Soil Analysis in Forensic Taphonomy: Chemical and Biological Effects of Buried Human Remains

SOIL ANALYSIS in FORENSIC TAPHONOMY Chemical and Biological Effects of

Buried Human Remains Edited by

Mark Tibbett and David O. Carter

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

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CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487‑2742 © 2008 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid‑free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number‑13: 978‑1‑4200‑6991‑4 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the conse‑ quences of their use. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978‑750‑8400. CCC is a not‑for‑profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging‑in‑Publication Data Soil analysis in forensic taphonomy : chemical and biological effects of buried human remains / editor(s), Mark Tibbett and David O. Carter. p. ; cm. Includes bibliographical references and index. ISBN 978‑1‑4200‑6991‑4 (alk. paper) 1. Forensic taphonomy. 2. Soils‑‑Analysis. 3. Soil microbiology. 4. Human decomposition. I. Tibbett, Mark. II. Carter, David O. [DNLM: 1. Forensic Anthropology‑‑methods. 2. Soil‑‑analysis. 3. Postmortem Changes. 4. Soil Microbiology. W 750 S6826 2008] RA1063.47.S65 2008 363.25‑‑dc22

2007045309

Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

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For Tammy, Jasmine, Courtney and Joseph. M.T.

For my father and mother, who encouraged curiosity and my desire to learn. And to Mike Madison and the Hepworth family, for showing me the importance of study. D.O.C.

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Contents

Preface Editors Contributors

1

vii ix xi

Nature, Distribution, and Origin of Soil Materials in the Forensic Comparison of Soils

1

Robert W. Fitzpatrick

2

Cadaver Decomposition and Soil: Processes

29

David O. Carter and Mark Tibbett

3

The Role of Soil Organisms in Terrestrial Decomposition

53

David W. Hopkins

4

Soil Fungi Associated with Graves and Latrines: Toward a Forensic Mycology

67

Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

5

The Role of Invertebrates in Terrestrial Decomposition: Forensic Applications

109

Ian R. Dadour and Michelle L. Harvey

6

The Decomposition of Hair in the Buried Body Environment

123

Andrew S. Wilson



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vi Contents

7

The Decomposition of Materials Associated with Buried Cadavers

153

Robert C. Janaway

8

Decomposition Chemistry in a Burial Environment

203

Shari L. Forbes

9

Potential Determinants of Postmortem and Postburial Interval of Buried Remains

225

Shari L. Forbes

10

Principles and Methodologies of Measuring Microbial Activity and Biomass in Soil

247

Phil C. Brookes

11

Methods of Characterizing and Fingerprinting Soils for Forensic Application

271

Lorna A. Dawson, Colin D. Campbell, Stephen Hillier, and Mark J. Brewer

Index

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Preface

Forensic taphonomy is an applied discipline that is coming of age. To date, however, the major advances in the field have been captured in publications that deal primarily with the cadaver and associated items rather than the grave itself. This book provides, for the first time, a collection of chapters from leading scientists in their fields that deal primarily with the burial environment. Our focus is on the processes of decomposition in soils, the decomposers in the soil, and the basic physiochemical composition of the soil as it relates to forensic science and taphonomy. The book aims to provide the reader with an up-to-date overview of fundamental scientific principles and methods used in forensic taphonomy from a soils-based perspective. Soils are the materials that make up most clandestine graves but are often given scant consideration. This is a shame, as soils can contain an enormous amount of information within them—if you know what to look for and how to find it. The purpose of this book is to illuminate this search for forensic information in the soils generally and at gravesites particularly. Of particular importance here is the detritusphere, the soil immediately around the cadaver. This soil is the most altered by the decomposition process and can contribute to the decomposition process. Many biological and chemical effects of buried human remains can be found here, and the analysis of soils around a cadaver for forensic use, though in its infancy, is progressing apace. The terrestrial environment has been much studied as a decomposition environment for materials of little forensic value, such as leaf litter or dead roots. These provide the basic methods and framework for studying and understanding decomposition of materials in soils. It is only in recent years that this has been applied to forensic taphonomy, in which studies have been conducted with mammalian tissues and cadavers. The burial environment is a complex and dynamic system of interdependent chemical, physical, and biological processes. These processes influence, and are influenced by, the inclusion of a body and its subsequent decay. Though this book deals with what is known in this context, much still remains to be discovered, understood, and applied to forensic science. We believe this book is timely, as soils are receiving increased attention as physical evidence. Thus far, the twenty-first century has seen an increase of peer-reviewed publications related to soils and forensic science of at least vii

