Stable isotope ratios as a tool in microbial forensics--Part 2. Isotopic variation among different growth media as a tool for sourcing origins of bacterial cells or spores

Since the anthrax attacks of 2001 the need for methods to trace the origins of microbial agents has become urgent. The stable isotope ratios of bacteria record information from both the nutrients and the water used to make their culture media and could potentially be used to provide information about their growth environment. We present a survey of carbon (C), nitrogen (N), and hydrogen (H) stable isotope ratios in 516 samples of bacteriological culture media. The observed variation was consistent with expected isotopic variation in the plant and animal products upon which the media are based. The variation is sufficient to translate into substantial isotope variation in cultures grown on different batches of media, and thus to allow investigators to determine whether seized media could have been used to produce seized bioweapons agents.     

Stable isotope ratios as a tool in microbial forensics--part 3. Effect of culturing on agar-containing growth media

Stable isotope ratios of hydrogen and oxygen in microbes have been shown to be functions of the corresponding isotope ratios of the water with which the culture medium was prepared, and thus to contain a potential geographic signal. Water can evaporate from agar (solid) media during culturing, changing its isotope ratios. Here we describe the effect of drying on the isotope ratios of water extracted from agar media and the H and O stable isotope ratios ratios of Bacillus subtilis spores cultured on agar. The delta2H vs delta18O relationship of water in Petri dish agar was surprisingly constant during evaporation regardless of the ambient relative humidity, making it possible to calculate the approximate isotope ratios of the original water, even in significantly evaporated agar. The H stable isotope ratios of spores cultured on agar remained relatively unchanged as the agar dried, but the O ratio became significantly enriched.     

Isotopic fractionation of carbon and nitrogen during the illicit processing of cocaine and heroin in South America

The forensic application of stable isotope analysis to cocaine and heroin for geolocation of exhibits must take into account the possible enrichment and/or depletion of 13C and 15N during the illicit manufacturing process. Continuous-flow elemental analysis-isotope ratio mass spectrometry was utilized to measure changes in the stable isotope ratios of carbon and nitrogen for both cocaine (N = 92) and heroin/morphine (N = 81) exhibits derived from illicit manufacturing processes utilized by South American clandestine chemists. In controlled settings in South America, there was no siginficiant carbon isotope fractionation during the conversion of cocaine base to cocaine HCI using current illict methodologies. In contrast, nitrogen isotope fractionation for this conversion was 1 per thousand. There was a kinetic carbon isotope ratio fractionation during the acetylation of Colombian morphine to heroin and as a result heroin exhibits will almost always have more negative delta13C values than the original morphine. There was an isotopic fractionation against 15N during the acetylation of morphine base to heroin base, but this effect was not expressed since all of the heroin base was precipitated during the manufacturing process. However, the clandestine process of converting a single batch of heroin base usually involved two consecutive crops of heroin HCl and the latter crop was isotopically depleted as expected from a Rayleigh distillation process. When heroin was deacetylated to morphine, the morphine produced resulted in delta13C values that were indistinguishable from the original morphine. The kinetic carbon isotope fractionation factor for the South American process of morphine acetylation was -1.8 per thousand, allowing calculation of the delta13C values of the acetic anhydride from deacetylated heroin delta13C values.     

