Preservation of hair stable isotope signatures during freezing and law enforcement evidence packaging

Accepted Manuscript Preservation of hair stable isotope signatures during freezing and law enforcement evidence packaging Gwyneth W. Gordon, Tiffany B. Saul, Dawnie Steadman, Daniel J. Wescott, Kelly Knudson PII: DOI: Reference:

S2468-1709(18)30030-4 https://doi.org/10.1016/j.forc.2018.10.004 FORC 126

To appear in:

Forensic Chemistry

Received Date: Revised Date: Accepted Date:

18 April 2018 3 October 2018 12 October 2018

Please cite this article as: G.W. Gordon, T.B. Saul, D. Steadman, D.J. Wescott, K. Knudson, Preservation of hair stable isotope signatures during freezing and law enforcement evidence packaging, Forensic Chemistry (2018), doi: https://doi.org/10.1016/j.forc.2018.10.004

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Preservation of hair stable isotope signatures during freezing and law enforcement evidence packaging Gwyneth W. Gordona,*, Tiffany B. Saulb, Dawnie Steadmanc, Daniel J. Wescottd and Kelly Knudsone

a

School of Earth & Space Exploration (SESE), Arizona State University, Tempe, AZ 85287-

1404 b

Forensic Institute for Research and Education, Middle Tennessee State University, 1301 East Main

Street, Box 89, Murfreesboro, TN 37132 c

Department of Anthropology, University of Tennessee, 502 Strong Hall, Knoxville, TN 37996-

0720 d

Department of Anthropology, Texas State University, 601 University Drive, San Marcos, TX

78666 e

School of Human Evolution and Social Change, Arizona State University, Tempe, AZ, 85287

*corresponding author [email protected], ORCiD 0000-0002-5157-0068

Manuscript in submission to Forensic Chemistry

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Abstract: Stable isotope signatures of bioelements are utilized for geolocation of unknown human remains. Hair in particular can generate a high-temporal resolution record of recent travel history, providing critical investigative leads. However, systematic studies of law enforcement packaging materials and evidence packaging protocols are needed, including the full range of sample types and conditions anticipated in casework. Arm 1 of this study examined the impact of freezing storage on hair samples using the FBI’s recommended storage materials (paper, plastic) and Mesa Police Department’s evidence packaging guidelines for varying periods of freezing storage (three weeks, five months). Hair studied was from individuals of different ancestry, including cosmetic treatments (relaxer, dyes), and exposure to decomposition fluids outdoors for up to 10 months. Arm 2 evaluated longer-term storage, comparing hair stored in a desiccator to hair frozen at -20°C for up to four years. Samples and certified standards were anonymized and randomized during sample preparation. To prevent cognitive bias from influencing interpretations, unblinding of samples only occurred after data correction and reduction were complete. Both the experimental and longer-term storage studies demonstrated 13C,  15N, and

18O values had no significant offsets between frozen samples and those stored at room temperature. However, there were small systematic offsets (+2 to +3 ‰) in  2H values, with frozen samples being enriched in 2H compared to controls. In a minority of samples frozen for > six months,  2H offsets of > 9 ‰ were observed, an amount that could impact the interpretation of an individual’s geographic travel history. Keywords: hair, stable isotope analysis, geolocation, isotope ratio mass spectrometry, evidence packaging

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1.

Introduction Isotope ratios of bioelements are valuable in identifying unknown human remains by

providing geographic constraints on an individual’s birthplace and residence history. The hydrogen, oxygen, carbon, and nitrogen isotopic ratios in tissues such as teeth, bone, and hair reflect the local sources of water and food consumed during tissue production [1, 2, 3, 4, 5]. Stable isotope analysis of hair, which grows at a known rate, can constrain an individual’s recent travel history, providing critical investigative leads [6, 7, 8, 9, 10, 11, 12]. While region of origin prediction from isotope ratios has long been used in anthropology research [13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25], law enforcement investigations have additional considerations such as chain of custody and storage conditions, so archaeological techniques must be demonstrated to be fit-for-purpose for forensic applications within law enforcement protocols [26]. The goal of this research was to test the fidelity of stable isotope ratios of hydrogen (H), oxygen (O), carbon (C), and nitrogen (N) in hair samples after storage according to common United States law enforcement evidence packaging guidelines and materials. The causes of isotope variation and the investigative information gained depend on the isotope system examined, and the fidelity of each isotope system during sample storage must be examined independently [26, 27]. Oxygen and hydrogen isotopes vary spatially on a global scale due to mass-dependent fractionation processes during evaporation and precipitation. Oxygen is probably the most commonly used stable isotope system, as oxygen isotope signatures can predict latitude of origin, as modified by altitude, distance from source water body and local water management practices [28, 29, 30, 31]. Oxygen isotopes are also impacted by metabolic reactions, which produce a substantial, but known, offset between drinking water and hair [8, 32, 33]. Hydrogen isotopes are more problematic, in part because of analytical challenges due to

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exchangeable hydrogen equilibrating with the humidity of the laboratory in which they are measured [34, 35]. In addition, the metabolic pathways of hydrogen incorporation into the keratin protein are more complex, and include additional sources from diet that are not represented in oxygen [32, 33]. Carbon isotopes reflect dietary intake, specifically the relative proportion of carbon from C3 (most vegetables, rice) or C4 (corn, sugar cane) photosynthetic mechanisms in an individual’s diet. Nitrogen isotopes reflect the amount and type of protein consumed [36, 37, 38]. If the isotope signature of hair present during sample collection is not preserved after storage, an investigation could mistakenly target the wrong geographic region for an individual’s history. Likewise, if sample reanalysis is required, any changes occurring during storage could endanger the court’s confidence in the analytical results. The fidelity of stable isotopes during storage can be compromised by numerous factors, including temperature, humidity, packaging materials used or duration of storage, while sample condition prior to storage may make some samples more susceptible to isotopic alteration than others. Furthermore, ancestry, genetic differences, and age impact many aspects of hair structure including the proportion of cortex, medulla, and cuticle; the abundance, type, and distribution of pigment granules; and physical properties such as hair swelling and cuticle integrity [39, 40]. Compounds such as explosives, drugs, and dyes can be differentially absorbed into hair depending on the ancestry of the hair donor [41, 42, 43, 44, 45, 46, 47], although some of these results are controversial [44, 48, 49]. Cosmetic hair treatments including straightening, bleaching, dyeing, and blow-drying compromise the integrity of hair making it potentially more susceptible to isotopic exchange. Dyeing has been shown to compromise the fidelity of  13C and  15N values in keratin during burial [50].

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Previous work in hair isotope analysis has typically involved well-preserved mummies in an archaeological context [51], or modern barbershop samples in a modern context [6, 8, 52]. Research on stable isotopes in human hair in association with decomposition fluids and environmental exposure has only recently been explored [53]. Studies on decomposed keratin samples have generally focused on morphological indicators of taphonomy, not on stable isotope preservation [54, 55, 56, 57, 58, 59, 60]. However, studies of wool keratin preservation during wet burial conditions showed significant change in hydrogen and oxygen isotopes, more modest changes in carbon and nitrogen isotopes at 80°C and elevated pressures [50], and large changes 87

Sr/86Sr ratios in buried samples [61], and found evidence for both kinetic and equilibrium

isotope exchange, highlighting the potential for isotopic changes depending on environmental and storage conditions. A previous study of isotope preservation during storage of human hair and nails found common storage materials and conditions can alter the isotope ratio of samples [27]. Fraser et al [27] examined hair and nails from a single individual, stored for either six weeks or six months in five different types of packaging materials including plastic, paper, tinfoil, and glass. They proposed that isotope exchange could occur because papers contain clay coatings or carbonate or acrylic stiffeners while plastic containers have plasticizers that may react with protein samples such as hair and nails. In addition, they concluded failure to properly seal samples may have led to inconsistent isotopic results with  15N values slightly higher in hair and nail samples stored for six weeks, but not for those stored for six months [27]. They observed no significant differences between controls and stored samples in  18O and  2H values, and the variation in

13C was within the expected range for a single individual over time.

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Standard protocol in studies of modern human hair of stable isotopic analysis of bioelements involves storage in a desiccator at room temperature [6, 8, 34, 35, 62], although some studies have been conducted following storage at room temperature in the dark [27]. However, most forensic case work comes from samples collected by law enforcement and evidence packaging protocols have never been validated as suitable for subsequent isotope analysis of hair. To identify an unknown individual, law enforcement will first investigate contextual information such as personal property, fingerprints, DNA, and a forensic anthropological profile. Stable isotopes will only be conducted if more conventional techniques have failed. Fraser et al [6] recommended glass storage containers for stable isotope analyses from hair and nails, but law enforcement guidelines for biological samples recommend paper to reduce moisture if hair is to be analyzed for DNA [63, p.19] or sealed plastic packaging for remains with fresh tissue [63, p.9]. Hair samples are typically frozen, both in casework of individual decedents as well as mass disasters. Hence, it would be beneficial to test any potential storage effects using the packaging that law enforcement and disaster relief organizations currently use. Several potential mechanisms can cause isotope ratios to shift during storage at sub-zero temperatures. First, freezing temperatures produce a very low humidity environment. Welldocumented issues exist with hydrogen isotopes if careful co-equilibration of samples and standards with local laboratory humidity is not performed prior to analysis [26, 35, 62, 64, 65]. Second, the process of freezing may cause structural damage to hair, making it more susceptible to isotopic exchange upon thawing. Third, if hair is frozen in association with decomposition fluids and soil, ice crystals can disrupt the structural integrity of the keratin protein and facilitate isotopic exchange between hair and fluids. There are no known studies of stable isotope

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preservation during freezing, as this has not been a significant concern in an archaeological context. Although case reports suggest that law enforcement packaging and storage may produce samples with accurate pre-storage isotope ratios [1, 2, 3], storage conditions for samples are frequently not described and more systematic validation is needed. To address these various concerns, this study examines stable isotope fidelity of storage of hair samples with diverse ancestry, cosmetic treatments, and exposure to decomposition fluids and insect activity. Experimental samples were stored at -20°C for three weeks or five months, and additional samples were analyzed to evaluate the effect of long-term storage up to four years. Packaging materials used were consistent with current law enforcement materials and evidence packaging protocols. This study also hopes to increase investigator awareness of the potential utility of stable isotope geolocation.

