PLAN NACIONAL DE PREVENCION Y ATENCION DE DESASTRES INDECI.

In order to support the pXRF data relating to prehistoric diet in four riverine sites in first millennia Peru, both bone collagen and bone apatite samples were prepared for processing. Only The Field Museum in Chicago permitted a limited number of individuals to be used for the destructive sampling process, and even then, protocols normally observed in the Laboratory of Archaeological Sciences at the University of South Florida gave way to what could be accomplished in a space provided in the basement of The Field Museum. There was no scale

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available for me to use, and it was requested that the tiniest of samples be taken. There was also no consistent bone type, such as a left rib, available for each individual. The collection I was presented contained inconsistent varieties of long bones, so a fragment of femur, fibula, or humerus were chosen when possible, while several tiny fragments of unknown origin were recovered from sample boxes; four small cranial fragments, one mandible fragment, and one rib completed the array. Since bones “remodel” at different rates throughout the body, consistency of bone type for such testing is always preferable. Under the constraints implied from tiniest of samples and a bottle cap of sand as my example of 10 grams, the initial weights of each of my samples ranged from half a gram to two and one-half grams. Ideally, the sample size should start at 10 grams or more. I physically cleaned the bone sections as well as possible with the soft brush and cloth that I had taken with me. I was to do no visible damage to any crania and had to choose from post-cranial samples that were less than ideal. Using a Dremel 5000 drill equipped with an engraving cutter, a small piece of bone was removed from each available sample. Powder created in the process of cutting the bone was reserved for use in the apatite samples. I was unable to ultrasonically clean anything since most of the tiny sample of each that came to Florida with me was already powdery bone shards from the process of cutting it from the larger specimen in the collection.

Fourier transform (FT) Raman spectroscopy was not available to me in analyzing the potential for quality collagen in my samples, but that is a powerful tool for future field studies, able to assure positive results when human osteological samples are chosen for destructive analysis (France et al. 2014; Pestle et al. 2015, 2014; Wilson et al. 1999). In addition to sampling bone from 44 individuals, tooth enamel was also carefully removed from teeth of 53 individuals, a few providing a root section and/or enamel from both first and third molars, for a total of 57

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samples; without the ability to ultrasonically clean the teeth before removing a portion of the enamel, determining viability of the mass spectrometry enamel apatite results is an issue confronted in the Discussion chapter.

The protocols established by Robert Tykot at the Laboratory of Archaeological Sciences at the University of South Florida were followed in the processing performed after returning to Florida.

6.3 Bone Collagen Preparation

Approximately 90 percent of the organic matrix of bone is collagen; as far as the human organism is concerned, this represents the main protein in the diet (McLean & Urist 1968; Price et al. 1985). Each small sample was assigned a USF number and a bone collagen spreadsheet was started. There was no evidence nor notation of any of the bones having been treated with preservatives, so steps that would have otherwise been required for that were unnecessary. A variety of methods of preparation for isotope analysis have been postulated over the years; in the USF Laboratory for Archaeological Science students have been trained in a method developed by Sealy and published by Sealy and van der Merwe (1986) (as cited in Schwarcz & Schoeninger 1991). The only alteration noted in the USF laboratory was the absence of the freeze-drying step immediately prior to exposure in the isotope ratio mass spectrometer (IRMS) (Schwarcz & Schoeninger 1991), replaced by long-term oven-drying. Small portions of bone were set aside for apatite and strontium testing. Because the sample sizes were so small, 2-dram vials were used for processing. Each vial was labeled with the USF number which had been assigned. Contaminants such as humic acids were removed by using 0.1 M sodium hydroxide (NaOH) for 24 hours. After pouring off the NaOH solution, each sample was rinsed in distilled water multiple times to assure

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neutrality. All pieces were uniformly pulverized at this point in an attempt to secure similar timing results for the following steps in the processing. The next step was demineralization and this was done in a 2 percent hydrochloric acid (HCl) solution for 72 hours; solution was poured off and replaced with fresh every 24 hours for the three days of this step in the process.