Using morphometric and analytical techniques to characterize elephant ivory

There is a need to characterize Asian elephant ivory and compare with African ivory for controlling illegal trade and implementation of national and international laws. In this paper, we characterize ivory of Asian and African elephants using Schreger angle measurements, elemental analysis {X-ray fluorescence (XRF), inductively coupled plasma-atomic emission spectroscopy (ICP-AES), and inductively coupled plasma-mass spectroscopy (ICP-MS)} and isotopic analysis. We recorded Schreger angle characteristics of elephant ivory at three different zones in ivory samples of African (n=12) and Asian (n=28) elephants. The Schreger angle ranged from 32 degrees to 145 degrees and 30 degrees to 153 degrees in Asian and African ivory, respectively. Elemental analysis (for Asian and African ivory) by XRF, ICP-AES and ICP-MS provided preliminary data. We attempted to ascertain source of origin of Asian elephant ivory similarly as in African ivory based on isotopes of carbon, nitrogen and strontium. We determined isotopic ratios of carbon (n=31) and nitrogen (n=31) corresponding to diet and rainfall, respectively. Reference ivory samples from five areas within India were analyzed using collagen and powder sample and the latter was found more suitable for forensic analysis. During our preliminary analysis, the range of delta13C values (-13.6+/-0.15 per thousand and -25.6+/-0.15 per thousand) and delta15N values (10.2+/-0.15 per thousand and 3.5+/-0.15 per thousand) were noted.     

Stable-isotope fingerprints of biological agents as forensic tools

Naturally occurring stable isotopes of light elements in chemical and biological agents may possess unique "stable-isotope fingerprints" depending on their sources and manufacturing processes. To test this hypothesis, two strains of bacteria (Bacillus globigii and Erwinia agglomerans) were grown under controlled laboratory conditions. We observed that cultured bacteria cells faithfully inherited the isotopic composition (hydrogen, carbon, and nitrogen) of media waters and substrates in predictable manners in terms of bacterial metabolism and that even bacterial cells of the same strain, which grew in media water and substrates of different isotopic compositions, have readily distinguishable isotopic signatures. These "stable-isotopic fingerprints" of chemical and biological agents can be used as forensic tools in the event of biochemical terrorist attacks.     

Multiple stable isotope characterization as a forensic tool to distinguish acid scavenger samples

Acid scavengers are frequently used as stabilizer compounds in a variety of applications. When used to stabilize volatile compounds such as nerve agents, the lower volatility and higher stability of acid scavengers make them more persistent in a post-event forensic setting. Compound-specific isotope analysis of carbon, nitrogen, and hydrogen in three acid-scavenging compounds (N,N-diethylaniline, tributylamine, and triethylamine) were used as a tool for distinguishing between different samples. Combined analysis of multiple isotopes improved sample resolution, for instance differentiation between triethylamine samples improved from 80% based on carbon alone to 96% when combining with additional isotope data. The compound-specific methods developed here can be applied to instances where these compounds are not pure, such as when mixed with an agent or when found as a residue. Effective sample matching can be crucial for linking compounds at multiple event sites or linking a supply inventory to an event.     

Forensic analysis of explosives using isotope ratio mass spectrometry (IRMS) — Discrimination of ammonium nitrate sources

An evaluation was undertaken to determine if isotope ratio mass spectrometry (IRMS) could assist in the investigation of complex forensic cases by providing a level of discrimination not achievable utilising traditional forensic techniques. The focus of the research was on ammonium nitrate (AN), a common oxidiser used in improvised explosive mixtures.The potential value of IRMS to attribute Australian AN samples to the manufacturing source was demonstrated through the development of a preliminary AN classification scheme based on nitrogen isotopes. Although the discrimination utilising nitrogen isotopes alone was limited and only relevant to samples from the three Australian manufacturers during the evaluated time period, the classification scheme has potential as an investigative aid.Combining oxygen and hydrogen stable isotope values permitted the differentiation of AN prills from three different Australian manufacturers. Samples from five different overseas sources could be differentiated utilising a combination of the nitrogen, oxygen and hydrogen isotope values. Limited differentiation between Australian and overseas prills was achieved for the samples analysed.The comparison of nitrogen isotope values from intact AN prill samples with those from post-blast AN prill residues highlighted that the nitrogen isotopic composition of the prills was not maintained post-blast; hence, limiting the technique to analysis of un-reacted explosive material.     