2. Material and methods The first arm of the study used a diverse selection of hair samples, obtained from local Phoenix area hair salons, intake samples from Anthropology Research Facility (ARF) at the University of Tennessee (Knoxville, TN), and samples exposed to decomposition and outdoor environmental conditions at both ARF and the Forensic Anthropology Research Facility (FARF) at Texas State University (San Marcos, TX). Each sample was divided into five aliquots. The treatment conditions for the aliquots included: A) a control sample immediately processed and stored in a desiccator, storage at -20°C, and experimental samples stored in B) a plastic clamshell container for three weeks and C) for five months, D) in butcher paper for three weeks and E) for five months. The specific packaging materials were selected after consultation with members of the Mesa, Arizona Police Department’s Crime Scene Unit regarding the collection and 7

preservation policies and procedures for hair discovered during forensic case work. Each frozen sample was subsequently packaged and sealed in accordance with Mesa Police Department’s Evidence section guidelines. Arm 2 of the study examined the impact of longer-term storage using paired samples from seven individuals at the University of Tennessee’s Anthropology Research Facility (ARF). Samples collected at donor intake were divided into two aliquots and placed in manila coin envelopes. One aliquot was stored in a desiccator, and the other was frozen, according to standard protocols at ARF. The storage periods ranged from 9 months to 4.1 years. To reduce any potential researcher bias, each sample aliquot was assigned a sample identification number with a random number generator prior to storage. The samples were then stored, cleaned, and processed blind, and all data reduction was performed without knowledge of the sample’s association with either control samples or other aliquots from the same sample. Only after all data analysis and correction were complete were data from sample aliquots reassociated. 2.1. Experimental sample selection Twenty samples were selected to maximize the range of sample types expected in the context of a forensic investigation (Table 1, Figure 1). These included pristine samples of African ancestry (n = 6), Asian ancestry (n = 2), and European ancestry (n = 12). Some samples also had cosmetic treatments (n = 7) including color treatment, with or without the use of relaxer. Salon samples (n = 14) were collected at four different retailers, catering to clientele of different ancestry (XanderLyn Salon and Hair Transformation in Scottsdale, AZ, Hùng’s Hair Design in Mesa, AZ, and a SuperCuts in Tempe, AZ). Ancestry of salon samples was assigned from morphology and visual appearance of the hair in combination with the demographics of the 8

observed clientele of the salon, but no information about the specific donors was available and ancestry was not validated. Six additional samples were collected from two human decomposition facilities (ARF and FARF) after unprotected exposure to the outdoor environment in association with decomposing human remains. Samples were selected to prioritize samples that had sufficient material to divide into five aliquots, as well as to maximize the diversity of samples in terms of climate, time of environmental exposure, and cosmetic treatment. Ancestry for individuals from the decomposition facilities was taken from the self-identification listings on donor forms prepared prior to death. Due to demographic constraints associated with donor selfselection, all samples with environmental exposure in proximity to decomposed remains were from individuals of European ancestry. Table 1. List of samples used in the study.1

sample identifier

Arm of study1

Source

Ancestry

known cosmetic treatment2

duration of environmental exposure3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

salon salon salon salon salon salon salon salon salon salon salon salon salon salon ARF ARF FARF

African African African African African African Asian Asian European European European European European European European European European

none none dyed relaxer + dyed relaxer + dyed relaxer none none dyed dyed dyed none none none dyed dyed none

1 day 5 days4 10 months

9

18 19 20 21 22 23 24 25 26 27

1

1 1 1 2 2 2 2 2 2 2

FARF FARF FARF ARF ARF ARF ARF ARF ARF ARF

European European European European European European European European European European

none none none none none none none none none none

1 week 1 week5 6 months -

All samples in Arm 1 of the study had control samples that were never frozen (Treatment A) and

were compared to four aliquots from the same sample that were frozen in different conditions (Treatments B-E). All samples in Arm 2 had matched aliquots split at the time of collection and either frozen at -20°C or stored at ambient temperature in a desiccator. 2Cosmetic color treatment was assumed if a strong color banding was visible near the root end, or if the hair appeared an unnatural color. Cosmetic relaxer treatment was determined by visual textural analysis. There may be additional cosmetic treatments to samples listed as no treatment if they could not be easily visually recognized. 3Samples that underwent environmental exposure were air-dried at the respective decomposition facility and stored at ambient temperature in dark conditions for up to six months until the beginning of the study. Samples were not cleaned prior to freezing with the exception of physical removal of the majority of maggots, to more closely simulate forensic casework conditions. No additional precautions were taken to prevent fungal or bacterial growth. 4Samples 15 and 16 were from the same individual, with sample 16 collected after several days of heavy rain. 5Samples 18 and 19 were from the same individual and collected at the same time. Sample 18 was sampled from one side of the head and appeared

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relatively clean and dry, while sample 19 was from the other side of the head and was immersed in a mixture of decomposition fluid and mud, with significant insect activity present.

[INSERT FIGURE 1 HERE] Figure 1. Photographs of some hair samples used in the freezing validation study, illustrating the diversity of hair condition and texture used. Photo A is of a sample from an elderly female donor at FARF, collected at intake. Photo B is from a FARF donor, one week after placement. Photo C is from a FARF donor, six months after placement. Photo D is a modern sample from a salon with a clientele of predominantly African ancestry.

2.2. Storage conditions for experimental samples (Arm 1) Samples were photographed before being divided into aliquots for experimental storage conditions. Samples (including control samples) were not cleaned prior to storage, so samples from decomposition facilities were frozen with dirt, soil, and dried decomposition fluids intact. Samples were packaged in either plastic clamshell boxes or white butcher paper. The plastic clamshell boxes (2” x 1 ¾”) are standard issue in the Mesa Police Department Crime Scene Unit and are commonly used to package small evidence items such as cartridge casings or cigarette butts. The white butcher paper is commonly used to preserve trace evidence on fabrics or clothing and was folded in a pharmacist’s fold to enclose the hair. The paper or plastic container was then put in a manila evidence envelope (9” x 6”) and sealed with evidence tape before being placed in a –20°C freezer for the specified time (Figure 2). A control aliquot of each sample (Treatment A) for each sample was stored at room temperature in butcher paper without

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the evidence envelope outer enclosure and cleaned, ground, and weighed in parallel with samples.

[INSERT FIGURE 2 HERE] Figure 2. Photograph of samples packaged according to local law enforcement evidence packaging guidelines in preparation for storage at –20°C. 2.3. Selection and storage for ARF samples (Arm 2) The experimental samples in the first arm of this study were intended to evaluate the stability of isotope ratios of hair samples for up to five months of storage. However, many forensic case examples may have been stored for much longer periods prior to submission for isotopic analysis. To study the fidelity of longer-term freezing storage, we compared paired aliquots from 10 individuals collected during intake stored in a desiccator and frozen from 8.7 months to 4.1 years. Existing protocol at the University of Tennessee’s Forensic Anthropology Center involves collecting two separate aliquots of hair during donor intake to maximize opportunities for future research. One portion is stored in a manila coin envelope in a desiccator, and the other is preserved for potential DNA analysis in a manila coin envelope at –20°C. Due to the inherently limited amount of the available potential samples, this study’s requirements substantially depleted the available samples for these individuals from the ARF collection. In order to preserve samples for future studies, only seven samples that had the largest amount of material available were selected for study inclusion. There were no known cosmetic treatments for any of the ARF samples, and all were from individuals of European ancestry. 2.4. Mechanical and chemical cleaning

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After removal from storage, samples from the decomposition facilities were mechanically cleaned by separating hair strands from any adhering soil or insect materials using a combination of tweezers and gentle rubbing with a clean Kimwipe. Samples were then placed in 50 mL glass beakers, and sufficient 2:1 chloroform to methanol cleaning solution was added to cover the sample [7]. Samples were then sonicated for 10 minutes, and the cleaning solution was decanted and discarded. This process was repeated at least twice, until the cleaning solution was clear and free of particulates. Samples were air-dried in a Class 100 laminar air-flow exhaust hood. To homogenize samples and prevent difficulties with static electricity, samples were ground in a SPEX Certiprep liquid nitrogen ball mill using a stainless-steel impactor and a plastic housing following the manufacturer’s guidelines for grinding of hair. Impactors and housing were cleaned between uses with repeated rinses using 18 M water and a flexible cleaning tool. Impactors were cleaned with 100% ethanol between uses. All cleaning, grinding, and capsule preparation were completed at the W.M. Keck Foundation Laboratory for Environmental Biogeochemistry (KFLEB) at Arizona State University. 2.5. Carbon and nitrogen isotope analyses Data are expressed as parts per thousand relative to a standard, using the equation:

with the international standard VPDB used for carbon isotope normalization [66, 67]. Nitrogen isotopes are similarly expressed as 15N values, using the standard AIR.

