

isotopes (86Sr and 204Pb) (Schwarcz & Schoeninger 1991) and serve as useful analytical

tools.

Eighty-one elements have stable isotopes of varying numbers (Herz & Garrison

1998). All of the biochemically important elements, with the exception of fluorine, have

more than one stable isotope (Schwarcz & Schoeninger 1991). Four of these; carbon,

oxygen, strontium, and lead; were examined in this study will be discussed in detail being

on page 6.









Measurements of stable isotopic ratios are performed by a mass spectrometer, an

instrument that determines the relative abundances of different isotopic masses in a

variety of elements (Thirlwall 1997). For carbon, the mass spectrometer determines the

raw ratio of 13C/12C, which it then compares to the ratio of a marine carbonate standard,

known as Pee Dee belemnite (PDB, now referred to as V-PDB, based on the Vienna

Convention; Hoefs 2004). The difference between the sample ratio and the V-PDB

standard ratio is what is known as the relative 13C content and is the value reported and

used for inferential purposes (van der Merwe 1982). The equation is as follows:

element = (ratiOsample/ratiOstd -1) x 1000%o = value in %o

613C 13C/12same x 1000%o (1-1)
C/ CV-PDB

This measure is denoted by the symbol 6 (delta) and measured in parts per mil (%o)

(van der Merwe 1982). If the hypothetical 13C/12C ratio of a sample was calculated as 12

per mil less than the V-PDB standard, the 613C value would be -12%o and considered

depleted compared to the sample. It is important to note that the V-PDB standard does

not equal zero (it equals 2.0671 x 10-6; Hoefs 2004) and results should not be interpreted

as deviations from the zero point.

Oxygen values for 180/160 are calculated similarly. When 6180 is calculated in

concert with 613C, the V-PDB standard is used along with a conversion factor (Dr. Jason

Curtis, personal communication). When isotopic calculations are performed singly or in

combination with hydrogen, the internationally accepted standard of standard mean ocean

water (SMOW or V-SMOW) is used (Hoefs 2004). The heavy isotopes of strontium and

lead are not generally normalized to a conventional standard, but instead, results are









expressed directly as ratios (Herz & Garrison 1998) and the standards are used for mass

spectrometer calibration adjustments.

Stable isotope standards have been drawn from a variety of sources over the years.

Some of the most commonly utilized in zoological and anthropological studies are listed

in Table 1-1.

Table 1-1. Stable isotope standard materials and calibrants.
Element Ratio Standard (Std) Std Notation Std Value
Hydrogen1 D/H (2H1H) Standard Mean SMOW or 155.76 x 10-6
Ocean Water V-SMOW
Carbon1 13C/12C Belemnitella PDB or V- 2067.1 x 10-6
Americana from PDB
the Cretaceous
Peedee formation,
South Carolina
Nitrogen1 15N/14N Air nitrogen N2 (atm) 3676.5 x 10-6
Oxygen' 1O/O60 Standard Mean SMOW or V- 2067.1 x 106
Ocean Water SMOW
also
Belemnitella PDB or V- 2067.1 x 10-6
Americana from PDB
the Cretaceous
Peedee formation,
South Carolina
Strontium2 7Sr/86Sr Strontium NBS-987 or 0.7045
carbonate/bulk NIST 987
earth
Lead3 208Pb/204Pb Lead metal wire NBS-981 or 36.696
207Pb/204Pb NIST 981 15.491
206pb/204pb 16.937
0.9146
207Pb/206Pb 2.1665
208Pb/206Pb
SFrom Hoefs (2004)
2 From Beard and Johnson (2000)
3 George Kamenov (2006)









Study Isotopes

Carbon

In 1968, Margaret Bender first reported that the major photosynthetic pathways of

plants manifest themselves in distinct carbon isotope ratios. This discovery served as the

catalyst for the multitude of carbon isotope studies documented in the literature today.

When interpreting carbon isotope signatures, one must harken back to the days of basic

biology class and discussions of the differences in the two major photosynthetic systems.

C3 photosynthesis occurs in the majority of cultivated and wild plants in temperate

regions (Schwarcz & Schoeninger 1991), such as wheat, rice, and barley, and produces

an initial three-carbon metabolite (van der Merwe 1982, Schwarcz & Schoeninger 1991,

MacFadden et al. 1999b). C4 photosynthesis, found in more drought-resistant plants,

produces an initial four-carbon compound in cultigens such as sugar cane, maize and

millet (van der Merwe 1982, Schwarcz & Schoeninger 1991, MacFadden et al. 1999b).

These different metabolic processes produce different isotopic ratios, which are then

incorporated into plant tissues. C4 plants exhibit more rapid carbon dioxide intake

leading to values between -9%o and -16%o. C3 plants on the other hand, have slow rates

of carbon dioxide uptake leading to values from -20%o to -35%o (van der Merwe 1982).

What makes carbon isotope analyses so powerful is that these to ranges do not

overlap. Intermediate values are found in plants utilizing a third photosynthetic pathway,

CAM or crassulacean acid metabolism (van der Merwe 1982, MacFadden et al. 1999b).

These plants are primarily succulents such as cactus and pineapple, and as such, they

neither factor significantly into most human diets nor the present research.

Plants demonstrate preferential uptake of 12C to 13C, thus they are depleted in 13C

compared to12C (Bender 1968). These two carbon species are differentially incorporated









into body tissues (i.e., they are fractionated in a characteristic manner) during digestive

processes (Durrance 1986). As a result of the differences in photosynthetic pathways in

plants, it is also possible to determine approximate proportions of C3 versus C4 plants in

an individual's diet based on the 613C value (Schwarcz & Schoeninger 1991).

Carbon isotopes also convey information regarding the use of marine foods in an

organism's diet. Marine animals present isotopic signatures intermediary to C3 and C4

food chains (Schoeninger & DeNiro 1984, Larsen et al. 1992). Marine mammals and fish

display 613C values that are enriched by roughly 6%o over animals that feed on C3

foodstuffs, and depleted by about 7%o compared to animals that feed on C4-based foods

(Schoeninger & DeNiro 1984). The best indicator of a reliance on marine food sources is

the information provided through a joint 613C and 815N analyses (Schoeninger & DeNiro

1984, Ambrose & Norr 1993).

Oxygen

Oxygen is the most abundant elemental component of the earth's crust (Herz &

Garrison 1998) and its isotopic ratios provide an indication of the point of origin of

remains. Isotopes of oxygen take the form of 160, 170, and 180 (Mattey 1997). Oxygen

is primarily incorporated into body tissues via atmospheric oxygen, water, and oxygen

bound in food (Sponheimer & Lee-Thorp 1999b). Because the 6180 value of atmospheric

oxygen is relatively constant, it is believed that oxygen isotopic signatures are primarily

representative of imbibed water, and to a lesser extent, the macronutrients found in

foodstuffs (Sponheimer & Lee-Thorp 1999b). The oxygen isotopes in water are

preserved in bone, teeth, and other tissues and are reflective of a particular environment

and climate, decreasing with increasing latitude, increasing altitude, and as you move

inland (Dupras & Schwarcz 2001, Kendall & Coplen 2001, Rubenstein & Hobson 2004).









Analytically available oxygen is present in both the phosphate and carbonate ions

of hydroxyapatite in the mineral phase of skeletal tissues. Most studies have examined

phosphate oxygen because the P-O chemical bond is much stronger than the C-O bone,

suggesting that phosphate oxygen is less susceptible to diagenesis than carbonate oxygen

(lacumin et al. 1996, Sponheimer & Lee-Thorp 1999b). Lengthy and harsh chemical

procedures are required to extract the phosphate oxygen from apatite however, while the

carbonate portion is easily obtained from the CO2 produced during mass spectrometry for

carbon isotopes (Sponheimer & Lee-Thorp 1999b). Bone carbonate has shown a strong

positive correlation to local meteoric water with an r2 value = 0.98 (lacumin et al. 1996).

Additionally, both carbonate and phosphate are better preserved by highly-mineralized

tooth enamel versus more porous dentin and bone phosphate (lacumin et al. 1996).

Strontium

Strontium has been used to characterize prehistoric mobility patterns since the mid

1980s (Budd et al. 2004, Millard et al. 2004). There are four stable isotopes of strontium:

"Sr, 17Sr, 86Sr, and 84Sr. Only 87S is the product of radioactive decay (radiogenic), being

a product of the beta decay of rubidium 87. This radioactive decay pair, 8Rb-87Sr, has

consequently produced distinctively different 87Sr abundances in different parts of the

earth over its history (Beard and Johnson 2000) that have proven quite valuable in tracing

the origin of matter to a particular locale.

Strontium signatures depend purely on local geology since they reflect the

underlying bedrock of a particular area. Strontium isotopic ratios vary with the age and

type of bedrock underlying the soil. So the quantity of strontium in a particular rock will

depend not only on the amount of rubidium parent material found in the rock, but the age

of the rock as well as the original amount of 87Sr present in the rock when it was formed.









Strontium varies in plant tissue with the age and type of geological substrate or bulk

composition. Older soils are more enriched compared to younger soils as are calcium-

rich soils compared to calcium-poor soils. Additionally, atmospheric deposition or dry

fall from natural sources can also affect strontium values (Beard and Johnson 2000).

Anthropogenic factors that can influence isotope ratios include nuclear fallout, airborne

pollution from fossil fuels, and land-use practices that expose bedrock (Rubenstein and

Hobson 2004).

Strontium is incorporated into human tissue following the calcium pathway

because this non-nutrient, non-toxic element has chemical properties similar to calcium

(Aberg et al. 1998). During nutrient uptake strontium often replaces calcium in bones

and therefore can be used to trace the flow of minerals from the soil through the food web

(Rubenstein and Hobson 2004). "Strontium concentrations in plants and animals are

controlled by trophic position, but the isotopic composition is invariant; that is, Sr does

not fractionate. Thus, bones and teeth in an individual will have different Sr abundances

but identical 87Sr/86Sr ratios" (Herz & Garrison 1998), with human enamel demonstrating

lower strontium content than bone (Price et al. 1994, Grupe et al. 1997, Beard & Johnson

2000). If food sources are local then, all participants in the food chain, regardless of what

tissue is sampled, should reflect the same isotopic signature.

Additionally, strontium abundance has commonly been examined to discriminate

between the meat and vegetable components in an organism's diet. Toots and Voorhies

(1965) published the seminal study in this area, discovering significant differences

(p-value <0.001) not only between the mean strontium concentrations for fossil Pliocene

carnivores and herbivores, but among the herbivorous grazers and browsers themselves.









The basis for this is that for each trophic level above the soil, there is a metabolic

discrimination against strontium in mammalian epithelium, as opposed to calcium

(Radosevich 1993). As one increases in trophic levels among the consumers, the

contribution from food sources to skeletal strontium is decreased at each step (Toots &

Voorhies 1965). Plants will retain 50-100% of the strontium found in the soil, with each

progressive trophic level exhibiting a reduction of 33% strontium over the lower level

(Radosevich 1993). Keep in mind that this refers to strontium abundance (or

concentration) and not the 687Sr value. So theoretically, someone such as a vegan should

have a higher strontium concentration than an ardent follower of the Adkins' diet, by

approximately 33%. Radosevich (1993) cautions against blindly accepting these

measurements however, without first considering factors such as parent material and soil

chemistry variation influencing plant uptake and physiological differentiation, as well as

behavioral changes in feeding strategies, trophic placement, and cultural practices.

Lead

"Lead is one of the most heavily utilized metals in human history" (Sangster et al.

2000). Lead has four naturally occurring stable isotopes: 204Pb, 206Pb, 207Pb, and 208Pb.

As previously discussed, the latter three isotopes are radiogenic. Because 204Pb is not

radiogenic, it serves as stable reference isotope (Sangster et al. 2000). Similar to

strontium, the isotopic composition of lead in a particular locale (or ore deposit) is

dependent upon four factors: 1) the length of time before lead was separated by

geological processes in the source reservoir; 2) the decay rate of the parent isotopes; 3)

the initial ratio of the abundance of the parent material to the abundance of lead in the

source reservoir; and 4) the initial isotopic constitution of the reservoir lead (Sangster et

al. 2000). The variations in parent isotope decay rates result in systematic differentials in









the ratios of 206Pb, 207Pb, and 208Pb to each other, as well as to 204Pb (Sangster et al.

2000). Most archaeological studies are based on the ratios of the radiogenic isotopes to

204Pb, whereas environmental studies tend to also form ratios from only the radiogenic

isotopes themselves (Dr. George Kamenov, personal communication). Additionally, lead

is favored by many researchers because like strontium, it does not exhibit fractionation in

nature (Stille & Shields 1997).

Lead is assimilated into skeletal elements in a similar manner to strontium, in that it

accumulates from the blood through calcium pathways and substitutes for calcium in the

carbonate hydroxyapatite fraction of hard tissues (Vogel et al. 1990). Juveniles exhibit a

higher propensity to absorb ingested lead than adults (Reinhard & Ghazi 1992), likely

due to the rapid modeling of bone occurring during the growth phase and because small

children tend to frequently put objects in their mouths. Lead particles are thought to enter

the body through ingestion, either through food stuffs/fluids or lead objects, or inhalation

(Gulson 1996). Environmental contamination by lead is found through mining

operations, waste dumps, emissions from lead smelting, coal combustion, and leaded

gasoline (Aberg et al. 1998). Furthermore, acid rain can transmit contamination from

emissions/combustion over great distances.

Fractionation

Prior to drawing conclusions regarding the delta value of a material, additional

issues such as fractionation effects must be factored in. Fractionation is the disparate

partitioning of isotopes between two substances or tissues (Hoefs 2004). Without it,

biological processes would be homogenous and some of the most powerful inferences in

isotopic analyses would not be possible.









Differential fractionation manifests itself in a variety of forms. One example is the

different rates carbon is fractionated as one progresses through the food chain. Carbon

found in the atmosphere is present with a near constant 13C/12C ratio of about 1:99

(Chisholm 1989). As plants incorporate carbon into their tissues during photosynthesis,

isotopic fractionation occurs altering the 13C/12C ratio. Since C3 and C4 photosynthetic

pathways differ chemically, they produce different degrees of fractionation. This is

beneficial, and in fact, essential in the case of carbon isotope studies, because the 613C

values can be utilized to classify between C3 and C4 plants and diets based on a complete

separation of approximately 14%o between groups allowing for discrimination between

them (DeNiro & Epstein 1978a, 1978b, Chisholm 1989, Ambrose & Norr 1993).

The selective metabolism and recombination of plant chemicals within organisms

feeding upon them, results in fractionation of elemental isotopes, leading to differences in

613C values between diet and bone collagen of primary consumers of +3%o to +5.3%o. An

additional fractionation factor of about +1%o must be accounted for as you increase in

trophic level (i.e., from primary to secondary consumer) (Schoeninger 1985, Chisolm

1989, Schoeninger 1989, Ambrose 1993). The 613C values of mammal hydroxyapatite

trend even further from the whole diet, with rats on experimentally controlled diets

showing an enrichment of +9.6%o (DeNiro & Epstein 1978b) and other mammals

displaying enrichments of+12%o to +13%o (Lee-Thorp et al. 1989). Additionally,

preferential uptake among different tissues within the same organism has been noted and

can further complicate matters, with animal muscle generally showing 613C values 3%o to

4%o less positive (-3%o to -4%o) compared to bone collagen (Schoeninger 1989).