Forensic analysis of explosives using isotope ratio mass spectrometry (IRMS) — Preliminary study on TATP and PETN

The application of isotopic techniques to investigations requiring the provision of evidence to a Court is limited. The objective of this research was to investigate the application of light stable isotopes and isotope ratio mass spectrometry (IRMS) to solve complex forensic cases by providing a level of discrimination not achievable utilising traditional forensic techniques.Due to the current threat of organic peroxide explosives, such as triacetone triperoxide (TATP), research was undertaken to determine the potential of IRMS to differentiate samples of TATP that had been manufactured utilising different starting materials and/or manufacturing processes. In addition, due to the prevalence of pentaerythritoltetranitrate (PETN) in detonators, detonating cord, and boosters, the potential of the IRMS technique to differentiate PETN samples from different sources was also investigated.Carbon isotope values were measured in fourteen TATP samples, with three definite groups appearing in the initial sample set based on the carbon data alone. Four additional TATP samples (in a second set of samples) were distinguishable utilising the carbon and hydrogen isotopic compositions individually, and also in combination with the oxygen isotope values. The 3D plot of the carbon, oxygen and hydrogen data demonstrated the clear discrimination of the four samples of TATP. The carbon and nitrogen isotope values measured from fifteen PETN samples, allowed samples from different sources to be readily discriminated.This paper demonstrates the successful application of IRMS to the analysis of explosives of forensic interest to assist in discriminating samples from different sources. This research represents a preliminary evaluation of the IRMS technique for the measurement of stable isotope values in TATP and PETN samples, and supports the dedication of resources for a full evaluation of this application in order to achieve Court reportable IRMS results.     

Assessing Carbon and Hydrogen Isotopic Fractionation of Diesel Fuel n-alkanes During Progressive Evaporation

Compound-specific isotope analysis offers potential for fingerprinting of diesel fuels, however, possible confounding effects of isotopic fractionation due to evaporation need to be assessed. This study measured the fractionation of the stable carbon and hydrogen isotopes in n-alkane compounds in neat diesel fuel during evaporation. Isotope ratios were measured using a continuous flow gas chromatograph/isotope ratio mass spectrometer. Diesel samples were progressively evaporated at 24 ± 2°C for 21 days. Increasing depletion of deuterium in nC₁₂–nC₁₇ alkanes in the remaining liquid with increasing carbon chain length was observed. Negligible carbon isotope fractionation was observed. Preferential vaporization was measured for the shorter chain n-alkanes and the trend decreased with increasing chain length. The decrease in δ²H values indicates the preferential vaporization of the isotopically heavier species consistent with available quantitative data for hydrocarbons. These results are most important in the application of stable isotope technology to forensic analysis of diesel.     

Geo-location of heroin and cocaine by stable isotope ratios

Analyses of the carbon and nitrogen stable isotope ratios in heroin and cocaine samples obtained from different geographic regions indicated stable isotope ratio combinations that were strongly correlated with geographic location. Further analyses of the isotope ratios of morphine derived from the deacetylation of heroin exhibited more pronounced isotopic differences among regions, increasing its potential as a tool for geo-location and for sample-to-sample comparison.     

Background survey of polyethylene in the Australian Capital Territory – A demonstration of variability in isotopic abundance values and their application to forensic casework

Plastics including adhesive tapes, cable ties, and packaging are common evidence types encountered in forensic investigations and casework. Traditional examination techniques such as Fourier Transform Infrared (FTIR) spectroscopy lack specificity and are unable to discern differences within the same polymer structures leaving the analyst with a generic identification. High quality manufacturing methods further amplify the limitations in detecting variability between samples. Isotope Ratio Mass Spectrometry (IRMS) has been shown to be a valuable technique in further discriminating plastics. Discrimination is achieved by analysing the relative abundances of stable isotopes within a sample, with differences detected in isotope ratios possibly attributed to the source of raw materials and fractionation during the manufacturing process. A survey of cling wraps and re-sealable zipper storage bags collected in the Australian Capital Territory was undertaken to assess the variability in carbon and hydrogen isotope ratios of different brands and samples. The results of this research are discussed, particularly with respect to within and between brand trends, and a case study is presented as an example of the value of including IRMS in a casework context.     