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Sample analysis was completed at the Stable Isotope Facility (SIF) at the University of California, Davis. A PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK) was used to measure 13C/12C and 15N/14N isotope ratios of ground hair. Hair samples of 1.06 mg ±5% were combusted in a tin capsule at 1000°C, while the reduction reactor was maintained at 650°C. N2 and CO2 were separated on a Carbosieve GC column at 65°C prior to introduction to the IRMS. Two-point scale normalization to stretch and shift values used standards G-18 Nylon 5 (known: ∂13C -27.72 ‰;  15N -10.3 ‰; measured:  13C -27.72 ± 0.05 ‰;  15N -10.31 ± 0.12 ‰) and G-21 Enriched Alanine (known:  13C +43.00 ‰;  15N +41.00 ‰; measured:  13C +43.02 ± 0.05 ‰;  15N +41.13 ± 0.08 ‰) as isotopic scale anchors, as they bracketed the isotopic range of samples [68, 69]. Analytical standards used for quality control included G-13 Bovine liver (known: 13C -21.7 ‰; 15N +7.72 ‰; measured:  13C -21.72 ± 0.06 ‰;  15N +7.73 ± 0.05 ‰), and G-20 Glutamic Acid (known:  13C -16.7 ‰;  15N -6.8 ‰; measured:  13C -16.63 ± 0.09 ‰;  15N -16.64 ± 0.09 ‰) (Table S1 and [70]). Since samples were measured at SIF, additional quality control was conducted to ensure that no artifacts were introduced due to sample weighing at the KFLEB facility. This included measuring two certified hair standards (USGS 42, Tibetan Human Hair and USGS 43, Indian Human Hair) and two in-house hair standards (ASU H Std 1 of African ancestry, Arizona and ASU H Std 2 of probable European ancestry, Arizona) as anonymized samples randomly interspersed with the experimental samples. The two in-house standards were selected to be materially and isotopically similar to the study samples. While USGS 41 and 42 are similar in elemental composition and protein structure to the study sample, they are isotopically quite different from the study samples. The use of in-house standards provided quality control on

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weighing, encapsulation, and handling processes from KFLEB, as well as ensuring that data reduction was completed without optimization for the blinded standards. The in-house standards had previously been analyzed at four independent laboratories in parallel with USGS 42 and 43 as anonymized samples. Complete measured values and comparison with certified values for USGS 42 and 43 and previously determined values H Std 1 and 2 for quality control for 13C and

15N values are listed in Table 1 of the supplemental material and at [70]. Ten percent of all samples were analyzed in triplicate, and the larger of the measured standard deviation of replicates or the median standard deviation of sample external reproducibility was used as sample error. The median standard deviation for triplicate samples for 13C values was 0.054 ‰, and the maximum was 0.096 ‰ (n=12). The median standard deviation for triplicate samples for 15N values was 0.049 ‰, and the maximum was 0.185 ‰ (n=12; Table S6). Precision (u(Rw)) was 0.17 ‰ for symbol13C and 0.21 ‰ for  15N measurements, based on the repeated measurements of calibration standards, check standards, and sample replicates and following the calculations of Szpak et al [71]. Accuracy (u(bias)) was 0.15 ‰ for  13C and 0.13 ‰ for  15N measurements, as calculated from the difference between the measured and known  values for the check standards. Using the equations in [71], the standard uncertainty (uc) is 0.20 ‰ for  13C and 0.23 ‰ for  15N measurements. Calculation of the analytical uncertainty is included in Table S7. 2.6. Oxygen isotope analyses Cleaned, powdered samples of 0.9 mg ±10 % were weighed into silver capsules for 18O measurement at KFLEB for measurement of 18O values at SIF. An Elementar PyroCube TC/EA (Total Combustion/ Elemental Analyzer; Elementar Analysensysteme GmbH, Hanau, Germany)

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interfaced to an IsoPrime VisION (Isoprime Ltd., Stockport, UK) was used to measure 18O values. Samples were decomposed to CO at 1400°C, and N2 was removed with an adsorption trap. To minimize water adsorption that could impact the analyses, samples were stored in a vacuum desiccator for at least three days prior to analysis, while the TCEA carousel is heated to 75°C. All samples and standards were treated in the same manner, following the principle of identical treatment. Two-point scale normalization to stretch and shift values to the VSMOW scale used standards IAEA 600 Caffeine (known  18O: -3.5 ‰, measured: -3.48 ± 0.12 ‰) and USGS 35 Sodium Nitrate (known  18O: +57.5 ± 0.6 ‰, measured: +56.8 ± 0.16 ‰) as isotopic scale anchors. Alanine was used for correction of the isotope composition for variations in the area under the curve and to calculate the elemental concentration. A nylon standard was used for drift correction by taking the difference from average for all the drift references throughout the run and applying the corresponding regression to all the sample data. USGS 42 Tibetan Human Hair (certified value +8.56 ±0.10 ‰, measured value of USGS 42 = +9.02 ±0.61 ‰) and USGS 43 Indian Human Hair (certified value +14.11 ±0.10 ‰, measured value of USGS 42 = +13.81 ± 1.13 ‰), weighed and encapsulated at SIF were run as analytical quality control; the relatively large error was caused by a weighing error that resulted in USGS 42 and 43 being smaller than the lowest sized calibration standard for one rack of analyses. An additional layer of quality control was utilized by providing USGS 42, USGS 43, ASU H Std 1, and ASU H Std 2 weighed and encapsulated at KFLEB as blind samples randomly interspersed with samples. These were in good agreement with known values (USGS 42 = +8.71 ±0.24 ‰, n=9; USGS 43 = +14.26 ±0.33 ‰, n=9; ASU H Std 1 +10.27 ±0.09 ‰, n=6; ASU H Std 2 = +10.55 ±0.32 ‰, n=6); see Table S2 or [70] for complete QA/QC results. According to

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an independent t-test, there was no significant difference between the means for USGS 42 and 43 encapsulated at SIF and KFLEB (see Supplemental section S1 for details of t-test results). The median standard deviation for triplicate samples for 18O was 0.41 ‰, and the maximum was 1.64 ‰ (n=12; Table S6). Precision (u(Rw)) was 0.30 ‰ for  18O measurements, based on the repeated measurements of calibration standards, check standards, and sample replicates and following the calculations of Szpak et al [71]. Accuracy (u(bias)) was 0.60 ‰ for

18O measurements, as calculated from the difference between the measured and known ∂ values for the check standards. Using the equations in [71], the standard uncertainty (uc) is 0.69 ‰ for

18O measurements. Details of the calculation of the analytical uncertainty is included in Table S7. 2.7. Hydrogen isotope analyses Cleaned, powdered samples of 1.22 mg ±10 % were weighed into silver capsules for 2H measurement at SIF. Aliquots for 2H measurement were independent from those for 18O measurement. Samples were co-equilibrated with keratin standards for at least 96 hours before analysis, as keratin is well known to have significant equilibration with local water vapor [26, 35, 62, 64, 65]. Samples were thermally decomposed to H2 at 1450°C in an elementar PyroCube TC/EA (Total Combustion/ Elemental Analyzer; Elementar Analysensysteme GmbH, Hanau, Germany) interfaced to an IsoPrime VisION (Isoprime Ltd., Stockport, UK). A polyethylene laboratory standard was used for correction of the isotope composition for variations in the area under the curve and to calculate the elemental concentration, while a lab keratin standard (trk, -39.3 ±2.7 ‰, n=121 during the analysis of standards) was used for drift correction as detailed above. Samples were isotopically normalized to matrix-matched certified standards

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USGS 42 and 43 (measured value of USGS 42 = -72.9 ±0.3 ‰, n=7, 1; certified value -72.9 ±2.2 ‰; measured value of USGS 43 = -44.4 ±0.4 ‰, n=7, 1; certified value -44.4 ±2.0 ‰). Values were referenced to VSMOW-SLAP. The United States Geological Survey released revised certificates for 2H values for USGS 42 and 43 on August 29th, 2016, and all data was normalized to the revised values as listed above. Because samples were encapsulated in Arizona and analyzed in California, additional aliquots of USGS 42 and 43 were also encapsulated at KFLEB, anonymized, and randomly interspersed with samples. This allowed direct comparison of data for USGS 42 and 43 prepared at both facilities. Comparison of values encapsulated at both facilities ensured 1) that the coequilibration technique for samples that were already encapsulated prior to receipt by the analyzing laboratory was effective, 2) there was no isotopic offset between samples and analytical standards at SIF, 3) ensured that no cross-contamination or isotopic offsets due to sample weighing, encapsulation, and packaging at KFLEB occurred, and 4) finally, ensured that data reduction procedures for the analytical run were not optimized around known certified standards, potentially at the expense of samples. Hence, providing USGS 42, USGS 43, ASU H Std 1, and ASU H Std 2 as blind samples randomly interspersed with samples was critical to ensure accurate and precise 2H values, and a relatively unique aspect of this study. The blinded standards (USGS 42 = -73.6 ±2.8 ‰, n=6, 1; USGS 43 = -45.1 ±3.6 ‰, n=6, 1) were in good agreement with both known values and in-run analytical standards for USGS 42 and 43 and with consensus values for ASU H Stds 1 and 2 (measured ASU H Std 1 -69.7 ±2.3 ‰, n=6, 1; consensus value -67.5±1.1 ‰; measured ASU H Std 2 = -68.8 ±2.7 ‰, n=6, 1; consensus value of -67.5 ±1.3 ‰; see Table 2 in the Supplemental Material or [70] for complete QA/QC results). According to an independent t-test, there was no significant difference between the means for