Oxygen undergoes fractionation due to environmental factors such as evaporation,

condensation, and freezing and is also strongly influenced by temperature and humidity

(Stille & Shields 1997, lacumin 1996, Hertz & Garrison 1998, Kendall & Coplen 2001).

This leads to differential isotope incorporation in plant tissues and is reflected in the

differing values of herbivores thought to be a result of foraging habits. For instance,

oxygen isotope ratios were found to vary by as much as 8%o to 9%o in herbivores based

on whether they were browsers or grazers (lacumin et al. 1996).

A difference of approximately 9%o has also been measured between the carbonate

and phosphate fractions of bone and teeth from a variety of mammals (lacumin et al.

1996) as well as marine invertebrate shells (Longinelli & Nuti 1973). This consistent

enrichment of carbonate 6180 values, regardless of the animal, seems fairly constant as

long as temperature remains within the range of 00 C to 370 C. Outside of this

temperature range, the fractionation is not as predictable (lacumin et al. 1996).

One reason strontium and lead analyses appear so attractive is the general

consensus that these elements do not undergo fractionation in nature. Strontium and lead

do not appear to exhibit this trend due to their significantly large atomic masses (Stille &

Shields 1997) versus the lighter isotopes such as carbon and oxygen. Such being the

case, comparisons can be drawn then utilizing organisms from different trophic levels as

well as between different tissues, without having to employ conversion factors.

Frequently Sampled Human Tissues

A variety of human tissues have proved useful in isotopic studies within the

anthropological disciplines in recent years. Tissues primarily available to forensic

anthropologists include bone, teeth, hair, desiccated skin, and finger/toenails; each









presenting its own benefits and drawbacks potential isotopic use and preserving records

of residency and diet at different points of the individual's life.

Bone

Bone is arguably the most utilized tissue in archaeological isotope studies

(Schwarcz & Schoeninger 1991). It is a composed of three primary constituents: 1)

water; 2) an inorganic mineral fraction (hydroxyapatite); and 3) an organic matrix

(Schwarcz & Schoeninger 1991). Bone isotope studies utilize both hydroxyapatite

(apatite) and collagen, which is found in the organic phase. Dry bone is composed of

approximately 70% inorganics and 30% organic (Katzenberg 2000). The overwhelming

majority of the inorganic phase is comprised of the protein collagen (85% to 90%)

(Katzenberg 2000).

Bone has a turnover rate of between 10-30 years (Ambrose 1993) owing to the fact

that different bone components remodel at different rates. On average, trabecular bone

remodels much more rapidly than its denser cortical counterpart (Teitelbaum 2000).

Regardless of the speed of turnover, it is clear that bone delta values slowly change

throughout an individual's life as stable isotopes are constantly incorporated into this

continually remodeled tissue.

Apatite is a calcium phosphate product of which the carbonate portion arises from

dissolved carbon dioxide (C02) in the blood plasma. Fractionation does occur between

these two reservoirs with the bone carbonate portion 613C value enriched by

approximately 12%o over plasma CO2 (DeNiro and Epstein 1978b). Bone carbonate

therefore reflects the total metabolic carbon pool found in an individual's diet,

incorporating carbon equally from all dietary energy sources and representing the

isotopic signature of the whole diet (DeNiro and Epstein 1978b, Ambrose & Norr 1993).









Collagen is the most abundant protein in the body (Champe & Harvey 1987),

constituting roughly one-quarter of all proteins occurring in mammals (Stryer 1975). The

collagen found in bone, dentin, skin, and tendon is molecularly similar and falls under the

category of Type I collagen (Schwarcz & Schoeninger 1991). Controlled experiments

using rats demonstrated that collagen underestimates the non-protein component of the

diet, but is an excellent representative of the protein portion because of its heavy nitrogen

constituent (Ambrose & Norr 1993, Tieszen & Fagre 1993). One difficulty with bone

collagen is that is does degrade over time, much more so than apatite.

Teeth

Teeth are especially useful in isotopic studies because of their robustness and

ability to survive in environs where bone would normally degrade. Unlike bone, tooth

enamel tends to be highly inert in terms of mineral exchange with the environment (Price

et al. 2002, Lee-Thorp & Sponheimer 2003), consequently they represent small, closed

systems. Because enamel is non-cellular and heavily mineralized with 96% or greater of

the weight of the enamel comprised of the inorganic constituent (Hillson 1996), it

withstands the effects of diagenesis very well and long preserves an accurate biogenic

isotopic signal (Lee-Thorp & Sponheimer 2003). Dentin and cement, on the other hand,

are much heavier in organic (roughly 20% and 25%, respectively) (Hillson 1996) and

much more susceptible to contamination.

Additionally, the inorganic nature of enamel, and specifically the apatite, reflects

the whole diet of the individual while the collagen in dentin, because of its high nitrogen

content, primarily mirrors the protein content of the diet (van der Merwe 1982, Harrison

and Katzenberg 2003). This is one of the drawbacks of using enamel. You cannot

analyze nitrogen isotopes.










Moreover, since teeth are genetically conservative, there is little variation in the

development and specifically, the period of mineralization of the tooth, although females

are slightly precocious in terms of dental formation, completing most stages of dental

growth before males (Fanning & Brown 1971, Hillson 1996). This observation was

confirmed by the 1976 study by Anderson et al. of the mineralization in permanent

dentition, although the authors state the degree of variability between the sexes has been

reported to be similar. Their calculations of the mean age of attainment of mineralization

in the adult teeth are presented below in Table 1-2.

Table 1-2. Mean age of completion of permanent crown mineralization.
1st 2nd 1st 2nd 1st 2nd 3rd
Incisor Incisor Canine Premolar Premolar Molar Molar Molar
Males
Maxillary 3.70.28 4.00.48 4.90.53 5.81.0 6.30.65 3.80.30 6.70.72 13.31.58
Mandibular 3.6+0.21 4.00.46 4.80.59 5.61.21 6.30.70 3.70.14 6.70.71 13.31.51
Females
Maxillary 3.60.14 3.80.40 4.1+0.49 5.10.56 5.90.65 no data 6.30.66 12.71.49
Mandibular 3.60.20 3.70.28 4.1+0.49 5.00.54 5.90.74 no data 6.30.66 12.81.63
Source: Anderson et al. (1976)

With in- and outflow of materials ceasing once amelogenesis is complete,

examining the permanent enamel provides a snapshot of the nutritional ecology of that

individual during the period of crown mineralization for that specific tooth. Dentin

primarily is laid down during and after amelogenesis, thus the bulk of it is formed during

childhood. Secondary dentin lines the pulp chamber and has a slow, continued formation

during adulthood, with turnover rates similar to bone (Hillson 1996).

Sampling can be done in bulk, which will average the isotope value for the entire

tissue component, or serially. In serial analyses, very specific regions of the enamel or

dentin, corresponding to even finer time periods, are sampled and compared. This takes

much greater skill in drilling and one must be sure what point in the individual's life the









area represents, but this method can also allow even finer resolution of dietary studies

over a period of years.

Hair

Several studies have turned to hair as an alternative sampling tissue (van der

Merwe et al. 1993, Yoshingaga et al. 1996, O'Connell & Hedges 1999, White et al. 1999,

Bonnichsen et al. 2001, Ayliffe et al. 2004, Cryan et al. 2004, West et al. 2004, Roy et al.

2005). Hair holds a great untapped potential in forensic isotope work. Often, hair masses

are found in association with skeletal remains. It is extremely durable, proving insoluble

to a variety of fluids, and can remain intact for thousands of years (Bonnichsen et al.

2001). The shaft is sheathed in a cuticle, a hard protective covering that is resistant to

chemical and microbial insult (Lubec et al. 1987, Macko et al. 1999b).

Because of its hardiness and the fact that the average human sheds 50-100 hairs a

day (Macko et al. 1999b), sampling is easy and essentially non-invasive. Non-keratinous

material, such as the root (bulb) is not normally sampled (Ayliffe et al. 2004) because of

its signature is not reflective of the shaft. Hair is also readily renewable, growing roughly

1 cm/month in humans (Yoshinaga et al. 1996), with isotope shifts demonstrating about

an 8-day delay (in the case of beard hair) from change of diet to hair exposure from the

follicle (Sharp et al. 2003). The isotopic composition of hair offers information

concerning an individual's diet and recent geolocational background. Thus a section of

hair provides a snapshot of an individual's nutritional ecology at a particular point in time

and a chronological record of the same along its length (White et al. 1999, Roy et al.

2005).

Hair is easier to isotopically analyze than bone and only very small samples (much

less than bone) are required (Cryan et al. 2004, Roy et al. 2005). Hair is made of









approximately 95% keratin, the proteinacious component (Taylor et al. 1995). Because

of its high protein content, hair requires minimal chemical processing and no chemical

purification. Conversely, bone and dentin collagen must be chemically extracted and

purified before the protein fraction can be analyzed (Ambrose 1993).

Roy et al. (2005) produced consistent carbon and nitrogen isotope values with

human hair specimen weights as low as 100 rig, corresponding to a length of 2 cm of

hair. The authors expect that strand lengths as small as 5 mm could be analyzed without

significant loss of precision when determining 613C alone, as nitrogen was the limiting

factor in their study.

The 613C values of hair keratin correlate well with that of total dietary protein, with

keratin being enriched by +1%o to +4.8%o relative to protein in the diet and depleted by

-2%o to -3%o compared to bone collagen (DeNiro & Epstein 1978b, Ambrose & Norr

1993, Tieszen & Fagre 1993, Yoshinaga 1996) in lab animals and contemporary humans.

Carbon isotope signatures from hair keratin and bone collagen are related but cannot be

directly equated (O'Connell & Hedges 1999).

Fingernails and Toenails

Like hair, finger- and toenails also offer a non-invasive way to examine isotopic

values in both the living and dead. The keratin composition of human nail material

makes them an excellent source of collagen values, as well as a variety of elemental

isotopes. They also provide a recent geolocational reference for a specific individual

with a whole nail representing approximately 6 months of growth in adults (note: authors

did not state the length of the nail) (Fraser et al. 2006) and 2 to 3 months growth from

cuticle to fingertip in infants (Fuller et al. 2006a). While some state that hair and

fingernail values are "similar" to each other (O'Connel et al. 2001, Fuller et al. 2006a), it









has been noted that nails are depleted in 13C and 180 compared to hair by mean values of

and -0.55%o and -1.6%o, respectively (Fraser et al. 2006).

Fogel et al. (1989, 1997) published the details of a landmark study examining the

weaning of modern infants as reflected in the differences in 615N between mothers and

infants. Fingernails prove an excellent medium for studying diets in modem infants

because they are metabolically inert, resistant to degradation, and have such a fast

synthesis rate (Fuller et al. 2006a). What the authors found was that the isotopic values

of the babies' fingernails were enriched in 15N by approximately +3%o from that of their

mothers, indicating that the infants were feeding at a higher trophic level than their

mothers. This was confirmed by Fuller et al. (2006a), who found infant 613C values

enriched by +1%o over their mothers' and 15N enrichment of +1.7%o to +2.8%o compared

to maternal values (for more detail, see Chapter 2). Such conclusions have been

extrapolated to bone and tooth isotopic analyses of weaning practices of archeological

populations (Schurr 1997, Herring et al. 1998, Schurr 1998, Wright & Schwarcz 1998,

Wright & Schwarcz 1999, Dupras et al. 2001, Mays et al. 2002, Clayton et al. 2006,

Fuller et al. 2006b).

Skin

Often desiccated skin is adherent to bone on remains submitted for forensic

analysis. This skin serves as an additional potential reservoir for isotopic values and can

be relatively easily removed from associated bone. Carbon turnover rates for skin and

hair are much faster than for bone, giving these tissues the ability to confer information

regarding diet and provenance much closer to death than hard tissues. Skin has an

estimated carbon turnover rate of roughly 15 days (Tieszen et al. 1983). The integrity of

skin after the decomposition process takes hold however is suspect, as skin appears









highly susceptible to contamination (White et al. 1999). For those studies in which viable

skin samples were obtained, relative to hair sample 613C values, skin appears to be

consistently depleted from -0.2%o to -2.7%o (White & Schwarcz 1994, White et al. 1999)

Complications

Radosevich (1993) aptly states that a reason for uncritical acceptance of methods or

assumptions is often the simple desire for a new technique to work. Stable isotope

analyses have seemingly been hailed as near-omniscient and people may turn a blind eye

to the limitations of such methods. On the other hand, modeling biological systems is an

extremely complex undertaking. There are times when a reductionist approach can

overwhelm the model in minutiae; where accounting for all the potentials of error

eclipses the actual data. All factors with the potential for confounding the data need to be

explored and understood, but often a relative weight can be assigned to them so the

model is not overloaded. There are much potential for error in stable isotope analyses;

but, as long as they are recognized apriori and dealt with, isotope ratios can provide

valuable insight into past and present systems.

Diagenesis

After the initial glow wore off following the popularization of stable isotope

techniques, researchers began to find chinks in the analytical armor. Often confusing or

contrary results were obtained leaving researchers to scratch their heads as to what it all

meant and if isotope studies were really worth all the hype. In 1981, A. Sillen was one of

the first to propose that perhaps post-depositional contamination, or diagenesis, was

responsible for at least a portion of this noise, but the effects of diagenesis were largely

ignored or dismissed in most studies (Price et al. 1992).









Diagenesis is a subset of the study of the postmortem processes which can affect

bone appearance and integrity, commonly known as taphonomy (literally meaning the

"laws of burial;" Sandford 1992). These processes take both physical and chemical

forms. When diagenesis in an anthropological context is discussed, it is in reference to

the postmortem alterations in the chemical constituents and physical properties of bone

following deposition in soil. Diagenesis takes the form of both contamination and

leaching and arises from several different mechanisms (Sandford 1992).

The dense mineralization of enamel affords teeth a great measure of protection

against effects, but it is important to keep in mind that no skeletal element is impervious

to postmortem modification. The porous structure of bone however, makes it susceptible

to infiltration by foreign elements, especially when it has been physically degraded. The

intrinsic skeletal chemistry and microstructure of osseous tissue therefore leads to a

dynamic relationship between it and the environment in which it is interred (Sandford

1992).

Mary Sandford (1992) lists several different means by which the environment

interacts with the structure of bone, leading to alteration:

Elements may be precipitated as discrete "void-filling" mineral phases in
the small cracks and pores of bone.

Soluble ions present in soils may be exchanged for those that normally
occupy lattice positions in bone hydroxyapatite.