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viii Preface

one third from the last decade of the twentieth century. We hope that this book will provide a solid foundation for forensic taphonomists, anthropologists, soil scientists, entomologists, bacteriologists, and mycologists who aim to use the processes of cadaver decomposition in terrestrial ecosystems to solve crime. Mark Tibbett David O. Carter

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Editors

Mark Tibbett, Ph.D. is a soil micro­ biologist with a long-standing interest in decomposition processes in terrestrial ecosystems. He has worked in many of the world’s ecoregions including tropical, Mediterranean, temperate, boreal, and polar ecosystems. His interests in forensic taphonomy arose from a research activity in organic nutrient patch dynamics in soils, the principles of which he has applied to forensic science. Dr. Tibbett is currently director of the Centre for Land Rehabilitation at the University of Western Australia. David O. Carter, Ph.D. is an assistant professor of forensic science at the University of Nebraska-Lincoln, where he teaches courses in forensic science and coordinates the undergraduate degree program in forensic science. Dr. Carter earned a master of science in forensic archaeology from Bournemouth University, U.K. (2001) and a Ph.D. from James Cook University, Australia (2005). He investigates the processes associated with cadaver decomposition in terrestrial ecosystems with a focus on the fate of cadaver-derived carbon, nitrogen, and phosphorus to develop methods for the estimation of postmortem interval and the identification of clandestine graves.

ix

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Contributors

Mark J. Brewer

Shari L. Forbes

Phil C. Brookes

Michelle L. Harvey

Colin D. Campbell

Stephen Hillier

David O. Carter

David W. Hopkins

Biomathematics and Statistics Scotland The Macaulay Institute Craigiebuckler, Aberdeen United Kingdom

Faculty of Science University of Ontario Institute of Technology Oshawa, Ontario

Agriculture and Environment Division IACR-Rothamsted Experimental Station Harpenden, Hertfordshire United Kingdom

School of Biological Sciences University of Portsmouth Portsmouth, Hampshire United Kingdom

Soils Group The Macaulay Institute Craigiebuckler, Aberdeen United Kingdom

Soils Group The Macaulay Institute Craigiebuckler, Aberdeen United Kingdom

Department of Entomology College of Agricultural Sciences and Natural Resources University of Nebraska-Lincoln Lincoln, Nebraska

Scottish Crop Research Institute Invergowrie Dundee, Scotland United Kingdom

Robert C. Janaway

Ian R. Dadour

Centre for Forensic Science University of Western Australia Crawley, Western Australia Australia

Archaeological Sciences School of Life Sciences University of Bradford Bradford, West Yorkshire United Kingdom

Lorna A. Dawson

Naohiko Sagara

Soils Group The Macaulay Institute Craigiebuckler, Aberdeen United Kingdom

Professor Emeritus Kyoto University Kyoto, Japan

Mark Tibbett

Robert W. Fitzpatrick

Centre for Land Rehabilitation University of Western Australia Crawley, Western Australia Australia

Centre for Australian Forensic Soil Science/CSIRO Land and Water Glen Osmond, South Australia Australia

xi

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xii Contributors

Andrew S. Wilson

Archaeological Sciences School of Life Sciences University of Bradford Bradford, West Yorkshire United Kingdom

Takashi Yamanaka

Forestry and Forest Products Research Institute Ibaraki, Japan

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Nature, Distribution, and Origin of Soil Materials in the Forensic Comparison of Soils