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USGS 42 and 43 encapsulated at SIF and KFLEB (see Supplemental section S1 for details of test results). Ten percent of all samples were run in triplicate, and the larger of the standard deviation of replicates or the median standard deviation of sample external reproducibility was used as sample error. The median standard deviation for triplicate samples for 2H was 3.86 ‰, and the maximum was 4.76 ‰ (n=12). Precision (u(Rw)) was 2.36 ‰ for 2H measurements, based on the repeated measurements of calibration standards, check standards, and sample replicates and following the calculations of Szpak et al [71]. Accuracy (u(bias)) was 2.18 ‰ for 2H measurements, as calculated from the difference between the measured and known  values for the check standards. Using the equations in [71], the standard uncertainty (uc) is 3.21 ‰ for 2H measurements; only standards and samples of hair keratin were used in calculating the standard uncertainty to most closely represent the uncertainty expected for samples. Details of the calculation of the analytical uncertainty is included in Table S7. 2.8. Statistical analyses Statistical analyses were completed using SPSS 24 (IBM Corporation, USA). To compare the effect of storage treatment, a repeated measures ANOVA with Bonferroni post-hoc tests was used to analyze any differences between the control and the four frozen treatments for the experimental samples. Due to the limited number of samples, the paired hair samples from the ARF were compared using the Wilcoxon Rank Sum test. 3.

Results and Discussion

19

Two storage experiments were carried out to examine both the effect of packaging materials on A) a week-to-month time scale (“Arm 1”) and B) on longer-term storage over years (“Arm 2”). 3.1. Experimental results The complete analytical measurements for samples are presented in Supplemental Table 3 and are also freely available at Mendeley Data [70]. Results are graphically illustrated in Figure 3 for the measured values of 13C, 15N, 18O, and 2H values, and for the offset between treatment and controls for 13C, 15N, 18O, and 2H values in Figure 4.

[INSERT FIGURE 3 HERE.] Figure 3. Each sample is shown with the storage treatments, in the following order: no storage, plastic clamshell for two weeks, plastic clamshell for five months, butcher paper for two weeks, butcher paper for five months, and sample mean. Data shown include measured values for A) 13CVPDB; B) 15NAIR; C)

18OVSMOW; and D) 2HVSMOW. Dotted lines are samples that underwent environmental exposure in either Texas or Tennessee prior to freezing. Solid lines are anonymized samples from salons in Arizona, although the travel history of the individuals sampled is unknown. Dashed lines are samples that had some form of cosmetic treatment (dyed, relaxer, or both) prior to freezing. Lines with both dashes and dots were dyed, but also underwent outdoor environmental exposure. Larger open symbols are outliers, as evaluated by points greater than 1.5 box-lengths from the edge of the box in box and whisker plots of the population. The standard uncertainty (uc) is 0.25 ‰, 0.18 ‰, 0.67 ‰, and 3.21 ‰ for 13CVPDB,

15NAIR, 18OVSMOW; and 2HVSMOW, respectively (Table S7).

[INSERT FIGURE 4 HERE.] 20

Figure 4. Differences between treatment and control for A) 13CVPDB; B) 15NAIR; C) 18OVSMOW; and D)

2HVSMOW. Dotted lines are samples that underwent environmental exposure in either Texas or Tennessee prior to freezing. Solid lines are anonymized samples from salons in Arizona, although the travel history of the individuals sampled is unknown. Dashed lines are samples that had some form of cosmetic treatment (dyed, relaxer, or both) prior to freezing. Lines with both dashes and dots were dyed, but also underwent outdoor environmental exposure. Larger open symbols are outliers, as evaluated by points greater than 1.5 box-lengths from the edge of the box in box and whisker plots of the population. Lines that fall above the horizontal line denoting zero are isotopically enriched in the heavy isotope in treatments compared to controls.

To more rigorously evaluate if plastic clamshell or butcher paper packaging induced an isotope change in the samples, the repeated measures one-way ANOVA was analyzed comparing the means for ∂13C, ∂15N, ∂18O, ∂2H values and weight percent C, N, O, H, and the C/N ratio, as well as examining for an interaction between ancestry (African, Asian, European), hair source (salon or decomposition facility), hair treatment (none, dyed, relaxer ±dyed) and storage. Repeated measures one-way ANOVA was used to avoid conflating the variation in the baseline values which significantly expanded the standard deviation for the population of 20 samples. For individual 9, the weight percent C and N were significantly different compared to the rest of the samples. However, the C/N ratio and the weight percent H and O (run on different capsules) were similar, so the most probable explanation was there was some loss of material from the capsule prior to measurement. The weight percent C and N for this individual were excluded from the statistical analysis.

21

Because elemental abundances have not been shown to be useful in geolocation, we do not further discuss the elemental abundances in the statistical considerations except in a basic way. However, having the elemental abundances may allow us to consider alternate mechanistic explanations for any changes in the isotope ratios, so this data is included in the tables. Prior to running the ANOVA, the data was examined for both outliers using boxplots and normality using Shapiro-Wilk test of normality, in two ways. The first was to look at the raw data, using a within subject variation of treatment / control. The second was to examine the differences between treatment and control. Inspection of a boxplot for values greater than 1.5 box-lengths from the edge of the box, revealed outliers for individuals 18 and 19 for 15N values in treatment B, while all other isotope measurements in the raw data had no outliers. For the differences between treatment and control, outliers included individual 5 for 13C values for treatment B – control and treatment C - control, individual 7 for 15N values for treatment C – control, individual 20 for 15N values for treatment D – control, individual 4 and 17 for 18O values for treatment B – control, individual 15 for 18O values for treatment C – control, individual 9 for 18O values for treatment D – control, individual 20 18O values for treatment E – control. Inspection of a boxplot for values greater than 3 box-lengths from the edge of the box, revealed an extreme outlier for individual 5 for 13C values for treatment B minus control. As indicated by the Shapiro-Wilk test of normality, treatment B was not normally distributed for the ∂13C value (p = 0.028), and  2H value (p = 0.049). For differences between controls and treatments, 13C values was not normally distributed for treatment B minus control

22

(p = 0.008) and treatment C minus control (p = 0.012). All other treatments and isotope measures for either the absolute value or the difference from control were normally distributed. There are several ways of dealing with outliers and non-normal distributions. The simplest is to exclude the outliers, but this can be problematic unless a specific reason can be shown why some points should be excluded. Another option includes running the non-parametric Friedman test, which is less effected by outliers and non-normal distribution. Alternatively, the dependent variable can be transformed to reduce outliers and make a more normal distribution or modifying the outlier value with one that is less extreme, although it maintains the data’s position in the data rankings [72]. Finally, the one-way ANOVA can be run if it can be show it does not substantially alter the conclusions. We compared these methods and concluded that there was not a substantial difference between them. Details of the findings of the nonparametric Friedman test are presented in the Section S3 of the Supplemental Material, while the results of the one-way repeated measures ANOVA with outliers removed are shown in Section S4 and Table S8 of the Supplemental Material. Including the outliers, Mauchley’s test for sphericity indicated that the assumption of sphericity had not been violated for  13C,  15N,  18O,  2H and weight percent O and H. However, the assumption of sphericity was violated for weight percent N (2(9) = 24.425, p = 0.004) and C/N ratio (2(9) = 20.126, p = 0.019). The Greenhouse-Geisser epsilon () was <0.75 (0.623, and 0.451, respectively), and was used to correct one-way repeated measures ANOVA for weight percent N, and C/N ratio, while no sphericity correction was used in reporting the results for all other measures as recommended by Field [73] and Howell [74]. The storage treatments did not produce any statistically significant isotopic differences for  13C (F(4, 44) = 2.244, p = 0.080),  15N (F(4, 44) = 0.397, p = 0.81), and  18O (F(4, 44) = 23

0.800, p = 0.532). There was a statistically significant difference for  2H (F(4, 44) = 2.713, P = 0.042, partial 2 = 0.169), There was no statistically significant effect of storage on ancestry (African / Asian / European), source (salon / ARF / FARF), or initial hair condition (none / dyed / relaxer ±dyed / decomposition facility), although the study is underpowered for determining significance for some subgroups. In post-hoc tests, Tukey HSD showed that there was a statistically significant difference between the salon samples and the decomposition facility samples in  13C. This is likely related to the diet and geographic residence of the individuals, as all the salon samples were from Arizona while the decomposition facility samples were from Tennessee and Texas. However, the weight percent hydrogen did not significantly change on average between treatment and control, suggesting that isotopic exchange between the storage environment and the sample occurred. This isotopic offset persisted despite co-equilibration of samples and standards in the analyzing laboratory, a protocol designed to ensure that easily exchangeable hydrogen is reset to local humidity, suggesting that the isotopic changes are beyond the easily exchangeable and are persistent. Aliquots of USGS 42 (n=8) and 43 (n=7) were prepared at KFLEB and sent as blind unknowns to the measuring laboratory. These aliquots were isotopically indistinguishable from the normalizing aliquots of USGS 42 and 43 prepared at SIF (see Supplemental Section S1). The samples were analyzed up to six months after sample preparation and storage in a desiccator, indicating that isotopic exchange is irreversible on this time scale. The offset was typically within or very close to one standard deviation of the change within the population. The average isotopic offset of 2-3 ‰ is small on the scale of expected natural variation and would not have changed the geographic interpretation of a forensic case. 24