Not every sample demonstrated complete demineralization in the 72 hours allotted. The samples in which the solution appeared clear and had no visible bubbling on the surface were rinsed four times and then left in the same vial until all of the samples were demineralized and had been at least rinsed four times with distilled water. At this point the first step of using 0.1 M NaOH to remove any residual humic acids was repeated for a 24 hour period. This solution was poured off and again the samples were rinsed. A defatting solution comprised of a mixture of methanol, chloroform, and distilled water at the ratio of 2:1:0.8 was poured over the samples. Again, the samples soaked for 24 hours, this time to strip the bones of any fat content. Defatting solution is one of the chemicals in the laboratory that is put in a waste jar for holding the pour- off for future safe disposal. Samples were again rinsed extra thoroughly with distilled water and while the protocol is for four rinses, my notes from the process indicate that a trace of the defatting odor was lingering still, so I used a fifth rinse.

Since I was already working in 2-dram vials, I did not need to transfer my samples. I did need to make sure that each of the USF numbers was still readable and several I relabeled for clarity. Open vials were then transferred into racks and set in the drying oven until thoroughly dry. Dual collagen samples for each individual were weighed into tin cups, averaging 0.8-1.2 mg of each, and the collagen yield was calculated. Ten of my 44 samples yielded no collagen, while a portion of those which were sent to the mass spectrometry lab at USF St. Petersburg were also determined to be of no value for the final report. Unfortunately, this is often the case with bone

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collagen and the problem of diagenesis in Peruvian bioarchaeology. All data were then updated in the bone collagen spreadsheet.

The bone remnants, called collagen pseudomorphs, were transferred to Ethan Goddard, the mass spectrometry Laboratory Director at USF St. Petersburg. A CHN analyzer coupled with a Finnigan MAT stable isotope ratio mass spectrometer was used to analyze the collagen pseudomorphs for δ13_{C and δ}15_{N in continuous flow mode. The integrity of the collagen samples} was then confirmed by collagen yields and C:N ratios. Ratios are included in the Tables of descriptive statistics which appear in the following chapter.

6.4 Bone Apatite Preparation

Carbonate apatite is part of the inorganic bone matrix. Each small sample which had been parsed out from The Field Museum’s microsamples was assigned a USF number and a bone apatite spreadsheet was started. Again, the steps normally involved relating to the ultrasonic cleaning and drying were skipped since there were no samples large enough to process. What I had left from separating samples for collagen was mostly already bone powder. This was ground with a mortar and pestle to assure all of the particles were crushed. A 2-dram vial became the recipient of approximately 10 mg of bone powder which was weighed out and then labeled. One ml of 2 percent bleach solution (sodium hypochlorite) was added as a primary step for the removal of collagen, bacterial proteins, and humates. Samples were left to soak for 72 hours. On the third day following the soaking the samples were centrifuged, bleach solution was pipetted off in most cases, poured off in a few, and the bleach was replaced with distilled water.

131 Figure 6.5 Pouring off defatting solution

This process was repeated four additional times. Upon completion of the fifth rinse the samples were placed in the drying oven. Once dry, the samples were weighed. One ml of 1.0 M buffered acetic acid/sodium acetate solution was used to process the bone powder for the next step, to remove non-biogenic carbonates, and this was left to soak for 24 hours. Again, samples were centrifuged, a pipette was used to extract the acetic acid/sodium buffer solution without accidently depleting the sample size with an erroneous pour-off, and the solution was replaced with distilled water. This process was repeated four times. The drying oven was preheated to temperature and when all of the samples were sufficiently rinsed, the bone powder was placed in the drying oven. Once dry, again the samples were weighed.

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Final weights ranged from 0.6 to 8.9 mg; only one sample was too small to continue the process. For all others 1.0 mg of the bone powder was weighed, and each was transferred to a 2- dram vial. All data was then updated in the bone apatite spreadsheet. The yields from each stage of the pretreatment process are used to assess the integrity of the bone apatite and enamel samples. Finally, apatite analysis was done on a ThermoFisher MAT253 IRMS coupled to a GasBench-II + continuous-flow interface and the tiny samples were reacted with 600 μl 104% H3PO4 @ 25̊ C for 24 hours in the USF St. Petersburg Paleolab.