1

Robert W. Fitzpatrick Contents 1.1  Introduction................................................................................................... 1 1.2  Nature of Soils Relevant to Forensic Soil Science and Human Decomposition Processes............................................................................. 3 1.3  Brief History of Forensic Soil Science......................................................... 4 1.4  Soil Origin, Classification, and Distribution............................................. 6 1.5  Spatial Scale and Pedogenic Processes..................................................... 10 1.6  Relationship between Soil Type and Scale: Regional and Global..........11 1.7  Most Favored Techniques Used by Forensic Soil Scientists....................11 1.7.1  Theory of Making Comparisons between Soil Samples............ 12 1.7.2  Approaches and Methods for Making Comparisons between Soil Samples..................................................................... 12 1.7.2.1  Soil Color.......................................................................... 13 1.7.2.2  Soil Consistence...............................................................14 1.7.2.3  Soil Texture...................................................................... 15 1.7.2.4  Soil Structure................................................................... 20 1.7.2.5  Segregations and Coarse Fragments............................ 20 1.8  Petrographic and Other Advanced Techniques and Instruments....... 21 1.9  Conclusions.................................................................................................. 25 References............................................................................................................... 25

1.1  Introduction Soils mean different things to different people. Soil scientists view soils as being made up of differently sized mineral particles (i.e., sand, silt, and clay) and organic matter. They have complex biological, chemical, physical, and mineralogical properties that are always changing with time. Agronomists, farmers, and gardeners, on the other hand, see soil as a medium for growing 

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Robert W. Fitzpatrick

crops, pastures, and plants primarily in the top 50 cm of the earth’s surface. Engineers regard soil as material to build on and excavate and are usually concerned primarily with moisture conditions and the ability of soil to become compacted to support structures. However, some people regard soil as dirt or mud because it makes them “dirty” when they make contact with it. What do soils do for us? Soils provide a physical and chemical setting for gases, nutrients, and water. They also exchange heat for living organisms. In fact, biological activity, diversity, and productivity depend on the specific properties of soil. Soils also distribute surface water, causing runoff or infiltration, storage, and deep drainage. Consequently, water and solute flow on the earth’s surface is primarily controlled by soils. Soil acts as sinks and filters, reducing contaminants that affect the quality of water and other resources. It also provides many construction materials (e.g., bricks) and is the foundation for urban and recreational facilities. In addition, soils are usually involved in the burial of human, animal, or plant remains in cemeteries or special kinds of landfills. Large-scale cadaver or plant decomposition processes are typically associated with such burial facilities. According to Dent, Forbes, and Stuart (2004) the discussion of human decomposition in soils has been largely untreated in detail, and the fragments available are often incomplete. The application of approaches and methods developed in pedology now are recognized by microbiologists, archaeologists, and forensic scientists as crucial to the understanding of human decomposition processes, burial site location, and questions relating to soil taphonomy. Pedology (from the Greek pedon = soil) is the soil science discipline concerned primarily with understanding the variety of soils and their distribution and is most directly concerned with the key questions concerning sampling, descriptions, and interpretations of soils from crime scenes. Pedologists are primarily interested in the way the five soil forming factors (i.e., parent material, climate, topography, organisms, and time) affect the properties of present and past (paleopedology) soils in both its natural and disturbed state. Soil surveyors, on the other hand, are interested in describing and classifying soils (using different National and International Soil Classifications Systems) and then mapping them, usually on aerial photographs with the aid of remote sensing techniques and geographic information systems (GIS). Forensic soil scientists (or forensic geologists) are more specifically concerned with disturbed or moved soils (usually by human activity) and sometimes with comparing them to natural soils or by matching them with soil databases to help locate the scene of crimes. Forensic soil scientists usually obtain soil samples from crime or polluted scenes and nearby suspected control sites from which soil may have been transported, by vehicle, foot, or shovel. Soil properties are diverse, and this diversity may actually enable forensic soil scientists to use soils as evidence with more certainty in criminal and environmental investigations.