There was no systematic difference in isotope values during freezing between the plastic clamshell and butcher paper conditions, or between three weeks and five months. This suggests the hydrogen isotopic exchange occurred relatively soon after initiation of freezing. Although the isotopic exchange rates decrease at lower temperatures, the magnitude of the isotopic fractionation typically increases. Although the majority of the hydrogen isotope exchange may well occur during the initial cooling period, isotope exchange may continue at slower rates – but larger magnitudes - over even longer periods. Previous work found no hydrogen isotopic offset in hair and nails stored at room temperature for up to six months in various packaging materials [27]. This is in contrast to our result of small, but systematic biases in hydrogen isotopes after freezing, suggesting the conformation of the keratin protein may be irreversibly modified during freezing. Table 2. Means and standard deviations by storage condition1

 13C

wt%C

 15N

wt%N

C/N

 18O

wt%O

 2H

wt%H

Mean

-17.63

46.07

+9.26

14.97

3.08

+11.28

22.52

-63.1

5.59



0.89

0.82

0.55

0.53

0.09

1.57

0.57

9.0

0.10

Plastic clamshell, 3 weeks (“B”)

Mean

-17.66

45.87

+9.24

14.99

3.06

+11.24

22.26

-59.7

5.47



0.95

0.89

0.64

0.42

0.10

1.60

0.46

9.3

0.14

Plastic clamshell, 6 months (“C”)

Mean

-17.60

44.81

+9.26

14.74

3.04

+11.20

22.03

-60.3

5.48

 Mean

0.91

0.62

0.56

0.39

0.07

1.65

0.92

10.2

0.15

-17.61

45.61

+9.27

14.92

3.06

+11.22

22.55

-60.6

5.53

 Mean

0.90

1.26

0.54

0.51

0.09

1.69

0.61

10.3

0.13

-17.60

44.42

+9.26

14.61

3.04

+11.20

21.89

-59.9

5.44



0.89

1.10

0.58

0.46

0.07

1.66

0.73

8.6

0.10

Storage condition Control (“A”)

Butcher paper, 3 weeks (“D”) Butcher paper, 6 months (“E”) 1

from the pairwise output in the one-way repeated measures ANOVA calculations.

25

Table 3. Means and standard deviations of the differences between controls and treatment samples by storage condition1

Storage condition

13C

wt %C

15N

wt %N

C/N

18O

wt %O

wt

2H

%H

Plastic clamshell, 3 weeks

Mean

+0.03

+0.24

+0.05

-0.05

+0.03

+0.07

+0.23

-3.32

+0.14*



0.19

1.20

0.18

0.32

0.07

0.28

0.60

4.70

0.15

Plastic clamshell, 6 months

Mean

-0.06

+1.31*

+0.10

+0.21*

+0.05

+0.11

+0.56

-3.05

+0.14

 Mean

0.11

1.11

0.14

0.34

0.04

0.35

0.82

4.76

0.13

-0.03

+0.39

0.00

+0.03

+0.02

+0.14

-0.04

-1.77

+0.06

 Mean

0.15

0.77

0.17

0.30

0.06

0.42

0.91

4.52

1.23

-0.04

+3.55

+0.02

+0.96

+0.05

+0.11

+0.59*

-3.63*

+0.15*



0.11

1.10

0.14

0.37

0.05

0.43

0.63

3.24

0.14

Butcher paper, 3 weeks Butcher paper, 6 months 1

from the pairwise output in the one-way repeated measures ANOVA calculations.  is the

standard deviation of the isotope difference between the treatment and control for the 20 samples. An asterisk and bold font indicate p <0.05.

However, the calculations above could conceal systematic differences in isotope change during storage for a subset of samples. For instance, samples that have been exposed to decomposition fluids, mold, insect activity, and an outdoor environment are known to have compromised structural integrity [54, 55, 56, 58, 59, 60] and might well preserve pre-freezing isotope composition more poorly than salon samples. To explore the effect of sample type on preservation, data were recalculated by binning by ancestry, cosmetic treatment, and decompositional exposure (Table 4). Salon samples of binned by ancestry had no statistically significant isotope or elemental offsets between treatment and control F(72, 226) = 0.708, p = 0.957, Wilks’  = 0.291, partial 2

26

= 0.143, although the limited number of samples (n=6, 2, respectively) cautions against a generalized interpretation. Note that all four treatments were binned, so for a grouping with six samples, there are 24 offsets for each dependent measure between treatment and control used in the calculations. Binning data by source (salon, ARF, FARF) did show a statistically significant isotope or elemental offsets between treatment and control F(36, 136) = 1.570, p = 0.034, Wilks’  = 0.273, partial 2 = 0.277, although binning by cosmetic or environmental exposure at a decomposition facility (none, dyed, relaxer ±dyed, environmental) F(72, 226) = 1.084, p = 0.324, Wilks’  = 0.165, partial 2 = 0.8202. Four of the groupings had changes in 2H significant at the p = 0.05 level, although within or very close to one standard deviation of the population. All the groupings with statistically significant changes in 2H were entirely or predominantly of European ancestry. The European ancestry treatment samples had 13C and 15N values slightly isotopically positive compared to the control samples. However, the changes in 13C and 15N values were small, and less than the standard uncertainty (0.20 ‰ and 0.23 ‰, respectively). Table 4. Means and standard deviations for isotope differences between frozen samples and controls by initial sample type1

13C

wt

15N

%C African ancestry, salon, n=6 Asian ancestry, salon, n=2 European ancestry, salon, n=6

wt%

C/N

18O

N

wt%

2H

O

wt% H

Mean

-0.05

-0.43

0.00

-0.08

-0.01

-0.06

-0.31

+1.48

-0.06



0.18

1.54

0.15

0.36

0.07

0.31

0.81

5.25

0.15

Mean

0.00

-1.03

0.00

-0.26

-0.02

-0.14

-0.34

+2.25

-0.07

 Mean

0.17

0.81

0.32

0.26

0.01

0.17

0.79

3.62

0.12

+0.10*

-1.22

+0.04*

-0.23

-0.03

-0.13

-0.03

+4.06*

-0.13

0.96

0.016 0.08

0.75

<0.0001 3.93

0.13



<0.0001 0.08

0.33

0.03

0.35

27

Decomposition facilities, n=6 Salon samples, no known treatment, n=7 Salon samples, dyed, n=4 Salon samples, relaxer ±dyed, n=3

1

Mean

+0.01

-1.05

-0.05

-0.14

-0.05

-0.06

-0.62

+3.34* <0.0001

+0.10

 Mean

0.10

1.02

0.15

0.41

0.07

0.49

0.72

3.47

1.13

+0.03

-1.07

+0.01

-0.22

-0.03

-0.05

-0.26

+3.40*

-0.11 0.15

 Mean  Mean 

0.12

1.07

0.19

0.35

0.03

0.27

0.78

0.0004 4.72

+0.10*

-1.22

-0.02

-0.23

-0.04

-0.26

-0.25

+3.29*

-0.11 0.13

0.0004 0.10

0.93

0.12

0.31

0.03

0.32

0.78

0.005 4.45

-0.13

+0.14

+0.08*

+0.05

0.00

0.00

+0.02

+0.27

-0.05

1.61

0.02 0.12

0.27

0.09

0.32

0.81

3.98

0.14

0.22

All treatments were included in the calculations, so for each sample, four isotopic offsets were

included in the calculations. Hence, for row entries with n=6 samples, 24 isotope offsets between treatment and control were used in the calculations. The African, Asian and European ancestry hair samples included only salon samples. Samples from decomposition facilities were all of European ancestry. An asterisk and bold font indicate p <0.05 in pair-wise comparisons. For p values <0.05, the p-value is shown underneath the mean difference. Note that these results should be interpreted with caution as the number of samples in some categories are limited.

3.2. Paired frozen-desiccator samples stored at ARF (Arm 2) Aliquots stored in manila coin envelopes in a desiccator were compared to aliquots frozen as described in Methods to evaluate the effects of storage on longer time scales. As a reminder, although these samples were collected at the Anthropology Research Facility at the University of Tennessee, they were all collected in the early postmortem period and had no outdoor exposure history. The complete elemental and isotopic results for the stored samples are

28

presented in Supplemental Table 5 and online [70]. The differences between frozen and desiccator aliquots are shown in Table 5. Frozen samples had more positive  13C and  2H values compared to desiccator samples at the p=0.05 level. The magnitude and direction of the isotope difference between frozen and room temperature samples were the same between the experimental salon samples from European ancestry (Table 4) and the results shown in Table 5. However, the 13C difference was less than one standard deviation for the population, similar to the analytical error. This would not change the interpretation for a forensic case [75]. The 2H difference was less than two standard deviations for the population and was larger than the instrumental error but has implications for forensic practice as discussed below. Inspection of a boxplot for values greater than 1.5 box-lengths from the edge of the box, revealed outliers for the samples stored for 0.7 years for 13Cfrozen-ambient and the samples stored for 0.9 years for 18Ofrozen-ambient. All isotope offsets between frozen and desiccator samples were normally distributed, as determined by Shapiro-Wilk test of normality. For elemental concentrations and ratios, inspection of a boxplot for values greater than 1.5 box-lengths from the edge of the box, revealed outliers for samples stored for 0.7 years in weight percent nitrogen (frozen-ambient) and the sample stored for 2.8 years in weight percent oxygen (frozen-ambient). Inspection of a boxplot for values greater than 3 box-lengths from the edge of the box, revealed an extreme outlier for the samples stored 0.7 years in weight percent carbon (frozen-ambient). As determined by Shapiro-Wilk test of normality, the difference between frozen and ambient samples in weight percent carbon was not normally distributed (p = 0.002), while all other elemental concentrations and ratios were normally distributed.