6.5 Tooth Enamel Apatite Preparation

Teeth and bones have different histologies. While bone turnover is relatively rapid, with skeletal remodeling generally occurring over a decade or less, tooth enamel is inorganic and forms as the teeth are being developed. Tooth enamel was drilled at The Field Museum. The surface layer of tooth enamel was removed to eliminate possible contaminants prior to drilling the tooth for the powdered sample. A Dremel Minimite-750 cordless drill equipped with an engraving cutter was used. It requires approximately 10 mg of powder for a viable sample, but most of my microsamples were considerably less from the beginning, with initial weights starting as low as 3.3 mg. Enamel requires immersion in 2 percent sodium hypochlorite (NaOCl) for 24 hours to dissolve organic components. A centrifuge was used and then the bleach solution was extracted by pipette and replaced with distilled water. Each sample was rinsed, centrifuged, and pipetted off five times prior to the next step in the process. Once fully rinsed, samples were moved to a drying oven.

When fully dry, samples were reweighed. The removal of non-biogenic carbonates was done next over a 24 hour period in 1.0 M buffered acetic acid/sodium acetate solution. Again the

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centrifuge was used and the acetic acid/sodium acetate buffer solution was removed by pipette and replaced with distilled water. This step of centrifuge, pipette, and fresh rinse of distilled water was repeated four additional times. Samples were then placed in the drying oven for the final drying and, when fully dry, samples were again weighed. All data was then updated in the aforementioned spreadsheet. Final weights ranged from 0 to 8.5 mg; several samples were too tiny for further testing. The yields from each stage of the pretreatment process are used to assess the integrity of the apatite and enamel samples. Analyses of the oxygen and carbon isotopes derived from the archaeological hydroxyapatite carbonate (δ18Oca, δ13Cca) were completed using a ThermoFisher MAT253 IRMS coupled to a GasBench-II + continuous-flow interface and the samples were reacted with 600 μl 104% H3PO4 @ 25̊ C for 24 hours in the USF St. Petersburg Paleolab.

6.6 The Sample Tested

Crania (n=209) were analyzed for their trace mineral components using pXRF spectrometry; of these, 110 samples were representative of total samples originally collected in cemetery complexes from three riverine communities - Maranga/Aramburu in the Rimac Valley, Cerro del Oro in the Cañete Valley, and Nazca in the Nazca Valley. During the EIP, these locales likely belonged to two Peruvian coastal cultures, the Lima culture on the Central Coast and the Nasca culture, considered South Coast, although this site is actually 50 km inland from the Pacific Ocean. Since Cañete may have been unaffiliated and independent, or may have been affiliated with the Lima culture or with the Nasca culture, one goal is to see if evidence from the dietary signatures of these samples provides any clues to cultural affinities. This portion of the collection was provided by The Field Museum of Natural History in Chicago, gathered in the

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course of two expeditions in 1925 and 1926 by Alfred Kroeber, described earlier in this study. In addition, 98 crania amassed in 1910 and 1913 by Aleš Hrdlička for the National Museum, now known as the Smithsonian, were made available by the Smithsonian Research Center in Suitland, Maryland. This portion of the collection was excavated in the Chicama Valley on the North Coast and is representative of the array of human osteological remains from the Moche culture collection held at that institute.

In addition, 44 samples derived from human bone were also sampled for isotopic analysis, primarily from the post-cranial remains of a portion of those in The Field Museum’s Kroeber collection. Of the 44 processed, 24 were considered viable and their isotopic information follows. While overall these human osteological samples were recovered from 11 cemetery sites throughout the Nazca Valley, one cemetery in the Rimac Valley, and one cemetery in the Cañete Valley, only Cañete and four cemeteries from the Nazca Valley are represented in the final carbon isotope study due to factors related to small sample size, diagenesis, and other potential contaminants. Bone apatite results represent 11 of the 14 cemeteries. Another 58 samples of tooth enamel were also processed; of these, 12 samples are represented in the final study with direct relation to bone isotope ratios derived from those deemed within the statistical range of acceptability. All additional tooth enamel results determined to fall within the range of expectation, meaning appearing free of contaminants, are also included.