Bone apatite can "seed" formation of recrystallization through a variety of
means.

Microorganisms break down bone collagen releasing elements through its
dissolution and the action of acid metabolites on hydroxyapatite.









Additional extrinsic factors such as the chemical environment of the burial sight

and the properties of the enveloping sediment influence the incidence and rate of

processes as well. Soil pH is one of the most important variables that affect change in

bone. Gordon and Buikstra (1981) first quantified the relationship, determining that it is

strongly negatively correlated, thus as soil pH decreases, degradation of bone increases.

The authors also noted that skeletal age was significant as well, with juvenile bone being

more susceptible to decay.

Temperature, microorganismal activity, groundwater, and precipitation also play a

role, as does the local geochemical environment to include soil texture, mineralogy, and

organic content. Sandford (1992) also mentions further intrinsic factors bearing on

processes such as bone density, size, microstructure, and biochemistry.

Recent investigations have shed light on bone alteration leading to several

generalizations: 1) elements differ in their susceptibility to diagenesis; 2) certain

categories of bones are more susceptible to diagenesis--less bone density, greater

porosity, or large quantities of amorphous material may predispose certain classes of

bones, such as immature bone, to taphonomic processes; 3) denser cortical bone

withstands diagenesis much better than the lattice-like trabecullar bone; 4) Direction and

intensity of change is not necessarily temporally or spatially uniform (Sandford 1992); 5)

the color and condition of skeletal material can be used as a general indicator of the

degree of diagenesis (Carlson 1996). The more the color approximates the color of fresh

bone, the less likely it is to have undergone change.

The majority of changes seen in bone arise due to precipitation of authigenic

carbonate or other minerals, exchange reactions in original carbonate or phosphate, and









uptake or loss of various trace elements. Recrystallization can also occur, producing

various phosphate-containing compounds with trace levels of elements often replacing

calcium at higher concentrations than found in modern bone (Schoeninger et al. 2003).

The same processes that bring about diagenetic change are ones that will eventually

return bones the lithosphere. The overwhelming majority of all deposited skeletal

material disappears relatively quickly, especially if exposed to taphonomic factors such

as acidic soil, alternate wet/dry conditions, strong solar radiation, and/or injurious

invasion by microorganisms (Lee-Thorp 2002). If we as anthropologists are fortunate to

encounter remains in the first place, we should not be discouraged from utilizing isotopic

resources in attempting to uncover clues about the lifestyle of the individualss. We must

keep in mind that these processes are not uniform over space and time, and thus even old

remains can produce valuable results.

Questions still remain however, as to what measures can be taken to minimize the

impact of diagenesis on isotopic interpretation. So what is a researcher to do? The first

step is to attempt to determine if processes have occurred and to what extent. In reality,

these processes are always occurring, but whether they exact a measurable effect upon

bone is another question. To begin with, a scientist should ask themselves several

questions. The first is what is/are the elements) of interest? Studies show that isotopes

of such elements as strontium and lead are little changed in bone due to diagenetic means

(Beard and Johnson 2000, Carlson 2002), thus scientists should have greater latitude in

using bones that have been interred for any period of time. Do the bones belong to an

adult or child? Because smaller bones have greater surface area to volume ratios, they

are more susceptible to change since there is more surface area for processes to act upon.









The absolute volume of cortical bone is reduced in juveniles as well, as they are still

growing, so bones are less shielded from environmental assailants. What bones are

available for sampling? Remains higher in cortical bone preserve better, so if presented

with a few cranial vault fragments, a researcher may be wise to opt out of isotope

analysis versus if a femoral shaft is available. Also, intact bone is always preferable to

fragmentary bone.

One should also assess the environment the bones are interred in. Sandford (1992)

believes chemical analysis of soil is a mandatory requirement for gaining insight as to the

condition of bone. Soil samples should be recovered from feature fill in direct

association with bone (Gordon and Buikstra 1981). Samples can be prepared and pH

determined in situ utilizing a portable pH meter. These values can then be applied to

something similar to a regional variant of Gordon & Buikstra's (1981) regression

formulae for pH and state of preservation. (It is interesting that while the authors provide

several regression formulae, for example, in adult assemblages, preservation = -1.3(pH)

+12.5, there is no scale provided in which to interpret the preservation value.)

Further testing can compare total elemental concentrations of bone and associated

soil. Following the assumptions of the concentration gradient theory, significant

contamination of bone by soil is considerably less likely if soil concentrations of a

specific element are disproportionately different than those same elements in bone. If a

more homogenous elemental state has been reached between bone and soil, it is a good

indication that significant change has transpired (Sandford 1992, Carlson 1996).

Other factors such as temperature and exposure to water should also be accounted

for. It is well accepted that higher temperature leads to degradation of collagen and that









warm, moist habitats encourage microbial proliferation. Exposure to water can also lead

to increased rates of both contamination and leaching of minerals into the surrounding

soil. The best environs for the preservation of DNA are those that are cool, dark, and dry

(Smith 2005). That is because these same conditions optimize the resilience of the whole

bone complex, so isotopic fractions will be best preserved as well. Heavy bone erosion,

trauma, burning, associated human alterations such as boiling and internment/funeral

practices, and carnivore and rodent activity compromise the structural integrity of the

bone itself leaving it more vulnerable to processes.

Instrumental analyses can be completed as well to include electron microprobes

and x-ray diffraction (Sandford 1992), and backscatter scanning electron microscopy

(Collins et al. 2002). These methods attempt to look at the structure of bone and analyze

it for changes in crystalline architecture, chemical constituency, and microbial activity.

Analyses of collagen content of bone may also provide insight, since some have observed

low yield in collagen is often associated with aberrant stable isotope readings

(Katzenberg 1992).

Osteological comparisons can also be completed in conjunction with soil analyses

examining constancy in values (Sandford 1992). Intrabone comparisons look for

statistically significant correlations between elements and known contaminants or

"indicator elements." Interbone comparisons look for agreement with the assumption

that different types of bone, such as ribs and femora, should reflect varying degrees of

diagenesis. Interspecies comparisons can indicate activity when measured elemental

values vary from those predicted on the basis of dietary patterns. Additionally, if









interpopulational data were available as we are attempting to collect, congruency to

published values could be ascertained (Sandford 1992).

Further precautions are essential during sample preparation in the laboratory.

Standard protocols attempt to minimize effects of diagenesis through mechanical

abrasion, to physically remove contaminants from outer bone surfaces, and acid washing.

None of the aforementioned methods are fail safe, but their use enhances overall

understanding of the processes active in a certain area and attempts to circumvent

diagenetic effects by careful sampling selection and preparation.

Many subscribe to the notion that the longer a set of remains has been interred, the

greater the alteration to the material. It is unwise to use temporal criterion in isolation in

making a decision about employing isotopic analyses though. As in any scientific

situation, you must take measure of as many variables as possible in order to make the

most informed decision. Diagenesis is a complex mechanism and time is but one factor

that comes into play. Cases in the literature abound detailing the successful extraction of

viable isotopic material from fossils that would have proved opportunities lost if the

authors had decided against isotopic analyses simply because they were working with

very old material. Studies have examined diets in ancient, human mummies (White &

Schwarcz 1994, White et al. 1999) and Neolithic Icemen (Macko et al 1999a, 1999b;

Miller et al. 2003), and the diet and paleoecology ofAustralopithecus africanus (van der

Merwe et al. 2003) and 5 million-year-old horses (MacFadden et al. 1999b), to cite but a

few. Differential preservation is a rule, rather than an exception and thus each interment

must be individually assessed for the appropriateness of isotopic analyses.









Anthropogenic Contamination

All organisms alter their environment. Human beings are unique though, in that we

are the only species on the planet that is actually altering the basic conditions of life on

Earth (Vitousek et al. 1997). We have altered landscapes, climate, and biogeochemical

cycles. Many of the wastes generated by our industrial metabolism play no useful role in

nature, cannot be recycled (i.e., nuclear waste) or overwhelm the current processing

capabilities of the biosphere (McMichael 2001). The ecological footprint of the human

species is enormous. Everything we do leaves traces of our kind behind.

This anthropogenic effect extends to isotopic signal variation. Industrial pollution

is implicated in the changing of isotopic values when contemporary populations are

compared to paleological assemblages and can outright alter or mask the isotopic

signatures a researcher is attempting to interpret. This can complicate analyses and lead

to false conclusions if not identified. To account for this, several correction factors have

been established to ease temporal analyses. Because nearly all of the anthropological

work done with stable isotopes has been in bioarchaeological contexts, these corrections

are essential in drawing conclusions.

The carbon isotope ecology of terrestrial systems is controlled by atmospheric

carbon dioxide (van der Merwe et al. 2000). This has changed dramatically in the years

since the Industrial Revolution, with fossil fuel emissions altering the 13C/12C ratio of the

atmosphere by -1.5%o in the last 150 years. (van Klinken et al. 2000). To correct for this

change in 613C values, "Industrial Effect" (van der Merwe et al. 2000) or "fossil fuel

effect" (van Klinken et al. 2000) calibrations must be factored into results, normally by

adding 1.5%o to convert modern samples to pre-industrial values (van Klinken et al.

2000).









We also have significantly altered the lead content of certain environs. Budd et al.

(2000) state, "It is widely believed that the contamination of the atmosphere by

anthropogenic lead has led to far greater human exposure today than that which prevailed

in the distant past, but this has proved difficult to quantify." A marked increase in the

mobilization of lead in Europe and North America occurred after industrialization.

Drilling of Greenland ice-cores has revealed a ten-fold rise in lead concentration, with

rates skyrocketing from roughly 10 parts per billion (ppb) to 100 ppb in the last 100 years

(McMichael 2001). This is due primarily to environmental contamination due to the use

of leaded gasoline (which is still utilized in many nations), lead-based pigments and

compounds, lead-acid batteries, and through mining operations, soldering, and coal

combustion (Sangster et al. 2000).

Global Economy

Today's global economy has the potential to homogenize biogeochemical

signatures in contemporary people. Because of world-wide trade, especially when it

comes to food importation, what people eat may not necessarily reflect where they came

from. Strontium values are especially vulnerable to being washed out by the effects of

the global food market. Archaeological research does not usually concern itself with

such matters because food tended to be locally grown and consumed. After the Industrial

Revolution and the establishment of global trade networks, food in the U.S. was very

rarely grown in the localities were people lived. So, on a trip to the refrigerator one may

find bananas from Guatemala, grapes from Chile, and free range, grass-fed beef from

Argentina.

Increasing consumption of bottled water from non-local sources further

complicates matters, affecting not only strontium values, but oxygen and hydrogen as






29


well. This situation may be further complicated by the importation of fertilizer produced

in foreign countries (Price et al. 2002). Such soil additives will affect not only plant

intake but run-off will affect, and may significantly change, the isotopic values of

groundwater (Bohlke & Horan 2000).














CHAPTER 2
APPLICATIONS OF STABLE ISOTOPE ANALYSES

Examples of the varied usages of isotopes in the literature abound. Stable isotope

analyses are an extremely effective means of recreating paleoecology (e.g., Amundson et

al. 1997, Cerling et al. 1997), tracking animal movements (e.g., Burton and Koch, 1999

Rubenstein & Hobson 2004), assessing migratory patterns of humans (e.g., Beard and

Johnson 2000, Dupras and Schwarcz 2001), and determining diet (e.g., DeNiro & Epstein

1978, van der Merwe 1982, MacFadden et al. 1999b). Within anthropology, stable

isotope analyses have been primarily relegated to realm of archaeology, but by applying

the technologies currently used in geology, paleontological and modern zoology, and

archaeology to forensic science, an effective means for presumptive identification

emerges.

Tracing Studies

One exciting application of stable isotopes that transcends disciplinary bounds is

that of tracing studies. In a tracing study, an element is introduced into a system with a

known delta value and tracked through the system or at the termination of certain

processes to see how that element normally moves through the system. This approach is

frequently used in clinical nutrition studies to understand the uptake of various nutrients

(see Abrams 1999 for a review). Stable isotopes offer many benefits over more

traditional radioactive approaches in that they present little of a safety concern for

pregnant women or children and are less difficult and less expensive to remove than

radioactive wastes (Abrams 1999).









For instance, isotopic tracer studies were used to measure the efficiency of zinc

utilization at different doses. Patients were given labeled zinc solutions, and then urine

samples were collected to determine absorption rates. Based on this approach the study

concluded aqueous zinc doses greater than 20mg resulted in quite small and diminishing

increases in absorptivity (Tran et al. 2004). Magnesium tracer studies demonstrated that

absorption of isotopically labeled magnesium could be accurately monitored through

urine sampling versus more invasive blood and fecal sampling methods (Sabatier 2003).

Additionally, tracer studies in Nigerian children with rickets determined that those with

the disease did not express impaired abilities to absorb calcium when compared to

healthy counterparts, although fractional calcium absorption did increase after resolution

of the active disease (Graff et al. 2004.) Stable isotopes were even utilized to measure

calcium metabolism of two cosmonauts and one astronaut aboard the Mir space station

prior to, during, and after a 3-month spaceflight (Smith et al. 1999). Further non-human

trials utilized three diets of different isotopic compositions to determine the turnover time

of carbon isotopes in horse tail hair West et al. 2004) and tail hair and breath CO2

(Ayliffe et al. 2004). These baseline studies could then be applied to other wildlife

studies in an attempt to understand the dietary history of mammals

Ecological studies have also utilized isotope tracers to examine nutrient flow in

various systems. One recent study added isotopically-labeled nitrogen to a creek for 6

weeks and monitored 1N in dissolved, aquatic, and terrestrial riparian food web

components. High levels of incorporation of the tracer into the tissues of resident

organisms led researchers to believe that streams within undisturbed primary forests may

be highly efficient at uptake and retention of nitrogen (Ashkenas 2004). Another project









examined root turnover in relation to forest net primary production by fumigating a stand

with labeled 13C in the form of 13CO2 over a 5-year period, then sampling fine roots.

Their results suggest that root production and turnover in forests have likely been

overestimated and that sequestration of anthropogenic atmospheric carbon in forest soils

may be lower than currently believed (Matamala et al. 2003).

Fractionation Studies

Fractionation studies have proven quite illustrative in a variety of genres as well.

Examination of carbon and nitrogen stable isotopes has yielded greater understanding of

the decompositional processes found within soil organic matter (Kramer et al. 2003).

Fractionation studies have also proven useful in attempting to measure the contribution of

gluconeogenesis to glucose production in humans. Here, body water was enriched with

2H20 and the ratio of 2H bound to carbon-5 versus carbon-2 of blood glucose was

measured (Katanik et al. 2003). Oxygen isotope fractionation has also been employed in

niche separation studies of African rain forest primates occupying overlapping

microhabitats. Oxygen isotope ratios from bone carbonate were positively correlated

with relative dependence of leaves in the diet, a fact obscured by carbon isotope analyses

(Carter 2003). A final study led to the discovery of what is commonly known as the

"canopy effect" (van der Merwe &Medina 1991). Van der Merwe and Medina

discovered the re-use of plant-fractionated, respired CO2 in dense vegetation can cause

systematic bias between plant and animal species living on the forest floor versus those

living in the forest canopy and open environments (also in van Klinken et al. 2000). Due

to the "canopy effect," the 613C value of atmospheric CO2 is lowest near the forest floor.