Stable carbon and nitrogen isotope ratios of sodium and potassium cyanide as a forensic signature

Sodium and potassium cyanide are highly toxic, produced in large amounts by the chemical industry, and linked to numerous high-profile crimes. The U.S. Centers for Disease Control and Prevention has identified cyanide as one of the most probable agents to be used in a chemical terrorism event. We investigated whether stable C and N isotopic content of sodium and potassium cyanide could serve as a forensic signature for sample matching, using a collection of 65 cyanide samples. Upon analysis, a few of the cyanide samples displayed nonhomogeneous isotopic content associated with degradation to a carbonate salt and loss of hydrogen cyanide. Most samples had highly reproducible isotope content. Of the 65 cyanide samples, >95% could be properly matched based on C and N isotope ratios, with a false match rate <3%. These results suggest that stable C and N isotope ratios are a useful forensic signature for matching cyanide samples.     

Resident and Nonresident Fingernail Isotopes Reveal Diet and Travel Patterns,,

Global travel has increased, and having a diagnostic tool to distinguish residents from visitors would be valuable. This study examined stable isotope biomarkers of fingernail tissues of resident (n = 26) and nonresident (n = 22) participants in Salt Lake City (SLC), UT, from 2015 to 2016. The purpose of this research was to determine whether fingernail isotopes could be used for reconstructing geolocation movements and to examine the convergence in nonresident fingernail isotopes to that of the resident signal following their arrival to SLC. Resident isotope values defined a baseline to make comparisons to. Initial nonresident hydrogen and oxygen isotope values were correlated with precipitation isotopes of their prior location. Fingernail isotope turnover rates were rapid and nonresident isotopes were indistinguishable from residents after ~71–90 days. The results of our study highlight the utility of stable isotope measurements of fingernail clippings to examine travel history reconstruction that could aid in identification of human remains.     

Stable isotope models to predict geographic origin and cultivation conditions of marijuana

Here we describe stable isotope based models using hydrogen and carbon isotope ratios to predict geographic region-of-origin and growth environment for marijuana, with the intent of applying these models to analyses of marijuana trafficking in the USA. The models were developed on the basis of eradication specimens and border specimens seized throughout the USA. We tested reliability of the geographic region-of-origin and growth environment models with a “blind” set of 60 marijuana eradication specimens obtained from counties throughout the USA. The two geographic region-of-origin model predictions were 60–67% reliable and cultivation environment model predictions were 86% accurate for the blind specimens. We demonstrate here that stable isotope ratio analysis of marijuana seizures can significantly improve our understanding of marijuana distribution networks and it is for that purpose that these models were developed.     

Effect of ethanol on cardiac single sodium channel gating

Alcohol in modest and higher doses has the potential to induce cardiac arrhythmias. The most famous alcohol-related arrhythmia is the "holiday heart syndrome". Furthermore, there is a clear association between excessive alcohol consumption and the risk of sudden cardiac death. However, the acute effects of ethanol on arrhythmia induction are not well understood. The effect of ethanol on single cardiac sodium channels has not been studied yet. To elucidate the effect of ethanol on human cardiac sodium channels we performed a patch clamp study in HEK-293 cells overexpressing the human cardiac sodium channel. We used HEK-293 cells overexpressing the human cardiac sodium channel (Na(1.5)). Single channel gating was investigated by the cell-attached patch clamp technique. Sodium channel currents were elicited by depolarizing pulses from -120 to -20mV for a duration of 150ms. Single channel availability, open probability and peak average current were assessed baseline and after addition of ethanol in increasing concentrations (0.50 per thousand (10.9mM), 1.00 per thousand (21.7mM), 2.00 per thousand (43.5mM) and 4.00 per thousand (87.0mM)). We found a concentration-dependent reduction of open probability which was statistically significant at 2.00 per thousand ethanol (66.5+/-14% of control). At higher concentrations (4.00 per thousand) also availability decreased to 66.5+/-11.0% of control. This resulted in a significant decrease of peak average current at 2.00 per thousand and at 4.00 per thousand ethanol (61.8+/-7.4 and 53.0+/-8.2% of control). For the first time the present study demonstrates acute inhibitory effects of ethanol on single cardiac sodium channel gating and provides one potential mechanism for the well known clinical observation that ethanol triggers supraventricular and ventricular arrhythmias.     