29

Because there was no a priori reason to exclude these outliers, a paired t-test was analyzed, with the results presented in Table 4. To evaluate the impact of the two outliers, the analysis was also run with the outliers removed, and those results are presented in the supplemental material. The same parameters that showed statistical significance during analysis including all data were the same parameters that showed statistical significance after outlier exclusion, and no additional parameters were statistically significant after outlier removal (Section S5). Data are mean ± standard deviation, unless otherwise stated. The only statistically significant isotopic difference between the frozen and ambient samples was in the 2H value. The frozen samples (-58.25 ±6.19‰) were enriched in 1H compared to the samples stored in a desiccator (-63.51 ±5.69‰), with a mean 2H value of +5.26‰ (95% CI, +1.99 to +8.52‰), t(6) = 3.94, p = 0.008, d = 1.48. For elemental concentrations and ratios, the carbon to nitrogen ratio had a statistically significant offset, with C/N ratios in the frozen samples (3.05 ±0.02) depleted in carbon compared to the ambient desiccator samples (3.09 ±0.02), with a mean C/N ratio of -0.05 (95% CI, -0.08 to -0.01), t(6) = -3.459, p = 0.013, d = 1.30. The hydrogen concentration in the frozen samples (5.30 ±0.10% H) was depleted in hydrogen compared to the ambient samples (5.45 ±0.09% H), with a mean difference in concentration of -0.15% (95% CI, -0.23 to -0.06% H), t(6) = -4.347, p = 0.005, d = 1.64. The O/H ratios in the frozen samples (4.37 ±0.10) also reflected the depletion in hydrogen compared to the ambient samples (4.23 ±0.08), with a mean offset in the oxygen to hydrogen ratio of 0.15 (95% CI, 0.05 to 0.25), t(6) = 3.572, p = 0.012, d = 1.35. In order to examine if storage time was a predictive factor in the changes between frozen and ambient samples, linear regressions of the different offset variables and storage time were 30

examined, although these should be interpreted with caution due to the limited number of samples. Several parameters appeared to have linear correlations with storage time, but later proved to not be statistically significant. Visual inspection of a scatterplot of 13C values and storage time indicated a linear relationship between the variables and there was independence of residuals, as assessed by a Durbin-Watson statistic of 2.104. Residuals were normally distributed as assessed by visual inspection of a normal probability plot. Storage time accounted for 49.0% of the variation in 13C values with adjusted R2 = 38.8%. However, storage time was not statistically significant predictor of 13C values F(1, 5) = 4.80, p = 0.080. Visual inspection of a scatterplot between 2H and storage time also indicated a linear relationship between the variables. There was independence of residuals, as assessed by a Durbin-Watson statistic of 1.881 and residuals were normally distributed as assessed by visual inspection of a normal probability plot. Storage time accounted for 22.4% of the variation in 2H values with adjusted R2 = 6.8%. However, storage time was not a statistically significant predictor of 2H values F(1, 5) = 1.44, p = 0.2849. Visual inspection of a scatterplot between the change in  between frozen and ambient samples and storage time also indicated a linear relationship between the variables. There was independence of residuals, as assessed by a Durbin-Watson statistic of 1.303 and residuals were normally distributed as assessed by visual inspection of a normal probability plot. Storage time accounted for 54.7% of the variation in the change inweight percent hydrogen between frozen and ambient samples with adjusted R2 = 45.7%. However, storage time was not statistically significant predictor of the change inweight percent hydrogen between frozen and ambient samples F(1, 5) = 6.04, p = 0.057. Visual inspection of the scatterplot between 15N, 18O values, the change in weight percent carbon, nitrogen, oxygen concentration, carbon to nitrogen ratio, and oxygen to hydrogen 31

ratio indicated no linear relationship with storage time. In addition, storage time was not a statistically significant predictor of any of these parameters using linear regression. For forensic case work, the 13C, 15N, and 18O values of frozen samples would all give similar geographic and dietary information compared to pre-stored samples.

Table 5. Elemental and isotopic differences between frozen and ambient samples from ARF1

wt

Years in storage

13C

4.1

+0.28

-1.11

+0.02

2.8

+0.18

-0.24

1.9

+0.22

1.6

wt%

-0.15

-0.05

+0.09

+0.55

+10.5

-0.30

0.34

-0.17

+0.31

-0.08

+0.51

-0.63

+4.8

-0.19

0.03

-0.79

+0.07

+0.10

-0.08

+0.26

+0.60

+4.1

-0.13

0.22

+0.04

-0.61

+0.29

-0.17

-0.01

+0.37

+0.01

+3.3

-0.08

0.07

1.0

+0.15

-0.79

-0.15

+0.14

-0.02

-0.24

+0.25

+3.7

-0.06

0.09

0.9

+0.12

-0.79

+0.11

+0.14

-0.08

-0.69

+0.21

+0.8

-0.06

0.09

0.7

-0.15

+1.96

+0.07

+0.66

-0.01

+0.08

+0.02

+9.7

-0.21

0.18

average

+0.12

-0.30

+0.03

+0.12

-0.05*

+0.07

+0.14

+5.25*

-0.15*

+0.15*

0.008

0.005

0.012

median

+0.15

-0.79

+0.07

+0.14

-0.05

+0.12

+0.21

+4.1

-0.13

+0.09



0.14

1.03

0.16

0.29

0.04

0.53

0.41

3.53

0.09

0.11

N

0.013

O

2H

wt%

18O

1

15N

wt%

C/N

%C

H

O/H

calculations using paired t-tests. An asterisk and bold font indicate the two-tailed significance

value p <0.05, while italics denotes an outlier.

3.3 The potential for inaccuracy in geographic origin prediction Forensic case work typically involves analysis of a single individual, rather than of large populations. The probability of an inaccurate prediction of geographic origins for a single

32

individual depends on A) the magnitude of isotopic offset needed to cause geographic misinterpretation and/or B) the variance in a local population. While both of these questions are outside the scope of the current work, considering the context of previous research is beneficial in evaluating the importance of the current findings. For example, if an individual was from central Montana, a +10 ‰ offset in  2H values due to freezing storage would predict someone was from as far south as northern Arizona or as far east as Minnesota (cf Figure 3A in Ehleringer et al [8]). Of the 80 treatment-control pairs in the experimental portion of the study, 48 had an isotopic difference of >5 ‰, while only one treatment-control pair had an isotopic difference of >10 ‰. Five of the seven samples stored for >0.7 years at ARF had differences <5.0 ‰, while the other two had 2H differences of <10 ‰. For samples with the largest isotopic offsets of >+5 ‰, a small but systematic bias was caused in the geographic interpretation of the individual’s travel history. In the United States, the bias would make it appear that an individual came from further south or east than they actually did [8]. The inaccuracy in the prediction of geographic residence depends on the isotopic gradient for a region. For areas with steep 2H isotope gradients, the inaccuracy will be less than areas with gradual isotope gradients. The inaccuracy is not simply imprecision but has a bias with frozen samples enriched in 2H compared to non-stored samples. Hence, for hair samples known to be frozen, it may be appropriate expand the error estimate to  measurements for minimum potential ∂2H values. Despite the publication of maps showing  2H variations in hair and drinking water [8], the majority of researchers in the field of stable isotope geolocation use oxygen, not hydrogen isotopes, for region of origin prediction. This is due to both analytical and metabolic 33

complicating factors that make hydrogen isotope signatures more challenging to interpret. This study supports the fidelity of oxygen isotopes for region of origin determinations. 4. Conclusion This study highlights the importance of validating storage conditions for stable isotope analysis. Law enforcement evidence packaging materials and protocols currently assume samples are being frozen for DNA analysis, with stable isotopes analysis of bioelements employed only if other investigative techniques are exhausted. In addition, when validating isotopic analyses for forensic purposes, it is critical to examine the range of sample types and conditions anticipated for case work. To prevent cognitive bias from influencing interpretations, samples and certified standards were anonymized and randomized at the beginning of sample preparation, and unblinding of sample identification occurred only after all data correction and reduction were complete. Within the constraints of the range of samples, storage materials, and storage time here investigated, 13C, 15N, and 18O signatures in hair are fit-for-purpose for geolocation and dietary constraints, providing valuable investigative leads in homicides and identification of human remains. 2H values in frozen hair samples may be systematically enriched in 2H by several per mil, and this effect must be considered when interpreting isotope data. Analytical artifacts due to evidence collection, storage, and analyses preceding stable isotope analyses of bioelements should be evaluated in coordination with law enforcement to ensure systematic studies are as relevant as possible to their intended purpose. Including all relevant stakeholders in research design makes the research more relevant and useful.