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Forensic Comparison of Soils



Identification of soil differences by using various soil attributes is the first step for using soil information to help police and environmental investigators at crime scenes (i.e., including exhumations) and polluted sites, respectively. Unfortunately, pedologists often use quite difficult and convoluted terminology in soil classification (taxonomy) and for producing soil maps that are hard to understand or that will have little apparent relevance in forensic investigations. Pedological terminology is often difficult to understand, and a special education is needed to interpret it easily and meaningfully. A variety of unique terms is often used in soil reports and in legends to soil maps. However, work in the field and in the laboratory carried out by pedologists involves an assessment of a wealth of mainly soil morphological features that can readily be interpreted in relation to soil processes and so allows soils to be forensically compared. This applied aspect is often obscured by preoccupation with using different national and international soil classification systems, especially for the “nonpedologist,” so it is time to revisit the science of pedology and to reemphasize its interpretive value to forensic science. In recent years pedologists have developed several user-friendly special-purpose classification systems, covering for example the following variety of practical issues: (1) engineering applications (e.g., optical fiber cable and pipe installations); (2) minesoils; (3) soils used for viticulture and forestry; (4) saline and acid sulfate soils (links to policy and jurisdiction); (5) topdressing materials; (6) urban planning; and (7) mineral exploration (e.g., Fitzpatrick et al. 2003). These special-purpose or technical classification systems all involve soil assessment criteria and recommendations for soil management practices to end users. This chapter therefore has two principal objectives: 1. To review some established concepts and standard terminologies used in pedology that have practical relevance to forensic science and to insoil human decomposition processes 2. To provide a brief example of the use of some pedological and related mineralogical methods in the forensic comparison of soils

1.2 Nature of Soils Relevant to Forensic Soil Science and Human Decomposition Processes In 1910 the French scientist Edmond Locard, inspired by the Adventures of Sherlock Holmes, postulated the fundamental principle on which forensic science and trace evidence is based, namely, “The Locard Exchange Principle” (Chisum and Turvey 2000). When two things come into contact, physical components can be exchanged. For example, the exchange can take the

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form of soil from a location transferring to shoes of a person who walked through a particular area. These types of transfers are referred to as primary transfers. Once a trace material has transferred, any subsequent moves of that material are referred to as secondary transfers. These secondary transfer materials can also be significant in evaluating the nature and sources of contact. Hence, the surface of soils can provide information linking persons to crime scenes. The following key issues are especially important in forensic soil examination because the diversity of soil strongly depends on topography and climate, plus anthropogenic contaminants: • Forensic soil examination can be complex because of the diversity and in-homogeneity of soil samples. However, such diversity and complexity enables forensic examiners to distinguish between soil samples, which may appear to be similar. • A major problem in forensic soil examination is the limitation in the discrimination power of the standard and nonstandard procedures and methods. No standard forensic soil examination method exists. The main reasons for this are that examination of soil is concerned with detection of both (1) naturally occurring soils (e.g., minerals, organic matter, soil animals, included rock fragments); and (2) manufactured materials in soils such as ions and fragments from different anthropogenic environments (e.g., synthetic fertilizers with nitrate, phosphate, and sulfate; artifacts or objects containing lead from glass, paint chips, asphalt, brick fragments, cinders) whose presence may impart soil with characteristics that will make it unique to a particular location. In addition, fine soil material may often only occur in small quantities, especially in the examination of materials from (1) the crime scene such as in Figure 1.1a, which shows a very small amount of yellowish-gray soil adhering to a suspects shoe, and (2) the control site such as in Figure 1.1b, which shows the complex diversity and in homogeneity of the soil sample from the bank of a river (Fitzpatrick, Raven, and Forrester 2007). The yellowish-gray soil at the control site comprises a mixture of 95% coarse gravel and rock fragments and only 5% clay and silt (< 50 µm fraction).

1.3  Brief History of Forensic Soil Science On a Prussian railroad in April 1856, a barrel that contained silver coins was found on arrival at its destination to have been emptied and refilled with sand. A soil scientist acquired samples of sand from stations along lines of railway and used a light microscope to match the sand to the station from which the sand must have come (Science and Art 1856). This is arguably the

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Forensic Comparison of Soils

Whole Soil

Sieved Smaller Sized Fractions < 100 µm Sieves

 Soil Morphology - All Samples • Soil Munsell Colour, Structure, Texture, Consistence • Stereo Binocular Microscopy Mineral and Organic Composition - All Samples • Mid IR Spectroscopy (450–8000 cm-1) Drifts Diffuse Reflectance Infrared Fourier Transform Spectral Analyses • Magnetic Susceptibility • X-ray Powder Diffraction (XRD)