Forensic utility of carbon isotope ratio variations in PVC tape backings

Forensic interest in adhesive tapes with polyvinyl chloride (PVC) backings (electrical tape) derives from their use in a variety of illicit activities. Due to the range of physical characteristics, chemical compositions, and homogeneity within a single roll of tape, traditional microscopic and chemical analyses can offer a high degree of discrimination between tapes, permitting the assessment of potential associations between evidentiary tape samples. The carbon isotope ratios of tapes could provide additional discrimination among tape samples. To evaluate whether carbon isotope ratios may be able to increase discrimination of electrical tapes, particularly with regards to different rolls of tape of the same product, we assessed the δ(13)C values of backings from 87 rolls of PVC-based black electrical tape (~20 brands, >60 products) Prior to analysis, adhesives were removed to prevent contamination by adhering debris, and plasticizers were extracted because of concern over their potential mobility. This result is consistent with each of these tapes having approximately the same plasticizer δ(13)C value and proportion of carbon in these plasticizers. The δ(13)C values of the 87 PVC tape backings ranged between -23.5 and -41.3 (‰, V-PDB), with negligible carbon isotopic variation within single rolls of tape, yet large variations among tape brands and tape products. Within this tape population, carbon isotope ratios permitted an average exclusion power of 93.7%, using a window of +/-0.3‰; the combination of carbon isotope ratio measurement with additional chemical and physical analyses raises the discrimination power to over 98.9%, with only 41 out of a possible 3741 pairs of tape samples being indistinguishable. There was a linear relationship between the δ(13)C value of tape backings and the change in δ(13)C value with the extraction of plasticizers. Analyses of pre- and post-blast tape sample pairs show that carbon isotope signatures are within 0.3‰ of pre-blast values, indicating that carbon isotope values are largely preserved during an explosion.     

USGS42 and USGS43: human-hair stable hydrogen and oxygen isotopic reference materials and analytical methods for forensic science and implications for published measurement results

Because there are no internationally distributed stable hydrogen and oxygen isotopic reference materials of human hair, the U.S. Geological Survey (USGS) has prepared two such materials, USGS42 and USGS43. These reference materials span values commonly encountered in human hair stable isotope analysis and are isotopically homogeneous at sample sizes larger than 0.2 mg. USGS42 and USGS43 human-hair isotopic reference materials are intended for calibration of δ(2)H and δ(18)O measurements of unknown human hair by quantifying (1) drift with time, (2) mass-dependent isotopic fractionation, and (3) isotope-ratio-scale contraction. While they are intended for measurements of the stable isotopes of hydrogen and oxygen, they also are suitable for measurements of the stable isotopes of carbon, nitrogen, and sulfur in human and mammalian hair. Preliminary isotopic compositions of the non-exchangeable fractions of these materials are USGS42(Tibetan hair)δ(2)H(VSMOW-SLAP) = -78.5 ± 2.3‰ (n = 62) and δ(18)O(VSMOW-SLAP) = +8.56 ± 0.10‰ (n = 18) USGS42(Indian hair)δ(2)H(VSMOW-SLAP) = -50.3 ± 2.8‰ (n = 64) and δ(18)O(VSMOW-SLAP) = +14.11 ± 0.10‰ (n = 18). Using recommended analytical protocols presented herein for δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) measurements, the least squares fit regression of 11 human hair reference materials is δ(2)H(VSMOW-SLAP) = 6.085δ(2)O(VSMOW-SLAP) - 136.0‰ with an R-square value of 0.95. The δ(2)H difference between the calibrated results of human hair in this investigation and a commonly accepted human-hair relationship is a remarkable 34‰. It is critical that readers pay attention to the δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) of isotopic reference materials in publications, and they need to adjust the δ(2)H(VSMOW-SLAP) and δ(18)O(VSMOW-SLAP) measurement results of human hair in previous publications, as needed, to ensure all results on are on the same scales.     