"Leaves fixing this 13C-depleted CO2 have lower 613C values than those higher up in the

canopy. Combined with the effects of low light intensity, high humidity and high CO2









concentrations on water use efficiency, this creates a vertical dine in leaf 13C values"

(Ambrose 1993).

Zoology and Ecology

Within zoology and ecology, the examples of stable isotope use seem limitless.

One of the first such studies examined carbon ratios of two sympatric fossil hyrax

species, determining one a was browser, based on the C3-like signature these animals

displayed, while the other was chiefly a grazer, feeding on tropical grasses, which utilized

a C4 photosynthetic pathway (DeNiro & Epstein 1978a). Similar studies have shed new

light on the diet and ecology of 5-million-year-old horses (MacFadden et al. 1999b) and

Cenozoic sirenians from Florida (MacFadden et al. 2004). Stable isotopes have proven

especially insightful for scientists attempting to determine feeding strategies of marine

organisms, and in fact, "Most work on mammal and reptile movements using stable

isotopes has been done in the marine environment" (Rubenstein & Hobson 2004).

Carbon isotopes have been used to determine food sources for Red Sea barnacles

(Achituv et al. 1997) and examine photosymbiosis in fossil mollusks (Jones et al. 1988).

Delta 13C and 615N were useful in assessing not only the foraging strategies of Pacific

pinnipeds (Burton & Koch 1999), but tracking their migratory movements as well and

have been used in dietary studies of North Atlantic bottlenose dolphins (Walker et al.

1999). Moreover, adult female loggerhead turtles were sampled from around Japan to

determine the relationship between body size and feeding habitats (Hatase et al. 2002).

Claws (Bearhop et al. 2003) and feathers (Rubenstein et al. 2002, Bowen et al.

2005) have also been utilized to determine diets and habitat use of migratory birds whose

summering and wintering grounds are separated by thousands of kilometers; so too has

hair been examined in bats for evidence of seasonal molt and long-distance migration









(Cryan et al. 2004). Wing membranes from monarch butterflies have been sampled for

hydrogen and carbon isotopes to identify natal regions within the United States and

revealed that 13 discrete wintering colonies in Mexico were fairly well mixed as to the

origins of the individuals (Wassenaar & Hobson 1998). Biogeochemical fingerprints of

African elephant bone have been assessed to determine change in diet and habitat use

(Koch et al. 1995). Isotopic analyses have even been extended to determining the

allocation of reproductive resources in butterflies (O'Brien et al. 2004) and assessing

prey quality in predatory spiders (Oelbermann & Scheu 2002).

Stable isotope ratios also allow us a glimpse into the past. Based on 613C enamel

values of worldwide fossil mammals and modern endemic and zoo-housed African

mammals, Cerling et al. (1997) has postulated that between 8 and 6 million years ago,

there was a global shift to increased C4 plant biomass and a corresponding decrease in

atmospheric carbon dioxide. This has implications today as increasing levels of

atmospheric carbon dioxide could bring about a major biotic alteration towards a world

dominated by C3 plants, which would have widespread ecological consequences. Carbon

values have also been analyzed from ancient pollen in attempts to reconstruct

paleovegetational and paleoclimatic conditions with the hope of someday tracing the

origin of the C4 photosynthetic pathway (Amundson et al. 1997).

One further study has taken an innovative approach to tying stable isotopes to

hominin evolution. Wynn (2004) examined paleosols of Turkana Basin, Kenya, and

ascertained that modern hominins evolved during a period of waxing and waning

diversity of savanna-adapted fauna in an environment that trended towards increasing

aridity. Those hominins best suited to generalization of resources were the most capable









of surviving through evolutionary "pruning events" as savanna ecosystems changed

through time.

Mention of these zoological and ecological studies does not even scratch the

surface as to the diversity of isotopic studies that have been and are continuing to be

conducted within these disciplines. Isotope use in these fields is gaining momentum and

results generally enjoy widespread acceptance, ensuring their continued use far into the

future.

Archaeology

Within anthropology, stable isotope analyses have primarily been relegated to the

realm of archaeology. Here, they have been used extensively to answer a litany of

questions in a variety of contexts concerning the human experience.

Diet Assessment

Introduction of maize

In archaeological contexts, stable isotopes have been used extensively to infer diet,

mobility patterns, and origins of material culture. When considering diet, a great amount

of effort has been expended attempting to determine when exactly maize became

prominent dietary component in various human populations (Vogel & van der Merwe

1977, van der Merwe & Vogel 1978, DeNiro & Epstein 1978b, Farnsworth et al. 1985,

Norr 1995). In fact, the majority of early archaeological stable isotope studies were

aimed at resolving the temporal and geographic origins of maize introduction, especially

into North America (Schwarcz & Schoeninger 1991). Striking changes in 613C values of

collagen resulted from the introduction of maize into human dietary patterns. These

values markedly decreased from roughly -21.4%o to -12.0%o during the period of A.D.

1000-1200 indicating that proportion of carbon from C4 plants went from 0 to more than









70% in some individuals (van der Merwe & Vogel 1978, van der Merwe 1982). This

agrarian shift also had other implications, with the development of permanent settlements

and an abandonment of the hunter/gatherer life strategy and all associated changes

inherent in the transition to a sedentary lifestyle. Not all agree on the timing of the

introduction of maize to North America though, with individuals such as Farnsworth et

al. (1985) concluding that maize was incorporated into human diets much earlier than

indicated in the fossil record. Today, the consensus seems to be that there is a temporal

variation in the conversion to maize agriculture within North America (Schwarcz &

Schoeninger 1991). Age effects in a prehistoric maize horticultural population (Ontario

Iroquois) have also been examined, with significantly higher 613C values found in infants

and young children suggesting a weaning diet high in maize (Katzenberg et al. 1993).

Isotopic dietary studies have been applied to fossils as old as Australopithecus

africanus, where individuals demonstrated an unusually varied diet, a large portion of

which was C4-based (Sponheimer & Lee-Thorp 1999a, van der Merwe et al. 2003).

From this information, the authors speculate that by about 3 million years ago, hominins

had become savanna foragers for a significant part of their diet. Based on carbon and

nitrogen stable isotope values, the diet of a Neolithic Alpine "Ice Man" was determined

to likely be primarily vegetarian, at least in the period closest to his death, based upon

hair values (Macko et al. 1999a).

Stable isotope dietary studies have also been performed on individuals from other

Mesolithic/Neolithic sites (Krigbaum 2003, Richards et al. 2003, Milner et al. 2004), the

Bronze Age in Northern Jordan (Al-Shorman 2004), prehistoric Chile (Macko et al.

1999b), preclassic and historic Mayan Belize (White & Schwarcz 1989, Tykot et al.









1996), ancient Egypt (Macko et al. 1999b, While et al. 1999) and Sudan (White &

Schwarcz 1994), prehistoric South Africa (Sealy et al. 1992, Lee-Thorp et al. 1993), and

prehistoric Micronesia (Ambrose et al. 1997). In addition, indigenous Easter Islanders

(Fogel et al. 1997), additional Native North American groups throughout time (Price et

al. 1985, Larsen et al. 1992, Fogel et al. 1997, Hedman et al. 2002, Roy et al. 2005,

Yerkes 2005), and colonists from the Chesapeake area (Ubelaker & Owsley 2003) have

also been examined.

Weaning practices

One significant aspect of diet that has received much recent attention is that of

infant feeding. Breastfeeding practices, to include weaning, have wide implications for

population dynamics in earlier human groups (Mays et al. 2002). Breastfeeding is a

major determinant of fecundity and interval between births in societies lacking reliable

artificial contraceptive measures, (Vitzthum 1994, as cited in Mays et al. 2002) and thus,

can be a major factor in determining life histories of certain population groups.

Ultimately, the success of infant feeding will have far-reaching impacts in terms of

population health and growth, for it is the essential first step for realizing adulthood.

Bone chemistry has been critical in this area for archaeological interpretation of

remains. In 1989, Fogel et al. published a groundbreaking study comparing the

fingernails of mothers and newborns from birth through weaning, to determine the utility

of using isotopes for such analyses. Fetuses and newborns have a 615N roughly

equivalent to that of their mothers (Herring et al. 1998, Mays 2000). This makes sense

because a fetus receives nutrition through materials exchanged across the maternal and

fetal circulatory flows in the placenta. Once born and breastfeeding begins, neonates

change their trophic stratigraphy. They effectively become carnivores relative to their









mother (Ambrose 1993). Nursing infants are feeding at one trophic level above their

lactating mothers and hence, should show an enriched 615N level of +2%o to +4%o over

their mothers (Fogel et al. 1989, Fogel et al. 1997). This is exactly what Fogel et al.

found. During the period of breastfeeding, they measured infant 15N ratios approximately

+3%o higher than their mothers (Fogel et al. 1989, Fogel et al. 1997). These results were

further confirmed by Fuller et al. (2006a), who found infant 615N values enriched by

+1.7%o to +2.8%o compared to maternal values. As a child is weaned from its mother's

breast, its 615N level will begin to fall back towards a standard adult average, because the

shift from milk proteins to proteins obtained from solid foods registers as a decrease in

15N bone collagen values (Wright and Schwarcz 1998, Fuller et al. 2006b).

Since Fogel et al. (1989), numerous researchers have applied their findings to

various archeological assemblages ranging from mid-Holocene South Africa (Clayton et

al. 2006), the Roman period of Egypt (27 BC to AD 395) (Dupras et al. 2001), Mediaeval

England (Mays et al. 2002), pre-contact North America (Schurr 1997) to 19th century

Ontario (Herring et al. 1998). Wright and Schwarcz (1998, 1999) have taken a slightly

different slant by including oxygen isotopes in their analyses of prehistoric Guatemalans.

Their studies are based on the fact that, "Human breast milk is formed from the body

water pool and, thus, is heavier in 6180 than the water imbibed by a lactating mother"

(Wright & Schwarcz 1998). Infants who only breastfeed are enriched in their oxygen

ratios compared to their mothers, because of the mothers' metabolic processing of the

water incorporated into breast milk (Wright & Schwarcz 1998, Wright & Schwarcz

1999). Additionally, many studies have incorporated carbon delta values with the

standard nitrogen values to determine the approximate ages of supplementary food









consumption by children in their respective populations, providing further validation of

the conclusions drawn from nitrogen data. (Wright & Schwarcz 1998, Clayton et al.

2005, Fuller et al. 2006b)

Following in the footsteps of Fogel et al. (1989), many more contemporary studies

have been carried out in the hopes of applying the results to archaeological work.

O'Connell et al. (2001) compared pairings of hair keratin and bone collagen taken from

patients undergoing orthopedic surgery in the United Kingdom as well as pairings of nail

and hair keratin from living subjects to examine the utility of applying similar 613C and

615N results to archaeological work. Lead isotopes in modem people have also been

examined to determine comparative lead loads and digenetic effects in prehistoric teeth

(Budd et al 1998, Budd et al. 2000), with Budd et al. (2000) concluding that Neolithic

human enamel lead values were only an order of magnitude lower than modern juveniles.

Region of Origin

Paleodiet analyses have also been applied to detect human mobility since the mid-

1980s (Sealy & van der Merwe 1985, 1986). Such studies are predicated upon the notion

that individuals practicing seasonal migration from coastal to inland areas should have

similar 613C values, while those permanently inhabiting such diverse areas should

demonstrate distinct carbon ratios (Sealy & van der Merwe 1985, 1986). In addition to

carbon, there are a wide variety of isotopes that can be drawn on to infer information

concerning human migration. Schwarcz et al. (1991) were the first to demonstrate the

use of bone phosphate 6180 to in attempting to identify the geographical origin of 28

soldiers from the War of 1812, interred in the Snake Hill cemetery, New York. Their

findings of uniformity among 6180 values indicated the group all spent a major portion of

their lives living in the same geographical area. These values differed however from









oxygen isotope analyses performed on interments in southwestern Ontario and Antietam,

Maryland. Dupras and Schwarcz (2001) used oxygen isotopes to distinguish immigrants

from native peoples from a third-century cemetery in the Dakhleh Oasis, Egypt, and both

oxygen and strontium isotopes have been used to determine the geographic origin of

remains found from Viking occupation-era graves in Great Britain (Budd et al. 2004).

Strontium isotope ratios have been used extensively in transhumance studies from

Neolithic Europe (Grupe 1997, Budd et al. 2000, Bentley et al. 2002, Bentley et al. 2003,

Muller et al. 2003, Bentley et al. 2004) as well as Bronze Age and Romano-British sites

(Budd et al. 2000a, Montgomery et al. 2005, Fuller et al. 2006b), and prehistoric and

historic South Africa (Sealy et al. 1995). Two studies used strontium isotopes to

discriminate between immigrants and life-long residents of 14th century Grasshopper

Pueblo, Arizona (Price et al. 1994, Beard & Johnson 2000). Beard and Johnson (2000)

determined local strontium values by analyzing local field mice. Individuals outside of

this range were deemed immigrants to the area, with those having the greatest 687Sr

differences being the most recent additions to that area. Aberg et al. (1998) demonstrated

that strontium and lead isotopes could definitively distinguish between west coastal and

rural inhabitants of Medieval Norway. The authors further concluded that Medieval

residents subsisted on local products while contemporary people relied on imported or

industrially processed food to a greater degree.

Carlson (1996) discovered that lead isotope values corresponding to different

sources of anthropogenic and natural lead can indicate cultural affinity among Native

Americans and fur traders buried in a 19th century fur trade cemetery. Montgomery et al.

(2005) found lead to be a bit ambiguous, with results suggesting that lead isotopes









provide dissimilar types of information depending on what era is being examined. In

some instances it seemed to serve as a geographical marker, while in others it served

better as a cultural indicator.

Material Culture

Not all archaeological applications of isotopic signatures are anatomically-based.

The origins of various forms of material culture have also been traced using these

techniques and well as additional dietary analyses. The earliest attempt to determine

provenance through isotope use was attempted in 1965 by Robert Brill and colleagues on

lead and glass artifacts (Brill & Wampler 1967, Herz & Garrison 1998). Not only were

lead objects associated with specific mining regions in antiquity, but samples separated

by nearly a millennium in time were found to have virtually identical lead isotopic

signatures and are believed to have come from the same mine (Brill & Wampler 1967).

The source quarries of ancient marbles have been interpreted through 613C and 6180

values (Craig & Craig 1972) and today, an extensive database exists for the isotope

values of principle classical quarries so marble items can now often be associated with

the areas in which they originated (Herz & Garrison 1998). Oxygen values have traced

emerald trade routes from the Gallo-Roman period through the 18th century (Giuliani et

al. 2000) and the mining locations of lead artifacts, such as musket balls and coils, found

among Omaha Native Americans have been identified (Reinhard & Ghazi 1992).