6.7 Sampling Strategy

The initial collection of bone and tooth enamel from The Field Museum was based on choosing a representative sample of the Kroeber collection from the valleys associated with the

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rivers Rimac, Cañete, and the Rio Grande de Nazca Drainage. While distinct cemeteries were not noted for the Lima/Aramburu or Cañete/Cerro del Oro sites in the museum database, they were listed as such for Nasca. Therefore, choices were made to include samples from as many distinct burial sites as possible. Choice was also given to maintaining some degree of parity in sex and age representation, as well as an approximation of placement within the temporal realms under study, although some cases existed where the database listed sex as indeterminate while a notation of male or female was penciled onto the bones. In these cases, I have used undetermined in this study. Juveniles and most individuals identified as subadults are also listed as sex undetermined. I also had to work within the constraints of reasonable possibility for the museum staff who were assigned to help me access the physical remains. I was not able to access the drawers in the cases in The Field Museum collection myself and was limited to sample the bones which were presented, not all offering the best choices for organic and mineral integrity.

Upon returning to The Field Museum for the latter elemental analysis using pXRF, the objective was to access as many of the original samples as possible, provided that a cranium was present, since it was solely crania that I had been given permission to examine and process at the Smithsonian Museum. Because of this choice, I also was able to be relatively uniform in the choice of positions selected for each perspective selected for pXRF spectrometry analysis. The frontal area up to the sagittal suture of the neurocranium was always the first selected site, except in the rare instance wherein both the neurocranium and splanchnocranium were missing. The second measurement was taken from either right or left parietal, dependent on the condition of each. The exception here were several samples from the Smithsonian collection which, due to fragility on sides, caused me to choose the occipital area for each of these second readings; several others were already in pieces with one calvarium included in the Smithsonian sample.

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Working with an assistant assigned to pull entire drawers related to the first visit’s sampling choices, my choices this round were dependent on the presence of a skull. Ultimately, this had an effect on both age and gender samples included, but I was not fully aware of this as the sampling was being done. Some skulls that appeared to be female were in fact in the database as non-sex-specific juveniles; in these cases, database recordings were used.

At the Smithsonian Museum I was given a key to the drawers for the Chicama portion of the Hrdlička collection myself. David Hunt had an area in the collections hall that was already setup for photography and he invited me to use it and to setup my tripod with pXRF equipment on an adjacent workbench. Recording as much data as I could while the pXRF was in its two- minute runs, I was able to pick and choose samples which gave me a composite sketch of the dynamics of the overall Chicama collection based solely on samples from two cabinets. Knowing that the stable isotope results from The Field’s Kroeber collection was my supporting evidence for the efficacy of an elemental analysis in determining ancient diet, I wanted to make sure that the Hrdlička collection included a mix of brachycephalic and dolichocephalic samples, with and without cranial vault modifications, and that sex and age variations within the collection were all included.

Samples with estimated dates that coincide with the Early Intermediate Period (200 BC- AD 700) and Middle Horizon (AD 700-1000) provide a sense of both the dietary similarities and differences as coastal cultures declined or transformed and new ones emerge in their stead.

6.8 Sampling Challenges

Sampling challenges were many. Multiple trips to Peru yielded no samples for export for destructive analysis. Demands of some American museums for their written requests were nearly

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as cumbersome as writing an NSF grant application, and my teaching and other responsibilities eventually led me to give up on both. My work herein is fully self-funded. Once I found a museum willing to provide me some samples for destructive analysis working under the constraints of their environment without the necessary equipment that was readily available in my home lab at USF made the microsampling a bit of a guessing game.