Forensic Analysis of Explosives Using Isotope Ratio Mass Spectrometry (IRMS)—Part 1: Instrument Validation of the DELTAplusXP IRMS for Bulk Nitrogen Isotope Ratio Measurements

Abstract: A significant amount of research has been conducted into the use of stable isotopes to assist in determining the origin of various materials. The research conducted in the forensic field shows the potential of isotope ratio mass spectrometry (IRMS) to provide a level of discrimination not achievable utilizing traditional forensic techniques. Despite the research there have been few, if any, publications addressing the validation and measurement uncertainty of the technique for forensic applications. This study, the first in a planned series, presents validation data for the measurement of bulk nitrogen isotope ratios in ammonium nitrate (AN) using the DELTA^plusXP (Thermo Finnigan) IRMS instrument equipped with a ConFlo III interface and FlashEA? 1112 elemental analyzer (EA). Appropriate laboratory standards, analytical methods and correction calculations were developed and evaluated. A validation protocol was developed in line with the guidelines provided by the National Association of Testing Authorities, Australia (NATA). Performance characteristics including: accuracy, precision/repeatability, reproducibility/ruggedness, robustness, linear range, and measurement uncertainty were evaluated for the measurement of nitrogen isotope ratios in AN. AN (99.5%) and ammonium thiocyanate (99.99+%) were determined to be the most suitable laboratory standards and were calibrated against international standards (certified reference materials). All performance characteristics were within an acceptable range when potential uncertainties, including the manufacturer’s uncertainty of the technique and standards, were taken into account. The experiments described in this article could be used as a model for validation of other instruments for similar purposes. Later studies in this series will address the more general issue of demonstrating that the IRMS technique is scientifically sound and fit‐for‐purpose in the forensic explosives analysis field.     

Sourcing Brazilian marijuana by applying IRMS analysis to seized samples

The stable carbon and nitrogen isotopic ratios were measured in marijuana samples (Cannabis sativa L.) seized by the law enforcement officers in the three Brazilian production sites: Pernambuco and Bahia (the country's Northeast known as Marijuana Polygon), Pará (North or Amazon region) and Mato Grosso do Sul (Midwest). These regions are regarded as different with respect to climate and water availability, factors which impact upon the isotope fractionations of these elements within plants. It was possible to differentiate samples from the dry regions (Marijuana Polygon) from those from Mato Grosso do Sul and Pará, that present heavier rainfall. The results were in agreement with the climatic conditions of the suspected regions of origin and this demonstrates that seized samples can be used to identify the isotopic signatures of marijuana from the main producing regions in Brazil.     

Stable hydrogen isotope analysis as a method to identify illegally trapped songbirds

We measured stable hydrogen isotope ratios in the primary feathers of two subspecies of goldfinches, confiscated by the Police and the Royal Society for the Prevention of Cruelty to Animals (RSPCA) on suspicion that they had been illegally taken from the wild. We found significant differences in the delta2H values of the two subspecies indicating that they were sourced from different geographical regions. Our results correlated with isotopic precipitation maps and with the known distribution of the two subspecies of goldfinch. We believe that this technique could be used by law enforcement agencies to determine the origin of birds in cases where the species or subspecies involved are geographically distinct.