Building materials such as the timbers for the prehistoric great houses of Chaco

Canyon, New Mexico, have been traced to their individual mountain growing areas

(English et al. 2001). Major constituents of prehistoric and historic diet have also been

accomplished through the analysis of cooking residues found on potsherds or within

intact kitchenware (Hastorf& DeNiro 1985, DeNiro 1987, Hart et al. 2003). When









carbon and nitrogen analyses are combined for proven plant encrustations, they can

distinguish among three plant groupings: 1) legumes; 2) non-leguminous C3 plants; and

C4 or CAM vegetation (Hastorf & DeNiro 1985).

Forensic Investigations

Stable isotope analyses have been applied to a wide variety of contexts within the

forensic sciences. Within Europe two major organizations have emerged to advance the

development and application of isotopic work in this field. The Forensic Isotope Ratio

Mass Spectrometry (FIRMS) network and the Natural Isotopes and Trace Elements in

Criminalistics and Environmental Forensics (NITECRIME) European Union Thematic

Network both aim to raise awareness of the benefits of isotopes to forensic investigations,

encourage collaboration, and develop and validate new methodologies (Benson et al.

2006).

Stable isotopes have shown great promise as an analytical asset in the war on drugs,

specifically in determining the origin of illicit narcotics. The 62H (also denoted as 6D),

613C, 615N of components extracted from 3,4-methylenedioxymethylamphetamine or

"ecstasy" have shown that individual tablets can be traced back to a common batch

(Carter et al. 2002). Carbon and nitrogen isotopes have been further used to link heroin

and cocaine samples to the four major geographic regions in which they are grown

(Mexico, Southwest Asia, Southeast Asia, and South America). Morphine, which is

derived from heroin, demonstrated the most pronounced regional difference (Ehleringer

et al. 1999). Further studies were able to determine the country of origin in 90% of 200

coca-leaf samples, the source material for cocaine, as deriving from Bolivia, Columbia,

or Peru (Ehleringer et al. 2000).









Isotopic techniques have been used by food and spirit regulatory agencies as well to

ensure quality control. There is an international concern with not only simple validation

of food label claims, but with food adulteration as well. One application is within the

beer industry (Brooks et al. 2002). The primary ingredients in beer are water, malted

barley, hops, and yeast. All other "non-essential" ingredients are called adjuncts. In

many nations, the use of unlabelled adjuncts is forbidden by law. Carbon delta values

have proven very effective at detecting adjuncts and testing brewers' claims as to the

purity of their ingredients (Brooks et al. 2002). Additionally, 613C values have proven

invaluable in determining whether forms of glycerol are animal or vegetal in origin

(Fronza et al. 1998) and 613C and 615N values of eggs have been used to establish

whether chickens were given animal or plant protein as feed (Rossmann 2001).

Furthermore, oxygen values have been utilized to verify the regional origin of dairy

products, especially certain cheeses, which must be produced from milk of a particular

region (Rossmann 2001).

Food adulteration is of concern to authorities because it is essentially the

misrepresentation of an altered foodstuff as an authentic product. Here, a premium food

product is extended or completely replaced with cheaper materials, yet fraudulently sold

as a higher-end item (Parker et al. 1998). Stable isotope analyses have established

themselves as a particularly usefully analytical methodology in fighting this trend. The

most advanced applications of stable isotope analyses are within wine quality control,

where the European Union has established an official wine stable isotope parameter

database (Rossmann 2001). Carbon isotopes can detect the addition of exogenous

glycerol deceptively added to wine to disguise poor quality (Calderone et al. 2004). They









have also been used to differentiate between whiskies and assist in authenticating specific

whisky products (Parker et al. 1998) and determine the botanical origin of Brazilian

brandies (Pissinatto et al. 1999). Isotopic fractionation of hydrogen and oxygen resulting

from juice concentration processes have also been documented and utilized to quantify

added sugars in orange and grape juice (Yunianta et al. 1995); while 613C values have

been used for over 20 years to control for the authenticity of honey (Rossmann 2001).

Stable isotope analysis has also been employed by criminal investigators in cases

involving the use of firearms (Stupian et al. 2001). Bullet individualization via lead

isotope analysis was first reported in 1975 (Stupian 1975). Lead isotopic information can

indicate whether a fatal bullet shared a common origin with a box of ammunition

collected from a suspect or provide a detective with a tool independent of standard

ballistic methods to potentially link bullets from multiple crime scenes (Stupian et al.

2001). In instances where there is a shoot-out with several types of firearms and/or

ammunition, it may even be possible to conclude which bullet and/or weapon caused a

particular gunshot entry (Zeichner et al. 2006).

Another forensic isotope breakthrough occurred in 1975, when Nissenbaum

reported 613C could distinguish between trinitrotoluene (TNT) samples originating from

different countries. Other areas of forensic isotope applications include connecting the

sources of automobile (Deconinck et al. 2006) and architectural paints (Reidy et al.

2005), packaging tapes (Carter et al. 2004), and glass fragments (Trejos et al. 2003) to

crime scenes.

Similar measures have been drawn upon to detect environmental toxins in soils,

waters, and plants. Isotopes can assist in identifying a geographical relationship between









a source and a spilled product, whether the contamination might be from an oil spill,

illegal dumping, pipeline breaks, or leaking storage tanks (Philip et al. 2003). For

instance, in the case of a crime scene, such applications may be able to link engine oil on

the victim of a hit and run with a particular vehicle (Philip et al. 2003). Source

identification of environmental perchlorate contamination has been performed with

chlorine and oxygen isotopes (Bohlke et al. 2005). Perchlorate, in even small amounts,

can adversely affect thyroid function by interfering with iodine uptake (Bohlke et al.

2005), but hopefully, by identifying the source of such chemicals, this form of pollution

can be stemmed.

These techniques have further been extended to biowarfare defense efforts. Horita

and Vass (2003) determined that cultured bacteria (Bacillus globigii and Erwinia

aglomerans) faithfully inherit the isotopic signature of hydrogen, carbon, and nitrogen

from the media waters and substrates they were grown on, proving "stable-isotope

fingerprint" can be created for chemical and biological agents. Because of these

properties, Kreuzer-Martin et al. (2003) were able to undertake sophisticated tracing

studies involving oxygen and hydrogen isotopes. Culture media was prepared with water

spiked with known isotopic quantities of hydrogen and oxygen. The 6180 and 6D found

within strains ofBacilus subtilis spores grown on this media were then traced back to

specific water sources establishing that the origin of microbes can be pinpointed to

particular areas based on the water content of the media on which they are grown.

Stable isotopes are also prominent in wildlife forensic issues. Several studies have

used a trivariate approach, combining carbon, nitrogen, and strontium isotope ratios to

create geolocational fingerprints for elephant ivory and bone (van der Merwe et al. 1990,









Vogel et al. 1990). It is hoped this will aid in conservation efforts by assisting in efforts

to stem the illegal trade of ivory. Similar goals are also being applied to the bounty of

information concerning animal migrations (Bowen et al. 2005).

While great advances have been made in the applications of stable isotope analyses

to the forensic sciences, human stable isotope studies in the medico-legal realm are

relatively recent phenomena. To date, very few studies examining stable isotope ratios as

they pertain to region of origin in contemporary human populations have been presented

or published. When examining the literature, it appears that the bulk of isotopic research

in modern humans is in the form of isotopic tracers for nutritional studies (see also

Abrams and Wong 2003, Mellon and Sandstrom 1996). Many, as previously discussed,

are also used as proxies for archaeological comparison (Fogel et al. 1989, O'Connell et

al. 2001, Fuller et al. 2006a).

Several studies have been conducted to investigate lead exposure and identify the

sources of lead absorbed in contemporary, living children by examining their deciduous

teeth (Alexander and Heaven 1993, Gulson & Wilson 1994, Gulson 1996) and other

tissues and excretions (i.e., blood and urine, Angle et al. 1995). Alexander and Heaven

(1993) measured 206Pb/207Pb ratios and lead abundance in teeth finding significant

difference among the lead isotope ratios. When compared against various environmental

sources of lead, the authors were able to identify differences in sources in northwest

England. While these studies were not utilized for geolocational purposes, they

nonetheless could be applied as such, (although anthropological studies tend to utilize

isotopes compared to 204Pb), and provide a good example of the multiple uses for isotope

data.









One weaning study went one step further than those previously discussed and has

exciting forensic potential. Fuller et al. (2006) analyzed bovine milk-based and soy-

based formulas to determine if unique isotopic signatures exist that could identify infants

being fed different forms of supplementation. The authors purchased seven different

formulas sold within California and found that while the 613C values overlapped between

formulas derived from cow's milk and soy, the soy products demonstrated significantly

lower 615N values. This again, is a reflection of trophic level effects in nitrogen values.

Fraser et al. (2006) have begun compiling a database of modern human hair and

nail values examining the stable isotopes of hydrogen, carbon, nitrogen and oxygen. The

authors sampled hair and fingernails from 20 individuals living in Belfast, Northern

Ireland for a minimum of 6 months as well as an additional 70 individuals from 9

countries representing 4 European nations, Syria, the United States, Australia, India, and

Sudan. They did not report having yet applied the database results to a forensic situation,

but preliminary data is at least at the ready should the need arise. Similarly, at the 3rd

European Academy of Forensic Science Meeting in Istanbul, Turkey, Cerling et al.

(2003) presented results of a multi-element study of modem human hair. The authors

discovered regional differences in the 6D, 613C, 615N, and 6180 values of long-time

residents of particular locations and appear to still be collecting samples.

Beard and Johnson (2000) were the first to demonstrate the utility of strontium

isotopes in a human forensic setting. In their paper, they determined the region of origin

of an illegally harvested deer using the 87Sr/86Sr ratio of antler, then also applied this

information in an attempt to differentiate between the teeth of three commingled

Americans associated with the Vietnam conflict. They were able to match the natal area









of one individual, but the two others presented overlapping values. If the study had also

utilized alternative isotope comparisons, perhaps the authors might have been able to

discern between the remaining two individuals.

Also, preliminary data for a study using strontium isotope values in an attempt to

determine the geolocational fingerprints for Mexican-borne individuals residing in the

U.S. was presented at the 2005 annual meeting of the American Academy of Forensic

Sciences (Juarez 2005). Several bay-area dental clinics provided the author with 25

permanent lst molars of individuals originating from four different Mexican states.

Samples were accompanied by information as to the subjects' regions of origin within

Mexico, their ages, and sex. Initial results indicate four specific ranges of strontium

isotope ratios, one for each of the four states involved in the study. Within-state variation

proved too great however, to discriminate location further.

Additionally, a presentation at the 2001 annual meeting of the American

Association of Physical Anthropologists addressed the use of strontium isotopes and its

applications in forensic science (Schutkowski et al. 2001). The abstract makes reference

to the presentation of a multi-regional sample demonstrating differences in regional and

local strontium isotope ratios. Bone and tooth signatures were examined to determine if

mismatches of individual values with local isotope ratios demonstrate changes in

domicile. The areas of study were likely western European, as the authors at the time of

publication practiced in the United Kingdom and Germany. Unfortunately, it appears this

data has yet to be published in a western source.

Gulson et al. (1997) detail a pilot study comparing the lead isotope values in teeth

of native Australians to those of Australian migrants from Eastern and Southern Europe









(Table 2-1). While the actual data presented by Gulson et al. are not particularly useful in

cases of American service members this paper does indicate lead isotope ratios have the

potential to discriminate region of origin.

As can be seen from this short review, isotopic analyses and applications serve a

wide variety of functions. The incredible inferential value of isotopic analyses in

anthropology is clear. Examples of the power of isotopic studies abound in the literature

and continued advances will only further solidify how essential their inclusion is within

an anthropologist's analytical toolbox.

Table 2-1. Mean and standard deviations for selected groups of immigrant teeth
(enamel).
Australia CIS* Yugoslavia Lebanon Poland
(n=29) (n=14) (n=13) (n=8) (n=6)
Mean 206Pb/204Pb 16.56 17.98 18.23 17.62 18.07
SD 0.17 0.06 0.15 0.29 0.20
Mean 207Pb/206Pb 0.9318 0.8664 0.8566 0.8825 0.8617
SD 0.0088 0.0033 0.0063 0.0136 0.0088
Source: Gulson et al. (1997)
*CIS denotes the former Soviet Union














CHAPTER 3
HUMAN FORENSIC IDENTIFICATION

Assuming that isotopic analyses do prove fruitful for forensic practitioners, this

technique will be added to a bounty of available measures for use in the personal

identification process. Those specializing in the forensic arts acknowledge that there is

stratification when it comes to the probative value of identification data. In attempting to

tease a name from a body, certain characteristics of the person will be much more unique

and individualizing than others. The most powerful measure of identification is a

positive identification, the essential component of which is the possession by the

decedent of unique characteristics (Ubelaker 2000). Because these characteristics are not

replicated in anyone else, they exclude all other individuals from consideration.

Even with the high resolution of DNA, the method of choice today for positive

identifications tends to be dental comparisons (Col. Brion Smith, personal

communication). Dental records are still consulted when available. The Computer-

Assisted Postmortem Identification system (CAPMI) is based on the presence of dental

restorations and has increased the efficiency of matching and comparing

antemortem/postmortem records (Friedman et al. 1989), especially in the case of mass

fatalities. Dental radiographic matches are much quicker and less costly than DNA

evaluations, although the number of individuals with no dental anomalies (Friedman et al.

1989, Col. Brion Smith, personal communication) is rising due to advances in dental

hygiene and medicine and mass fluorination of community water sources. In those cases









where the skin of the fingers is still intact, fingerprints may establish a positive

identification as well.

When these measures prove inconclusive, genetic fingerprinting utilizing nuclear

and mitochondrial DNA is another option. With the development of the polymerase

chain reaction procedure, which enabled rapid amplification of genetic material, and

lowered costs, DNA analysis is much more practical (Herrero 2003) than in days past.

Nuclear DNA is known to be a unique identifier (unless the subjects are identical twins).

Many investigators, including the Department of Defense (DoD), test 16 bands from the

available microsatellite loci pool (Col. Brion Smith, personal communication). From

experimental observations, the average odds that one band will be shared by any two

unrelated individuals is approximately 0.25 (Sudbery 2002). So the resolution of a 16-

band testing procedure is 0.2516 = 2.33 x 10-10; that is, there is a 0.000000000233 chance

that two unrelated individuals will share all 16 bands tested. Put another way, if you take

the reciprocal of this figure you see that there is a 1 in nearly 4.3 trillion chance that

someone unrelated has the same DNA profile. Since this number is considerably larger

than the world's population, nuclear DNA testing is said to provide for unique

identifications.