34

Acknowledgements: The authors appreciated permission to use evidence packaging materials from the Mesa Police Department. The authors gratefully acknowledge discussion of hair packaging techniques with the entire Crime Scene Unit, particularly Senior Crime Scene Specialists Christine Loewenhagen, Rebecca Winger, and Crime Scene Supervisors Kristal Kolhepp and Elizabeth Wiltrout at the Mesa Police Department. The authors appreciate salon samples provided by Gabrielle Lanoue of XanderLyn Salon in Scottsdale, Arizona, Michelle D. Knowles of Hair Transformation in Scottsdale, Arizona, SuperCuts in Tempe, Arizona, and Hùng’s Hair Design in Mesa, Arizona. This project was supported by Award No. 2014-DN-BXK002 funded by the National Institute of Justice, Office of Justice Programs, United States Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of the Department of Justice. The authors would also like to express gratitude for the detailed and highly constructive reviews from two anonymous reviewers.

References 1. 2.

3.

4.

5.

Meier-Augenstein, W. and I. Fraser, Forensic isotope analysis leads to identification of a mutilated murder victim. Science and Justice, 2008. 48: p. 153-159. Lehn, C., E. Mützel, and A. Rossmann, Multi-element stable isotope analysis of H, C, N, and S in hair and nails of contemporary human remains. International Journal of Legal Medicine, 2011. 125: p. 695-706. Rauch, E., et al., Origin assignment of unidentified corpses by use of stable isotope ratios of light (bio-) and heavy (geo-) elements - a case report. Forensic Science International, 2007. 168: p. 215-218. Font, L., et al., Provenancing of unidentified World War II casualties: Application of strontium and oxygen isotope analysis in tooth enamel. Science and Justice, 2014. 55(1): p. 10-17. West, J.B., et al., Isoscapes: Understanding movement, pattern, and process on Earth through isotope mapping. 2010.

35

6.

7. 8. 9.

10.

11.

12. 13.

14.

15.

16.

17. 18.

19.

20. 21. 22.

Chau, T.H., et al., Reconstruction of travel history using coupled δ18O and 87Sr/86Sr measurements of hair. Rapid Communications in Mass Spectrometry, 2017. 31: p. 583589. Tipple, B.J., et al., Isolation of strontium pools and isotope ratios in modern human hair. Analytica Chimica Acta, 2013. 798: p. 64-73. Ehleringer, J., et al., Hydrogen and oxygen isotope ratios in human hair are related to geography. Proceedings of the National Academy of Sciences, 2008. 105(8): p. 2788-93. Tipple, B.J., Isotope Analyses of Hair as a Trace Evidence Tool to Reconstruct Human Movements: Establishing the Effects of the “Human Ecosystem” on Strontium and Oxygen Isotope Ratios, D.o. Justice, Editor. 2016: Washington, DC. p. 91. Mant, M., A. Nagel, and T. Prowse, Investigating Residential History Using Stable Hydrogen and Oxygen Isotopes of Human Hair and Drinking Water. J Forensic Sci, 2016. 61(4): p. 884-91. Valenzuela, L.O., et al., Spatial distributions of carbon, nitrogen, and sulfur isotope ratios in human hair across the central United States. Rapid Communications in Mass Spectrometry, 2011. 25: p. 861-868. Valenzuela, L.O., et al., Dietary heterogeneity among Western industrialized countries reflected in the stable isotope ratios of human hair. PLoS One, 2012. 7: p. e34234. France, C.A.M., D.W. Owsley, and L.-A.C. Hayek, Stable isotope indicators of provenance and demographics in 18th and 19th century North Americans. Journal of Archaeological Science, 2014. 42: p. 356-366. Giblin, J.I., et al., Strontium isotope analysis and human mobility during the Neolithic and Copper Age: a case study from the Great Hungarian Plain. Journal of Archaeological Science, 2013. 40(1): p. 227-239. Katzenberg, M.A., Stable Isotope Analysis: A Tool for Studying Past Diet, Demography, and Life History, in Biological Anthropology of the Human Skeleton, M.A. Katzenberg and S.R. Saunders, Editors. 2008, John Wiley & Sons: Hoboken, New Jersey. p. 413-442. Blom, D.E. and K.J. Knudson, Tracing Tiwanaku Childhoods: A Bioarchaeological Study of Age and Identity in Tiwanaku Society, in Tracing Childhood: Bioarchaeological Investigations of Early Lives in Antiquity, J.L. Thompson, M. Alfonso-Durruty, and J.J. Crandall, Editors. 2014, University Press of Florida: Tallahasee, FL. p. 228-245. Knudson, K.J., et al., Migration and Viking Dublin: paleomobility and paleodiet through isotopic analyses. Journal of Archaeological Science, 2012. 39(2): p. 308-320. Knudson, K.J., et al., The geographic origins of Nasca trophy heads using strontium, oxygen, and carbon isotope data. Journal of Anthropological Archaeology, 2009. 28: p. 244-257. Marsteller, S.J., et al., Biogeochemical reconstructions of life histories as a method to assess regional interactions: Stable oxygen and radiogenic strontium isotopes and Late Intermediate Period mobility on the Central Peruvian Coast. Journal of Archaeological Science: Reports, 2017. 13: p. 535-546. Müller, W., et al., Origin and migration of the Alpine Iceman. Science, 2003. 302: p. 862866. O’Connell, T.C. and R.E.M. Hedges, Isotopic comparison of hair and bone: Archaeological Analyses. Journal of Archaeological Science, 1999. 26(6): p. 661-665. Pellegrini, M., et al., Tooth enamel oxygen "isoscapes" show a high degree of human mobility in prehistoric Britain. Sci Rep, 2016. 6: p. 34986.

36

23.

24.

25. 26. 27.

28. 29.

30.

31.

32.

33.

34.

35.

36. 37.

38.

39.

Price, T.D., G. Grupe, and P. Schröter, Reconstruction of migration patterns in the Bell Beaker period by stable strontium isotope analysis. Applied Geochemistry, 1994. 9: p. 413-417. Price, T.D., V. Tiesler, and J.H. Burton, Early African Diasporo in colonial Campeche, Mexico: strontium isotopic evidence. American Journal of Physical Anthropology, 2006. 130: p. 485-490. White, C.D. and H.P. Schwarcz, Temporal Trends for Stable Isotopes in Nubian Mummy Tissues. American Journal of Physical Anthropology, 1994. 93: p. 165-187. Meier-Augenstein, W., Stable Isotope Forensics: An Introduction to the Forensic Application of Stable Isotope Analysis. 2010, West Sussex, UK: Wiley-Blackwell. 271. Fraser, I., W. Meier-Augenstein, and R.M. Kalin, Stable isotope analysis of human hair and nail samples: The effects of storage on samples. Journal of Forensic Sciences, 2008. 53(1): p. 95-99. Ehleringer, J.R., et al., Urban water - a new frontier in isotope hydrology. Isotopes in Environmental and Health Studies, 2016. 52: p. 477-486. Good, S.P., et al., Patterns of local and non-local water resource use across the western United States determined by stable isotope intercomparisons. Water Resources Research, 2014. 50: p. 8034-8049. Jameel, Y., et al., Tap water isotope ratios reflect urban water system structure and dynamics across a semi-arid metropolitan area. Water Resources Research, 2016. 52: p. 5891-5910. Kennedy, C.D., G.J. Bowen, and J.R. Ehleringer, Temporal variation of oxygen isotope ratios (∂18O) in drinking water: implications for specifying location of origin with human scalp hair. Forensic Sci Int, 2011. 208(1-3): p. 156-66. Podlesak, D.W., et al., ∂2H and ∂18O of human body water: a GIS model to distinguish residents from non-residents in the contiguous USA. Isotopes Environ Health Stud, 2012. 48(2): p. 259-79. Podlesak, D.W., et al., Turnover of oxygen and hydrogen isotopes in the body water, CO2, hair and enamel of a small mammal. Geochimica et Cosmochimica Acta, 2008. 72: p. 19-35. Bowen, G.J., et al., Treatment methods for the determination of ∂2H and ∂18O of hair keratin by continuous-flow isotope-ratio mass spectrometry. Rapid Communications in Mass Spectrometry, 2005. 19: p. 2371-2378. Chesson, L.A., et al., Evaluating uncertainity in the calculation of non-exchangeable hydrogen fractions within organic materials. Rapid Communications in Mass Spectrometry, 2009. 23(9): p. 1275-1280. Hedges, R.E. and L.M. Reynard, Nitrogen isotopes and the trophic level of humans in archaeology. Journal of Archaeological Science, 2007. 34: p. 1240-1251. Mekota, A.-M., et al., Serial analysis of stable nitrogen and carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering from anorexia nervosa. Rapid Communications in Mass Spectrometry, 2006. 20: p. 1604-1610. O’Connell, T.C., et al., The diet-body offset in human nitrogen isotopic values: a controlled dietary study. American Journal of Physical Anthropology, 2012. 149: p. 426434. Ji, J.H., et al., The ethnic differences of the damage of hair and integral hair lipid after ultra violet radiation. Ann Dermatol, 2013. 25(1): p. 54-60.

37

40. 41. 42. 43. 44. 45. 46.

47. 48.

49.

50.

51.

52.

53. 54.

55. 56.

57. 58.

Franbourg, A., et al., Current research on ethnic hair. Journal of the American Academy of Dermatology, 2003. 48(6): p. S115-S119. Oxley, J.C., et al., Accumulation of explosives in hair - Part II: Factors affecting sorbtion. Journal of Forensic Sciences, 2007. 52(6): p. 1291-1296. Apelburg, B.J., et al., Racial differences in hair nicotine concentrations among smokers. Nicotine and Tobacco Research, 2012. 14(8): p. 933-941. Kidwell, D.A., E.H. Lee, and S.R. DeLauder, Evidence for bias in hair testing and procedures to correct bias. Forensic Science International, 2000. 107(1-3): p. 39-61. Kidwell, D.A., F.P. Smith, and A.R. Shepherd, Ethnic hair care products may increase false positives in hair drug testing. Forensic Sci Int, 2015. 257: p. 160-4. Henderson, G.L., et al., Incorporation of isotopically labeled cocaine into human hair: race as a factor. Journal of Analytical Toxicology, 1998. 22: p. 156-165. Ropero Miller, J.D. and P.R. Stout, Analysis of Cocaine Analytes in Human Hair II: Evaluation of Different Hair Color and Ethnicity Types, D.o. Justice, Editor. 2011, National Institute of Justice: Washington, DC. Yu, H., et al., Role of hair pigmentation in drug incorporation into hair. Forensic Sci Int, 2017. 281: p. 171-175. Hill, V., N. Stowe, and M. Schaffer, Comments on "Ethnic Hair Care Products May Increase False Positives in Hair Drug Testing" by Kidwell et al. Forensic Sci Int, 2016. 259: p. e48-50. Kidwell, D.A. and F.P. Smith, Companies should test the limits of their decontamination procedures--Reply to: Comments on "Ethnic hair care products may increase false positive in hair" V. Hill et al. Forensic Sci Int, 2016. 259: p. e51. Von Holstein, I.C.C., et al., Wet degradation of keratin proteins: linking amino acid, elemental and isotopic composition. Rapid Communications in Mass Spectrometry, 2014. 28: p. 2121-2123. Webb, E., C. White, and F. Longstaffe, Dietary shifting in the Nasca Region as inferred from the carbon- and nitrogen-isotope compositions of archaeological hair and bone. Journal of Archaeological Science, 2013. 40(1): p. 129-139. Bender, R.L., et al., Stable isotopes (carbon, nitrogen, sulfur), diet, and anthropometry in urban Colombian women: investigating socioeconomic differences. Am J Hum Biol, 2015. 27(2): p. 207-18. Gordon, G., et al., Taphonomy of Human Remains, NIJ Report 2014-DN-BX-K002. (submitted). p. 233. Wilson, A.S., The decomposition of hair in the buried body environment, in Soil Analysis in forensic taphonomy: Chemical and Biological Effects of Buried Human Remains, M. Tibbett and D.O. Carter, Editors. 2008, CRC Press. p. 123-151. Wilson, A.S., et al., Evaluating histological methods for assessing hair fibre degradation. Archaeometry, 2010. 52: p. 467-481. Wilson, A.S., et al., Selective biodegradation in hair shafts derived from archaeological, forensic and experimental contexts. British Journal of Dermatology, 2007. 157: p. 450457. Wilson, A.S., et al., Modelling the buried human body environment in upland climes using three contrasting field sites. Forensic Sciece International, 2007. 169: p. 6-18. Chang, B.S., et al., Ultramicroscopic observations on morphological changes in hair during 25 years of weathering. Forensic Science International, 2005. 151: p. 193-200.

38

59.

60.

61.

62.

63. 64.

65.

66.

67.

68.

69.

70.

71.

72.

73. 74.

Tridico, S.R., et al., Interpreting biological degradative processes acting on mammalian hair in the living and the dead: which ones are taphonomic? Proceedings of the Royal Society B: Biological Sciences, 2014. 281(1796): p. 20141755. Mazzarelli, D., et al., Splitting hairs: differentiating between entomological activity, taphonomy, and sharp force trauma on hair. Forensic Sci Med Pathol, 2015. 11(1): p. 104-10. Von Holstein, I.C.C., et al., An assessment of procedures to remove exogenous Sr before 87 Sr/86Sr analysis of wet archaeological wool textiles. Journal of Archaeological Science, 2015. 53: p. 84-93. Wassenaar, L.I. and K.A. Hobson, Comparative equilibration and online technique for determination of non-exchangeable hydrogen of keratins for use in animal migration studies. Isotopes in Environmental and Health Studies, 2003. 39(3): p. 211-217. Coleman, J., Handbook of Forensic Services, U.D.o. Justice, Editor. 2013, Federal Bureau of Investigation, Laboratory Division: Quantico, VA. p. 63. Meier-Augenstein, W., et al., An interlaboratory comparative study into sample preparation for both reproducible and repeatable forensic 2H isotope analysis of human hair by continuous flow isotope ratio mass spectrometry. Rapid Communications in Mass Spectrometry, 2011. 25: p. 3331-3338. Soto, D.X., et al., Re-evaluation of the hydrogen stable isotopic composition of keratin calibration standards for wildlife and forensic science applications. Rapid Commun Mass Spectrom, 2017. 31(14): p. 1193-1203. Roberts, P., et al., Calling all archaeologists: guidelines for terminology, methodology, data handling, and reporting when undertaking and reviewing stable isotope applications in archaeology. Rapid Commun Mass Spectrom, 2018. 32(5): p. 361-372. Bond, A.L. and K.A. Hobson, Reporting Stable-Isotope Ratios in Ecology: Recommended Terminology, Guidelines and Best Practices. Waterbirds, 2012. 35(2): p. 324-331. Coplen, T.B., Guidelines and recommended terms for expression of stable-isotope-ratio and gas-ratio measurement results. Rapid Communications in Mass Spectrometry, 2011. 25(17): p. 2538-2560. Coleman, M. and W. Meier-Augenstein, Ignoring IUPAC guidelines for measurement and reporting of stable isotope abundance values affects us all. Rapid Commun Mass Spectrom, 2014. 28(17): p. 1953-5. Gordon, G.W., et al., [dataset] Preservation of hair stable isotope signatures during freezing and law enforcement evidence packaging, M. Data, 2018, doi:10.17632/fd52wddwks.1. Szpak, P., J.Z. Metcalfe, and R.A. Macdonald, Best practices for calibrating and reporting stable isotope measurements in archaeology. Journal of Archaeological Science: Reports, 2017. 13: p. 609-616. Statistics, L. One-way repeated measures ANOVA using SPSS Statistics. Statistical tutorials and software guides 2015 June 10, 2018]; Available from: https://statistics.laerd.com. Field, A., Discovering Statistics with IBM SPSS. 2013, Newbury Park, CA: Sage Press. Howell, D.C., Statistical Methods for Psychology. 5th ed. 2002, Pacific Grove, CA: Duxbury.

39

75.

Bartelink, E.J., et al., Application of Stable Isotope Forensics for Predicting Region of Origin of Human Remains from Past Wars and Conflicts. Annals of Anthropological Practice, 2014. 38(1): p. 124-136.

40

Highlights:



Hair frozen in paper or plastic retained carbon, nitrogen, and oxygen isotope values



Hair frozen was enriched in 2H by 2-3 ‰ on average compared to controls



Some frozen samples were enriched in 2H by >10 ‰, impacting forensic interpretation



Law enforcement packaging is suitable for travel prediction from ∂13C, ∂15N, and ∂18O

41

A

B

C

D

-15.00 -15.50

11.00

A.

B.

10.50 -16.00 10.00

d 15NAIR (‰)

d 13CVPDB (‰)

-16.50 -17.00 -17.50 -18.00

9.50

9.00

-18.50 8.50 -19.00 -19.50 15.0 -15.00

8.00 -35.0

C.

-15.00

-40.0

14.0

-15.50

-15.50

-16.00 13.0

-16.00

-50.0

-16.50

d C2H d13 (‰)(‰) VSMOW VPDB

dd1318 OVSMOW CVPDB (‰)(‰)

D.

-45.0

12.0 -17.00

11.0

-17.50

-16.50 -55.0 -17.00 -60.0 -17.50 -65.0

10.0

-18.00

-18.00 -70.0

-18.50

-18.50 -75.0

9.0 -80.0 -19.00

-19.00

8.0 -19.50

-85.0 -19.50 Control

plastic clamshell, 3 plastic clamshell, 5 weeks months

butcher paper, 3 weeks

butcher paper, 5 months

Control

plastic clamshell, 3 plastic clamshell, 5 weeks months

butcher paper, 3 weeks

butcher paper, 5 months

0.50

0.20

0.40

0.10

0.30

0.00

0.20

-0.10

0.10

D 15N (‰)

D 13C (‰)

0.30

-0.20 -0.30

-0.10

-0.40

-0.20

-0.50

-0.30

-0.60

-0.40

-0.70

-0.50

1.50

15.00

0.30

0.30

1.00 0.20

0.20 10.00

0.10

0.10 2 Dd 13HCVSMOW (‰) (‰)

0.50 0.00

O (‰) D 13DC18(‰)

0.00

-0.10 0.00 -0.20 -0.30 -0.50

0.00 5.00 -0.10 -0.20 0.00 -0.30

-0.40

-0.40

-0.50 -1.00

-5.00 -0.50

-0.60

-0.60

-1.50 -0.70

-10.00 -0.70 plastic clamshell, 3 weeks - control

plastic clamshell, 5 months - control

butcher paper, 3 weeks - butcher paper, 5 months control - control

plastic clamshell, 3 weeks - control

plastic clamshell, 5 months - control

butcher paper, 3 weeks - butcher paper, 5 months control - control

=?

Hair collected at crime scene

Packaged as evidence, stored at -20°C

d13C, d15N, d18O, d2H by IRMS