This calculation is made with the assumptions that all individuals are unrelated and

that the chance that bands will be shared is the same for all people. In truth, people are

related and ethnic affinities may lead to higher rates of band sharing than among the

general world populace. Even so, after accounting for such complications, nuclear DNA

analyses are still considered positive and unambiguous identification (Sudbery 2002).









The resolution of mtDNA, on the other hand, is not as fine. Because mtDNA is

passed through maternal lineages only, recombination does not occur. Mutations aside,

this accounts for the integrity of mtDNA as it is passed from mother to child. This

constancy of code allows for familial tracing by comparing sequences of certain base pair

lengths among those who are maternally related. This is a very powerful tool indeed, and

allows for a distinctive discriminating function from nuclear DNA. The downside to it

though is that is cannot distinguish among relatives and can be preserved for generations,

leading to populations of people with the same or similar mtDNA profile (Col. Brion

Smith, personal communication).

Many also consider various forms of radiographic comparison to equate to positive

identification. This is especially true with the frontal sinus. The sinus becomes

radiographically visible between 7 and 9 years of age, and barring trauma or disease,

remains relatively unchanged throughout life (Ubelaker 1999). In a comparison

completed by Ubelaker (1999), the author noted than in a radiographic comparison of 35

radiographs (595 comparisons), no two frontal sinuses were alike. The number of

differences between individuals average to approximately 8, with a range of 3 to 15. If

additional antemortem radiographs exist documenting unique skeletal anomalies (i.e.,

pathology or trauma), these characteristics may also serve as a basis for positive ID.

One further skeletal anomaly for consideration is that of prosthetic devices (Burns

1999). While it may not be unique that an individual has a total knee replacement, what

will be unique is the serial number that is imprinted upon the prosthetic device along with

the manufacturer's emblem. Hospitals must document these serial numbers. With a little

detective work, the serial or lot numbers can be traced back to the manufacturer who in









turn, can direct an investigator to the hospital to which the device was sold (Warren

2003). Some also consider the comparison of still photos and the skull at the same angle,

or what is known as video superimposition, to be conclusive as well (Ubelaker 1999).

Hope for a positive ID can often prove frustrating and futile though, when there are

no reference samples on file for that individual. An individual must have antemortem

information available to compare against if a positive identification is to be achieved. So,

for instance, while DNA may have successfully been extracted from a set of remains, an

identification cannot be accomplished when there is no nuclear DNA on file or source

material available and no relatives of maternal lineage for the decedent can be located.

One step below a positive ID is exclusionary evidence for identification. When

remains are presented for identification, they will arrive from one of two environs, either

an open environment, or one which is closed (Warren 2003). Open environments are

those in which the person laying before you could be anyone in the world who was up

until recently, alive. For example, a body found in the woods could be an indigent, a

local, or a tourist from another country. In the case of a light aircraft crash however, the

potential for identification is much higher. If a passenger manifest was filed listing two

adult males and child of 12, and assuming it was correct, then there is the potential for an

exclusionary identification. The child will be easy to distinguish from the adults due to

developmental differences in the skeleton. If one of the adults is identified via

antemortem radiographs and the other has no antemortem comparison data, then the latter

would usually be identified by exclusionary methods, since ideally, in a closed system

there is no one else it could be.









Burns (1999) also lists identification by means of a preponderance of evidence.

This is often linked with tentative identifications, or what are also known as presumptive

identifications. There is much greater uncertainty with presumptive identifications

because they are based on evidence found associated with the body, such as personal

effects, and/or verbal testimony of witnesses, last known whereabouts of the body, and

familial recollections of undocumented conditions the individual may have suffered from

(Burns 1999). See Table 3-1 for a recap of identification measures.

Table 3-1. Forms of forensic identification.
Type of I.D. Basis for I.D.
Tentative identification Clothing
Possessions
Location of body
Verbal testimony
Identification by preponderance of Anomalies known by family or friends, but
evidence without the existence of written records
Identification by exclusion "Everyone else is identified and there is no
evidence that this is not the only person
still missing."
Positive identification Dental identification
Radiographic identification
Mummified fingerprints
Prosthetic identification
DNA analysis
Unique skeletal anomalies
Reproduced from Burns (1999)

Military Identification Measures

"Over the past 200 years, the United States has set the standard for the

identification and return of its servicemembers [sic] to their families" (AFIP 2004).

Since as early as the American Revolution, efforts have been made to recover, identify,

and provide individual burial for American military personnel (AFIP 2004). As the years

have progressed, standards and expectations for identification have increased and the

technology with which to do it has made sweeping advances.









The United States DoD employs all of the standard personnel identification

measures previously mentioned. What is unique about the military as a population

however, is that their physical attributes and markers are much better documented than

the general populace (i.e., they have much better antemortem records). Members have

fingerprints on file and flight crews have footprints documented as well. Meticulous

medical and dental records are fairly centralized. With few exceptions, blood cards are

on file for all current total force members in the case their DNA needs to be sequenced.

Individuating marks are noted such as scars, large birthmarks and moles, and tattoos as

well as information such as hair and eye color, race, stature, weight, and age.

Even so, such measures are not without their complications. Dental radiographs

are commonly not available of military members unaccounted for from previous

conflicts, especially World War II and the Korean War (Adams 2003a). The Office of

the Armed Forces Medical Examiner notes that greater than 5% of all service members

have no dental restorations, the primary means for dental identifications, and the number

is rising (AFIP 2004). In a study of 7030 living U.S. soldiers, it was revealed that 9%

had a full complement ofunrestored teeth (Friedman et al. 1989). In a pooled data set of

over 29,000 individuals from the Third national Health and Nutrition Examination

Survey (NHANES III) and the Tri-Service Comprehensive Oral Health Survey

(TSCOSO), Bradley Adams found that 12.77% had "perfect teeth" (2003b). Not only has

the number of dental restorations declined in younger individuals, but the complexity of

them has decreased as well (Friedman et al. 1989).

Historically, only 70% of service personnel actually have their fingerprints on file

with the Federal Bureau of Investigation with a further 15-30% of the fingerprint cards









submitted by the services rejected as "unclassifiable" (AFIP 2004). These numbers will

likely be reduced significantly though, with the wide-spread implementation of digital

fingerprinting DoD-wide, which instantly scans recorded images for acceptability

immediately after each individual print is taken. Additionally, radiographic analyses may

not be possible on highly fragmented remains (AFIP 2004). Identifications made based

on material evidence associated with remains can be very problematic as well.

Traditional items such as dog tags are not necessarily accurate either. As an example,

during current operations in Iraq and Afghanistan dog tags have been known to have been

blown off one individual and burned into the chest of another (Dr William Rodriguez,

personal communication).

On the leading edge of identification efforts for the U.S. government is the Armed

Forces DNA Identification Laboratory (AFDIL). AFDIL is the focal point for the DoD

in all matters concerning DNA identification efforts for military personnel and special

federal government projects. In addition to performing laboratory testing, AFDIL

manages the DoD DNA Registry. This function is responsible for maintaining blood

cards for DNA testing as well as providing administrative oversight of the database of all

sequenced data. Besides the Registry, AFDIL is also responsible for the DNA

Repository, which administers the AFDIL Family Reference Specimen database for

mtDNA matching when nuclear DNA is unavailable (AFIP 2004).

It is a common misconception that the military maintains DNA profiles on all its

personnel. It does not. Instead, AFDIL houses nearly 4.5 million blood stain cards for

active duty, reserve, guard component, retired military members and additional

specialized government personnel (Col. Brion Smith, personal communication).









According to Colonel Brion Smith, Chief Deputy Medical Examiner for the Forensic

DNA Division, Office of the Armed Forces Medical Examiner, there are two basic

reasoning behind the logic of this. The first is that it is cost prohibitive to perform DNA

analyses for every member of the armed forces. It is much less expensive to house blood

stain cards and generate the same information on an "as needed" basis. The second is

that storing the profiles of all who serve presents an ethical dilemma, especially when it

comes to who should be permitted access to the information and for what purposes. This

is further complicated by the fact that medical and dental records, to included DNA

information and blood cards, stay on file for 50 years after the service member retires

(Col. Brion Smith, personal communication).

An additional benefit of this system is it gives examiners the option aposteriori to

decide which test is best suited based on the conditions of the remains. If the body is in

good condition, nuclear DNA would be the preferred method. If the remains are charred

and disassociated, mtDNA might be most appropriate. Furthermore, a fully utilized

blood card can provide 30-40 punches, allowing less common tests such as Y short

tandem repeats to be completed (Col. Brion Smith, personal communication) or

providing the opportunity for future testing utilizing methods that have yet to be

developed or hit the mainstream. The beauty of this practice then is that technicians are

not restricted to only performing a form of analysis that matches the information present

in a data base so there is greater flexibility in analyses and hopefully the best method for

the materials available.

The utilization of both nuclear and mitochondrial DNA is dependent upon the

situation and essential to military identification. All individuals who die in current









combat, training, or in otherwise duty-related capacities are sampled for DNA analyses

upon intake to the Dover Air Force Base Port Mortuary (Col. Brion Smith, personal

communication), the DoD central receiving and processing center for all military

deceased. Even when other conventional methods of positive identification are available

such as radiographic dental comparisons, a DNA fingerprint will be generated. This will

delay returning a casualty to their families unless identity is questionable, but instead, is

performed to prevent questions surfacing at a later date as to correct identification and to

reassure family members that the body being returned to them is kin. (Col. Brion Smith,

personal communication).

Present Study

This project was established to test the utility of stable isotope analyses for

identification of region of origin for modern, unidentified, human skeletal material that

has poorly-documented or unknown provenience. Initial efforts have focused on the

approximately 1,800 service members who remain unaccounted for from the Vietnam

conflict. It is hoped however, that this information will eventually be refined to use in the

identification of all those who remain unaccounted for and for those potentially

recoverable from previous conflicts (Table 3-2).

Often, the true national origin of remains recovered by the Joint Prisoner of

War/Missing in Action Accounting Command (JPAC) is uncertain. In addition, it is not

uncommon for de-contextualized, poorly preserved and/or highly fragmented remains to

be unilaterally turned over to the Central Identification Laboratory (CIL) by a foreign

agency. CIL personnel attempt to determine whether remains are U.S. service









Table 3-2. Numbers of unaccounted for U.S. prisoners of war and/or those missing in
action.
Conflict Number Unaccounted For
World War II 78,000
(35,000 considered recoverable)
Korean Conflict 8,100
Vietnam War 1,800
Cold War 120
First Gulf War 1
Source: JPAC (2006)


personnel through a variety of means. The identification of unknown remains believed to

be missing U.S. service personnel is frequently hampered by high levels of degradation

and fragmentation as a result of circumstances of loss and subsequent taphonomic

regimes. These effects often combine to prevent effective DNA sampling strategies.

Teeth often prove excellent at distinguishing among the populations in questions. U.S.

military personnel had access to regular dental care. In countries such as Vietnam, this

was not the case for the majority of the population. In addition to untreated dental

insults, the occlusal surfaces of the molars and other teeth are frequently worn down from

the grit present in native diets exposing the underlying dentin (Mark Gleisner, personal

communication). The teeth then of modern Vietnamese often present similarly to

historical/prehistorical Native Americans. Every effort is also made to extract DNA from

a set of remains, although such efforts are often unsuccessful because of the poor state of

preservation.

Additionally, the number of U.S. casualties during the Vietnam conflict of Asian

ancestry was relatively small. In 1985, the DoD reported the number of "Mongolian"

fatalities in Southeast Asia occurring from the period of 1 January 1961 to 30 April 1975

or as a result of injuries sustained in operations during said period was 114 or 0.002%.









Those listed of "Malayan" ancestry who died under the same circumstances was 253 or

0.004% (Reports 1985). See Table 3-3 for a complete listing of casualties by race.

More importantly for this study, only 5 servicemen of Asian ancestry remain

unaccounted for out of 1,760 total (JPAC 2006). The complete racial breakdown for

service members still listed as missing in Southeast Asia can be found in Table 3-4.

Besides military members, 32 American civilians are also listed as missing in Southeast

Asia. The racial backgrounds of these individuals were unavailable, but it is interesting

to note that two missing civilians are female. All of the military members unaccounted

for are male.

Because of the very low likelihood of a U.S. service member being a female or of

Asian ancestry, biological profiles can be useful in excluding individuals from

consideration. This is assuming enough of the skeleton remains to create a biological

profile. When the biological information is combined with documented information

Table 3-3. United States casualties in Southeast Asia by race.
Race (reported by DoD) Total U.S. Casualties
"Caucasian" 49,951
"Black" 7,257
"Mongolian" 114
"American Indian" 226
"Malayan" 253
"Other/Unknown" 221
Total 58,022
Source: Reports (1985)

Table 3-4. United States military listed as unaccounted for in Southeast Asia by race.
Race (reported by JPAC) Total U.S. Military Missing
"White" 1,653
"Black" 92
"Asian/Pacific Islander" 5
"American Indian/Alaska Native" 2
"Other" 8
Total 1,760
Source: JPAC (2006)










concerning troop engagement and staging areas and locations of downed aircraft, remains

may be returned to the originating nation if the evidence points overwhelmingly to the

fact that the remains are not of an American. Unfortunately, such an assessment is an

extremely complicated venture and in a great many cases it is simply impossible to make

such a distinction.

This project was initiated in the hopes that the results will assist in resolving this

dilemma. A two-pronged approach for this study has been utilized based on the

operating hypotheses that: 1) discernable differences exist between the isotopic ratios

incorporated into American and Southeast Asian tooth enamel and that these differences

can be used to determine region of origin; and 2) regional differences in natal isotopic

signatures are also discernable within populations raised within the U.S.

Because of the paucity of data in contemporary studies, it is near impossible to

predict the likelihood of the ability of this study to distinguish natal Vietnamese from

American-born individuals. It is encouraging that Juarez (2005) found significant

variation among the strontium isotope values for Mexican-born peoples from four

different states, even with her limited sample. If historical, human, migratory studies

(Montgomery et al. 2005, Miller et al. 2003, Montgomery et al. 2000, Dupras &

Schwarcz 2001, Aberg et al. 1998) are any indicator though, there is a high probability

that the chosen stable isotopes will be able to discriminate between these two

populations. The reasoning behind this is that the geochemical properties of different

continental systems should vary significantly and this difference will be further

magnified by the fairly culturally distinct dietary practices of the two populations.









None of the studies mentioned in Chapter 2 examined stable isotope use in a

forensic context in any great depth. The largest sample size was Gulson et al. (1997)

with 68, but it was a combined pool of permanent and deciduous teeth. This study will

utilize approximately 300-600 total samples and thus will have greater power.

Furthermore, all studies make mention of overlapping isotopic values which makes

discrimination virtually impossible. It is hoped this tendency will be reduced by

introducing multi-element analyses to forensic work. Theoretically, a multivariate

approach should allow finer resolution, especially since the deposition of the elements

depends largely on very different factors: carbon isotope ratios are based on cultural food

preferences; oxygen on meteoric water, altitude, and distance from major bodies of water;

and strontium and lead reflect the underlying bedrock and soil.

Carbon isotope ratios reflect the photosynthetic pathways of ingested plants and

echo cultural food preferences. It is expected that individuals who have subsisted on a

traditional, rice-based (C3 plant) Southeast Asian diet will differ significantly in their

carbon isotope signature from individuals who have subsisted on a heavier corn-and

sugar-based (C4 plants) American diet. Wild rice in the U.S. has produced results

ranging from -26.3%o to -29.7%o (Hart et al. 2003) and purified rice starch has been

averaged to -26.6%o (Ambrose & Norr 1993). This contrasts markedly to maize (corn)

values varying between -14.0%o (van der Merwe 1982) and -11.84%o (Hart et al. 2003)

and purified cane sugar at -11.2%o (Ambrose et al. 1997). Americans also eat a large

variety of wheat products. Wheat is a C3 plant, but it is enriched compared to rice, with

bread wheat leaves measuring -23.7%o (van der Merwe 1982 ). It stands to reason then

that those relying on a rice-based diet, such as the Vietnamese, would exhibit more









negative carbon isotope values than their American counterparts, whose corn and sugar

constituents of the diet, will shift the carbon isotope values in a less negative direction.

Due to the fractionation effects highlighted in Chapter 1, one must keep in mind

that the reported values will not trend directly with plant values. Mammal hydroxyapatite

will demonstrate an enrichment of +9.6% to +13% (DeNiro & Epstein 1978b, Lee-Thorp

et al. 1989) over plant material. Mixtures of the dietary plant constituent will also affect

an organism's overall 613C value as well as dependency on marine food resources

(Schoeninger & DeNiro 1984).

Since the majority of state borders within the continental U.S. are not based on

geomorphologic formations, it is unlikely that regional identification will be as

straightforward. This should be partially ameliorated through a multi-signature approach.

Due to the novelty of this approach, it is difficult to say with any certainty how precise

regional identification of geopolitical origin will become. Based on the limited success

of Beard and Johnson (2000) however, it appears natal origin within the U.S. can be

narrowed down to a regional level based on major geological formations.

Because 613C values represent dietary intake, they will not indicate regional origins,

since modern diets are primarily culturally based. The stable isotope ratios for oxygen,

strontium, and lead on the other hand, are aptly suited for this task. It is assumed that

individuals from Alaska, Hawaii, and the American territories will be identifiable. The

geographical distances between these areas and the continental United States (CONUS)

are vast, with a variety of different, but interrelated, environmental factors influencing

oxygen isotope distribution such as latitude, temperature, altitude, coastal affinity,









precipitation patterns, and humidity (lacumin 1996, Hertz & Garrison 1998, Kendall &

Coplen 2001).

The geologic history of the major land masses nearly represents the 4.5 billion-year

history of the earth (Beard & Johnson 2000). Because of this, there are large differences

in the isotope compositions of different parts of the planet. relative to the analytical error

of the 87Sr/86Sr measurements (+ 0.00001 to +0.00003). Within the U.S., the ages of

crust varies from under 1 million years old in Hawaii to nearly 4 billion years old in areas

of Michigan and Minnesota (Beard & Johnson 2000). This age effect produces

significant variations in the strontium isotope composition within different regions of the

U.S. and is the basis for analytical techniques attempting to discern region of origin in

different peoples. Another strength of strontium is that its isotopes are thought to be little

influenced by fractionation (Toots & Voorhies 1965, Ambrose 1993, Carlson 1996, Hertz

& Garrison 1998, Beard & Johnson 2000, Budd et al. 2000) thus the isotope ratio remains

constant from soil to top carnivore as you move through the ecosystem. Soil samples can

then be checked against values to determine the geolocational origins of a tissue sample.

It is difficult to speculate whether strontium isotope analyses can identify natal

geolocation to the regional level in contemporary peoples. The analyses may seem fairly

straight forward on the surface, but there are underlying factors for modern man that may

inhibit its deductive power. Of primary concern is homogenization of strontium values

due to the global food trade.

An array of geological process are also responsible for the formation of these areas,

hence the bedrock composition is quite varied. Discerning among individuals reared

within the CONUS will likely prove more difficult, and overlapping values are expected.









By using the three different geologically-based isotopes in concert however, it is hoped

that general patterns will emerge.

In isolation, isotope delta values have limited evidentiary value and will rarely lead

to any form of identification. The same could be said of other bases of identification.

Clothing alone will not lead to a presumptive identification. Someone has to recognize

the clothing as belonging to the decedent before it has any realized significance. If the

geo-political region of origin for a set of remains could be ascertained however, it would

provide a direction in which to concentrate identification efforts. In mass disasters and in

closed environments, isotopes could be combined with other methods, leading to

exclusionary identifications or directing where to focus further analyses for potential

positive IDs. Such techniques are relatively inexpensive and quick. Isotopic analyses

can be performed for under $100 at the University of Florida and in the case of enamel,

can be completed in roughly 1 week's time. If stable isotope analyses are performed at

the onset of the identification process, it could save countless man-hours and dollars for

the military, preventing unnecessary analytical efforts if the remains are not deemed

American.














CHAPTER 4
MATERIALS AND METHODS

This study is groundbreaking in that it is the first of its kind to compile a reference

sample of isotopic values associated with known natal regions to be utilized in forensic

work. More importantly, the information gleaned from this study will be applied in

support of the Joint POW/MIA Accounting Command's mission to achieve the fullest

possible accounting of all Americans missing as a result of our nation's past conflicts.

A two-pronged approach for this project was utilized based on the operating

hypotheses that: 1) discernable differences exist between the isotopic ratios incorporated

into American and Southeast Asian tooth enamel and that these differences can be used to

determine region of origin; and 2) regional differences in natal isotopic signatures are

also discernable within populations raised within the U.S.

Teeth were utilized for this project because they are much more robust than bone

and little affected by diagenetic processes. This reduces the sample preparation time by

several days to a week. By only examining the enamel, isotopic values can be studies for

a known period of the subject's life, because in- and outflow of materials in enamel cease

at the termination of amelogenesis (Hillson 1996). It is also much easier to obtain

modern teeth than modem bone for sampling. When teeth are extracted, the standard

protocol is to dispose of them as biomedical waste, so there is little objection to obtaining

them for study. It is much more difficult to acquire samples of contemporary bone for

legal and cultural reasons. Objection is further fueled by the fact that isotopic sampling is

a destructive process.









Teeth are genetically conservative displaying little variation in the period of

mineralization of the tooth, although females are slightly precocious (Fanning & Brown

1971, Anderson et al. 1976, Hillson 1996), with Garn et al. reporting that females were in

advance of males by an average of 3% (1958). Different ethnic groups have shown

slightly different timing patterns as well, but all differences whether sex-related or ethnic,

equate to not much more than a few months between groups (Hillson 1996). This fact

should not impact this study however, as all crown mineralization is completed prior to

individuals being eligible for military service. All teeth supplied for this study had fully

completed amelogenesis.

Materials utilized in this study were supplied by three different institutions. The

Joint POW/MIA Accounting Command's Central Identification Laboratory (CIL),

Hickam Air Force Base, HI, permitted access to their "Mongoloid hold" collection for the

creation of an East Asian reference sample. The "Mongoloid hold" collection contains

remains of individuals recovered from East Asia or unilaterally turned over to the CIL,

whose governments have refused repatriation, once the remains were determined not to

belong to U.S. service personnel. Donated contemporary teeth and surveys completed by

their donors were also provided by the 10th Dental Squadron, United States Air Force

Academy (USAFA), Colorado Springs, CO and the Malcolm Randall Veterans Affairs

Medical Center (North Florida/South Georgia Veterans Health System) Dental Clinic,

henceforth referred to as the "VA," Gainesville, FL.

Dental Protocols

Prior to utilizing live subjects in this research, all appropriate permissions were

obtained and training completed (Appendix A). The USAFA Institutional Review Board

(IRB) granted IRB exempt status to this project (HQ USAFA IRB FAC2005026H). Prior









to conducting this study with the VA, the protocol was approved by the University of

Florida's Health Center Institutional Review Board (IRB-01 approval #474-2005), and

both the VA's Sub-Committee for Clinical Investigation and Research and Development

Committee. Additionally, a research template had to be created for incorporation into

each study participant's electronic medical record, via the VA's Computerized Patient

Record System (CPRS).

To further assist all parties engaged in this research, information binders were

distributed to both dental facilities. These packets included copies of all IRB and

committee approval letters, dental staff instructions, a subject identifier log, copies of all

required forms, a blank and completed, example survey, background information related

to this specific research project, pre-paid FedEx shipping forms (for USAFA), and a CD

with all electronic media on it (see Appendix A for a reproduction of the VA binder).

This project was essentially a piggy-back study attached to the normal patient

dental care of those individuals who are selected by USAFA and the VA for tooth

extraction(s) for valid medical reasons. The study, in and of itself, had no bearing on

whether an individual was selected for dental extraction(s). All patients scheduled for

dental extraction(s) during the study period were queried as to their willingness to

participate in the study. Complete inclusion of all consenting subjects cut down on bias

that would be introduced with nonrandom, arbitrary sampling by the dental staff. Dental

administration personnel proctored all forms. Upon receipt from the patient, they

reviewed the forms for completeness and verified birth date and sex with the subject's

dental records.









Patients, to include Air Force Academy cadets, active-duty military, and military

retirees and/or veterans, already identified for tooth extraction for oral health reasons,

were asked to participate in a brief survey (Figure 4-1) and donate their extracted teeth

for analysis. The survey and, in the case of the VA subjects, associated combined Health

Insurance Portability and Accountability Act/informed consent form (Appendix A) were

administered upon initial intake while the patient filled out requisite preoperative

paperwork. This paperwork was in addition to the normal documentation required for

dental procedures.

The HIPAA form was compulsory to protect participant health information.

Researchers must obtain patient authorization before they are allowed to disclose

protected health information. It was required in this instance because we were requesting

information such as location of residence and birth date, which cannot be ascertained

from observation alone. The informed consent form was required to secure subject

participation in the study. The form detailed the background, procedures, benefits and

risks of the research project, and obtained witnessed, signed consent of the individual that

they knowingly and voluntarily participated in the study. Dental staff were available to

answer any questions and an example of a completed questionnaire and study background

information (Appendix A) was made available. Survey completion and tooth donation

were the only requirements of subjects.

The data acquired from each subject included:

Date of birth
Sex
Race
Tobacco product use
Childhood diet








70



* Location of residence, birth to age 18
* Date of prior dental extraction at each facility (if applicable)


Thank you for your participation in this study. Its purpose is to provide a powerful new tool to assist in identifying our fallen
servicemen and women. The information you provide will be used to determine if geographic regions of the U.S. have specific
isotopic signatures that become incorporated into dental tissues. We will be looking at the mineral elements in your teeth. No
DNA analysis will be performed. When compared with isotopic signatures developed for geographic areas of Southeast Asia, it
is hoped the information will identify the origin of unknown remains recovered by JPAC's Central Identification Laboratory.
Additionally, the information gleaned from this study may prove useful in identifying remains recovered from further conflicts
such as World War II and encountered in mass disasters such as airliner crashes and the events of I 11 September 2001.
Instructions: Please fully answer all 8 questions. Incomplete data may exclude your teeth from the study. If you
have any questions, please ask your attending dental staff.

I) Date of birth (day/month/year)

2) Sex (circle one) Male Female

3) What race do you consider yourself?

4) Have you ever regularly used tobacco products (i.e. cigarette, chew, snuff)? Yes No
If yes, what products did/do you use, what was the time period, and what was the frequency (i.e. I pack a day)?
Tobacco Product Used From Used Until Frequency
(year) (ear)
1i.
2.
3.

5) Which of the following categories would you consider your childhood diet to the age of 18. Please circle only
one category unless you had a major diet shift. If so, please indicate the ages at which you followed each diet.
Meat Eater Vegetarian Vegan

6) What locations have you lived in, starting with birth and extending to age 18? Please be as specific as possible.
If you require more room, please use the back of this sheet.
From To
(year) (year) City State Country
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

7) Please indicate the approximate location of each of the above areas on the attached map. For area (1) write a (,
area (2) write a 8, etc. If you lived outside of the U.S. for any portion of your childhood, please disregard for the
extent of your domicile outside of the U.S. Do include the numbers for any corresponding time lived in the U.S.

8) Have you undergone any prior dental extractions at this facility within the past year? (circle one) Yes No

If yes, please indicate the date to the best ofyour recollection. (day/month/year)

Approved UF-IRB-01 474-2005


Figure 4-1. Joint POW/MIA Accounting Command survey.

























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The aim of the survey was to control for as many sources of error or variation as

possible in the data as well as create an isotopic mapping capability for natal region. All

questions, with the exception of the prior dental surgery question, were pertinent to the

study in that they account for factors that may possibly lead to differential maturation in

teeth or absorption/deposition of the various isotopes being studied.

The first item, date of birth, allowed for temporal comparison of specimens

between JPAC and VA samples versus USAFA samples. While dental development is

relatively genetically conservative, there is some minor variation in dental development

rates between sexes and major ethnic groupings. This information; date of birth, sex, and

self-perceived race; served as potential blocking factors during data analysis.

The effects tobacco use upon isotope analyses for teeth, have thus far not been

addressed in the literature. While enamel isotopic fates are locked in after amelogenesis

terminates, it is unknown whether tobacco use may trigger diagenetic changes within

teeth that may affect isotope values. It is commonly known to stain teeth, and may need

to be accounted for in preparation protocols and in interpretation of results.

Dates and locations of childhood residence were critical for making sense of the

oxygen, strontium, and lead stable isotope results. The validity of the residence

information was confirmed by individuals visually approximating these areas on a map.

It was also useful if someone could not remember the exact name of a town/city in which

they lived but did know approximately where it was in relation to neighboring areas.

The other questions served to control for potentially confounding variables. Dental

extraction history was necessary to prevent counting one individual who underwent









multiple extractions over multiple days as more than one subject. Survey questions were

limited to one page with the map encompassing a second page.

Pertinent information corresponding to each patient was also recorded by the dental

staff on each survey. Here, each facility assigned a unique subject identifier number to

each patient (i.e., VA-001). Additionally, the position in the arcade that each tooth came

from (tooth number) was noted according to the Universal/National System dental

numbering scheme for permanent dentition as well as the date of extraction.

Teeth were extracted following standard dental protocols for each facility. Care

was taken to preserve as much of the crown as possible. Each tooth was placed into its

own vial, which was labeled with the subject identifier number and tooth number, All

vials from a particular individual were then placed in a resealable bag and the bag stapled

to the associated survey. The surveys and teeth from USAFA were shipped via FedEx to

the C.A. Pound Human Identification Laboratory (CAPHIL). Surveys and teeth from the

VA were picked up weekly and CPRS updated by the author. Teeth provided by both

facilities were not stored in any solution or fixative.

Sampling

Teeth were selected for sampling using the following hierarchy. Those teeth whose

cessation of amelogenesis was most similarly timed with the third molars were preferred,

with other teeth chosen on a decreasing sliding scale (Table 4-1). The younger in an

individual's life crown completion occurred for a specific tooth, the less desirable the

tooth was for sampling. Additionally, molars were preferable as they have the largest

surface area available for enamel removal. As a matter of course, mandibular teeth were

chosen over maxillary teeth and right over left. Furthermore, for the East Asian reference

samples from the CIL, teeth still present in the alveoli were selected over loose teeth,









Table 4-1. Crown formation/tooth eruption.
Crown Initiation' Crown Completion2 Tooth Eruption3
Tooth (upper/lower) in yrs (upper/lower) in yrs (upper/lower) in yrs
3rd molar 7.0-10.0/7.0-10.0 13.3/13.3 17-21/17-21
2nd molar 2.5-3.0/2.5-3.0 6.7/6.7 12-13/11-13
2nd premolar 2.0-2.5/2.0-2.5 6.3/6.3 10-12/11-12
Ist premolar 1.5-2.0/1.5-2.0 5.8/5.6 10-11/10-12
Canine 0.3-0.4/0.3-0.4 4.9/4.8 11-12/9-10
2nd incisor 0.8-1.0/0.25-0.3 4.0/4.0 8-9/7-8
1st incisor 0.25-0.3/0.25-0.3 3.7/3.6 7-8/6-7
1st molar 0.0/0.0 3.8/3.7 6-7/6-7
range in Shour & Massler (1940)
2 mean values in Anderson et al. (1976)
range in ADA (1999)


since the actual tooth number could be verified more easily with the presence of the

associated bone. In nearly all CIL cases however, a full arcade was not present to choose

from, thus the best option according to the aforementioned sampling scheme was selected

based on the resources available.

Each individual was assigned a unique identifier, not each tooth. Therefore, if two

teeth were utilized from the same individual, the samples would be given the same

identifier, with an additional tooth number identifier. This numbering scheme prevented

inflation of actual individual numbers. Furthermore, due to potential intertooth variation

in stable isotope values, enamel was not combined from multiple teeth to achieve the

desired weight of enamel powder.



Central Identification Laboratory

The CIL is an American Society of Crime Laboratory Directors (ASCLD)-certified

crime lab. As a result, all sampling conducted at the CIL conformed to their standard

operating procedures, to ensure compliance with ASCLD requirements. Prior to









sampling, potential specimens were researched utilizing a list of "Mongoloid holds"

provided by Dr. Andrew Tyrrell, the Casualty Automated Recovery and Identification

System (CARIS) database, and a thorough personal investigation of the entire evidence

storage area. Once suitable specimens had been identified, individual accessions were

checked out from the evidence manager and transferred to the CIL autopsy suite for the

actual sampling.

Study identifiers with a "CIL" prefix and a three-digit suffix were associated with

lab accession numbers from the CIL Mongoloid hold collection. Two teeth, if available,

were selected from each accession following the above procedures. In all cases, at least

one intact and undisturbed tooth was left with the case in the event that future

identification efforts, such as DNA sequencing, were required. Each tooth selected for

sampling was assigned an additional sample number (01A or 02A) mirroring the lab's

DNA sampling procedures. Information cards for each tooth were created for photo

cataloging and provided the following information (Figure 4-2):

CIL accession number
Individual designator (if applicable)
Tooth number
Sample identifier
Date
"Isotope study"
Subject identifier number
Researcher's initials


From 14 June 2005 to 06 July 2005, a total of 112 teeth were sampled from 61

individuals believed to have originated from or been recovered from the following areas:

Vietnam (48 individuals); Cambodia (4 individuals); Laos (3 individuals); the Korean
























Figure 4-2. Pre-drilling photo of CIL-033 #19 with data card. Note: the accession
number is purposely, partially obscured.

peninsula (3 individuals); the Solomon Islands (2 individuals); and the Philippines (1

individual). Teeth were eased out of their respective alveoli by hand or drilled out, when

necessary, using an NSK UM50 TM slow-speed dental drill with either a #2 or #4 carbide

dental drill bur, taking care to minimize damage to each alveolus. A photo, to include an

information card, reference scale (ruler), and empty collection vial was taken of each

tooth to document what each element looked like prior to drilling (Figure 4-2). Separate

photos of the buccal or lingual and occlusal surfaces were taken. (In cases where the

teeth had to be drilled out of their respective alveoli, pictures of the unaltered arcades or

portions thereof were taken using the same format.)

Each tooth was then placed into a vial of 3%, household-use hydrogen peroxide

and cleaned via a Branson Bransonic 2510 tabletop ultrasonic cleaner for 30 minutes.

When finished, teeth were removed from the solution and manually cleaned with a

toothbrush. The enamel surface of the teeth was prepared for drilling by cleaning off

excess calculus, soil, and/or staining using the same apparatus and a #8 carbide dental

drill bur.









Samples of approximately 100 mg of pristine enamel were drilled off of each tooth

using the same set-up (see Appendix B for drilling data). Care was taken not to drill into

the dentine. Enamel powder was collected on creased weighing paper and transferred to

labeled 1.5 mL microcentrifuge tubes. The drilled tooth, collection vial, scale (ruler), and

information card were again photographed to document the end-stage condition of the

tooth and for chain of custody purposes. The teeth were then returned to their original

storage bag along with the information cards with the associated elements for that

particular accession number. The bag was resealed with evidence tape, and the tape

initialed and dated on both sides. The remains were then turned in to the evidence

manager. Drill burs and weighing paper were discarded after each use and the drill

cleaned of adherent enamel powder.

Chain of custody forms were completed for all specimens, transferring possession

of the enamel powder and any associated enamel chips to the author (Appendix B). The

microcentrifuge tubes containing the enamel specimens were then transported from CIL

to CAPHIL through the services of FedEx.

The author also attempted to gain access to human teeth from native populations

while performing duty-related activities in Vietnam from July and into August 2005.

Such efforts were abandoned however, when provincial officials stated that regional and

higher government officials would be required to approve any request to procure human

samples.

United States Air Force Academy and Veterans Affairs

The Air Force Academy collected surveys and a total of 948 teeth from 274

individuals between late August 2005 and late April 2006 (Table 4-2; see also Appendix

C for a list of survey results). Of these, one third molar was selected from each









Table 4-2. Isotope sampling matrix.
# # Total # Individuals Run Total # Teeth Run Total # Runs
Source Inds Teeth C O Sr Pb C O Sr Pb C O Sr Pb
CIL 61 112 61 61 36 36 64 64 36 36 65 65 36 42
USAFA 274 948 230 230 36 36 238 238 36 36 279 279 36 36

Total 335 1060 291 291 72 72 302 302 72 72 344 344 72 78

of 228 individuals for inclusion in the primary study. One tooth each from two

individuals of unknown natal region were utilized for additional testing examining the

necessity of using acetic acid to process the enamel. Samples originating from three

different individuals were not used because of experimenter error in labeling the samples

and erroneous or missing information provided by the subject. Furthermore, after sample

AFA-185 from USAFA, samples were selectively chosen to fill in the geographic gaps

until optimally, each state had a minimum of five individuals represented. This approach

was chosen to reduce costs. Additionally, individuals from duplicate cities or those

people born prior to 1980 were sampled as well. Collection of specimens from the VA

began in mid-February 2006 and is ongoing. Unfortunately, because of the low number

of teeth provided by the facility and the poor condition of these teeth (i.e., little to no

enamel present) no samples were run for the current study. Sample collection is still

ongoing though, with the hope that the teeth can be used at a later date.

Upon receipt at CAPHIL, teeth were soaked in 3% hydrogen peroxide in their

original vials for 2 days. Teeth were then rinsed of the hydrogen peroxide with tap water

and scrubbed with a toothbrush to remove surface contaminants, such as blood. Any

adherent periodontal tissue or accessible neurovascular bundles were also removed.

Clean teeth were allowed to air dry overnight in a ventilation hood and each tooth was

stored in a separate, clean, labeled, resealable, plastic bag.









All USAFA samples contained at least one third molar, with the majority of individuals

providing all four. Only third molars were run from this facility. Whole teeth, in the best

overall condition were preferentially selected for drilling. If multiple teeth from an

individual were of the same quality, sampling selection was based on the same criteria as

mentioned for the CIL samples: mandibular teeth were chosen over maxillary teeth and

right over left. Teeth exhibiting unusual crown anomalies or staining patterns and/or

teeth in which the author disagreed with the dental staff numbering were photographed

prior to drilling only. The remaining teeth were not photo-documented. Photo content

consisted of the tooth, subject identifier number, tooth number, and a scale (Figure 4-3).

Two photos, one of the buccal or lingual surface, and one of the occlusal surface were

taken. Teeth were cleaned in distilled water within individual capped vials with a

Branson Bransonic 1510 tabletop ultrasonic cleaner for 30 minutes. After air-drying,

teeth were cleaned of any surface contaminants to include alveolar bone remnants using a

NSK UM50 TM slow-speed dental drill with a #8 carbide dental drill bur. Samples of

approximately 100-200 mg of pristine enamel were drilled off of each tooth using the

same set-up. Care was taken not to drill into the dentine. Enamel powder was collected

on creased weighing paper and transferred to labeled 1.5 mL microcentrifuge tubes. Drill

bits, weighing paper, and latex gloves were discarded after drilling each tooth and the

drill cleaned of adherent enamel powder.
























Figure 4-3. Pre-drilling photo of AFA-093 #32.

Carbon and Oxygen Sample Preparation

Central Identification Laboratory Samples

Chemical preparation of the enamel powder was performed in the stable isotope

laboratory at the Florida Museum of Natural History, Gainesville, FL, according to the

protocol developed by Dr. Pennilynn Higgins, museum postdoctoral fellow. The powder

of one tooth from each individual was selected based on the integrity of the sample (i.e.,

whether there was the possibility of dentin or other contaminants mixed in with the

enamel) and greatest mass of powder available for analysis. Organic residues were

removed from the sample powder by adding 1 mL 30% hydrogen peroxide (H202) to

each microcentrifuge tube. Tubes were shaken utilizing a Thermolyne Maxi-Mix 1,

16700 mixer and the lids lifted up to prevent gas pressure build-up inside the tubes. The

opened vials were stored in a closed reaction cabinet. Samples were periodically shaken

with the mixer, every 1 to 2 days, to re-suspend the enamel powder that had settled at the

bottom of the vial.

On a weekly basis, the H202 was removed by centrifuging samples for 20 minutes

at 10,000 RPM in an Eppendorf 5415D microcentrifuge and pipetting off the H202.









Pipette tips were discarded between each sample to prevent cross-contamination. Fresh

H202 was then added following the same protocol. Samples were reshaken, lids opened,

and placed back in the reaction cabinet. The absence of escaping air bubbles from the

solution usually indicates the sample powder is finished reacting and ready for the next

phase of treatment. After consulting with Drs. Bruce MacFadden, Florida Museum of

Natural History, and John Krigbaum, University of Florida Department of Anthropology,

the samples were decanted of all H202 after 51 days in solution, even though nearly half

still appeared to be reacting. This was likely due to the large quantity of powder being

processed, with most enamel samples measuring 100 mg or greater. Samples were then

twice rinsed with 1 mL deionized water utilizing the same procedure for removing H202

(i.e., water added, then tubes shaken, centrifuged down, and decanted).

After rinsing the samples with deionized water and decanting all water from them,

secondary carbonates were removed via an acetic acid bath. Rinsed samples, free from

water, were bathed in 1 mL 0.1 N acetic acid, shaken, and allowed to sit for 30 minutes.

The acetic acid was pipetted off after centrifuging the microcentrifuge tubes at 10,000

RPM for 5 minutes. Samples were then twice rinsed with deionized water and the water

removed in the same manner as previously discussed. Samples were allowed to air-dry in

their open microcentrifuge tubes inside of a desiccator for 2 weeks. This ensured all

liquid had evaporated from the enamel powder.

An additional side test examining the necessity of performing the acetic acid step

was performed. Theoretically, teeth extracted from living subjects should not have to

undergo the acetic acid bath, because teeth in the living are not subject to diagenetic

changes associated with the build-up of secondary carbonates due to taphonomic factors.









The acetic acid bath was performed on all samples because there is no know precedence

to do without acetic acid for forensic isotope purposes. Eight USAFA and two CIL

samples were split in half with one half undergoing the full protocol previously outlined

and the second sample of each pair undergoing the H202 bath and rinses only. Values

were then compared to determine if this step of the protocol is indeed required.

Portions of the dried enamel measuring between 1.2 mg and 1.5 mg were then

loaded into stainless steel boats at the University of Florida, Department of Geological

Sciences, Light Isotope Laboratory. Each boat was placed into 1 of 44 numbered slots on

a brass tray (Figure 4-4) and the tray placed into a desiccator until they were run on the

laboratory's VG/Micromass (now GV Instruments) PRISM Series II isotope ratio mass

spectrometer with an Isocarb common acid bath preparation device. Load sheets were

accomplished for each tray listing the sample name and weight for each position in the

tray (Appendix D). All samples were loaded by the author. The PRISM was operated by

Dr. Jason Curtis and Kathy Curtis.

The first run was organized as follows: slots 1-4, standards ofNBS-19 measuring

between 60 gg and 120 gg; slots 5-20, alternating enamel powder and blank positions;


Figure 4-4. Loaded tray for PRISM mass spectrometer analysis.









slots 21-22, NBS-19 standard; slots 23-42, alternating enamel powder and blank

positions; slots 43-44, NBS-19 standard. This arrangement allowed for the analysis of

18 samples. Empty or blank positions were included in the first run to check for

contaminants within the samples. Mass spectrometer readings for the blank positions,

indicate leaching of slow-reacting sample into these positions and hence likely

contamination. Contamination tends to be much more of an issue with fossilized samples

versus modern or historical (Koch et al 1997). Because the first run ran clean with no

indication of contamination, the blanks were replaced with sample for all subsequent

runs. The sample line-up for all subsequent runs therefore was as follows: slots 1-4,

NBS-19 standard; slots 5-20, sample; slots 21-22, NBS-19 standard; slots 23-42,

sample; slots 43-44, NBS-19 standard. This arrangement allowed for the analysis of 36

samples for each run of the mass spectrometer.

United States Air Force Academy Samples

The Academy samples were prepared in the same manner as the CIL samples with

three exceptions. The first change to the processing protocol entailed reducing H202

exposure time to 24 hours. This change was made upon the recommendation of Dr.

Bruce MacFadden, Florida Museum of Natural History, and Dr. Pennilynn Higgins,

Stable Isotope Ratios in the Environment Analytical Laboratory, Department of Earth and

Environmental Sciences, University of Rochester. It was implemented because whole

teeth were cleaned for 2-3 days using a 3% H202 solution to remove external organic,

but more importantly, because teeth were collected directly from living subjects.

Organics associated with diagenetic transfer due to burial were not encountered with

these samples as they were with the CIL samples.