Selected Samples –Depending Upon Individual Circumstances

< 2 µm

Heavy Mineral Fractionation

Magnetic Fractionation

• Powder XRD, Petrographic Microscopy • Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) • X-ray Fluorescence (XRF), ICP-MS, Gandolfi or Debye Scherrer XRD • FTIR, Mass Spectrometry, NMR, Thermal Analysis (DTA, TGA, DSC) • pH, Electrical Conductivity, Exchangeable Cations, CEC, Organic Carbon, Charcoal • Synchrotron Analysis, Others

Figure 1.1 A systematic approach to discriminate soils for forensic soil examinations using soil morphology (e.g., thickness, color, consistency, texture, structure), organic matter, mineralogy, geochemistry (e.g., spectroscopy, magnetic susceptibility analyses), and wet chemical techniques (x-ray diffraction, XRD; inductively coupled plasma spectroscopy mass spectroscopy, ICP-MS; Fourier transform infrared spectroscopy, FTIR; nuclear magnetic resonance, NMR; differential thermal analysis, DTA; thermogravimetric analysis, TGA; differential scanning calorimetry, DSC; cation exchange capacity, CEC. (From Fitzpatrick, R. W., Raven, M., and McLaughlin, M. J., in R. W. Fitzpatrick (ed.), Proceedings of the First International Workshop on Criminal and Environmental Forensics, http://www.clw.csiro.au/cafss/, May 2006. With permission.)

very first documented case where a forensic comparison of soils was used to help police solve a crime. Then in 1887 Sir Arthur Conan Doyle (Doyle 1981, p. 22) published several fictional cases involving Sherlock Holmes such as “A Study in Scarlet” in Beeton’s Christmas Annual of London, where Holmes can “tell at a glance different soils from each other … has shown me splashes upon his trousers, and told me by their color and consistence in what part of London he had received them.” In 1891 in “The Five Orange Pips,” Holmes observed, “chalk-rich soil”

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Robert W. Fitzpatrick

on boots. This clearly indicates that Conan Doyle (Doyle 1981, p. 217) was well aware of the key soil morphological properties (e.g., color and consistence) and soil mineralogy (e.g., chalk) in forensic soil comparisons. For the first time, as stated in Murray and Tedrow (1975), forensic scientist George Popp successfully examined soil collected from clothing associated with the murder of a seamstress named Eva Disch. Several recent reviews covering mainly “forensic geology” have been compiled by Ruffell and McKinley (2004) and Murray (2004). The issue of human decomposition processes in soils and the need to take into account the knowledge of soil environmental factors have been reviewed by several researchers (e.g., Dent et al. 2004; Garrison 2003; Spennemann and Franke 1995). Forensic soil science is a relatively new activity that is strongly method oriented because it is mostly a technique-driven activity in the multidisciplinary areas of pedology, geochemistry, mineralogy, molecular biology, geophysics, archaeology, and forensic science. Consequently, it does not have an overabundance of past practitioners such as in the older disciplines like physics and chemistry.

1.4  Soil Origin, Classification, and Distribution Pedology has two broad purposes: (1) to describe and classify; and (2) to interpret soil differences with respect to their management or use requirements. An appropriate definition of pedology is the area of earth science responsible for the quantification of factors and processes associated with soil formation (Wilding 1994). This includes the analysis of quality, distribution, and spatial variability of soils from micro- to megascopic scales (Wilding 1994). This definition introduces the phrase “extent, distribution, spatial variability, and interpretation” in a general way. It is fair to presume, though, that extent, distribution, spatial variability, and interpretation, for the pedologist, includes primarily the descriptive aspects of the science—the field and laboratory descriptions of soil attributes such as presence and degree of development of particular soil features (e.g., soil color, mottling) and the interpretive aspects of those attributes (e.g., soil in relation to drainage class or wetness). This description and its interpretation can then be explained in relation to the forensic comparison of soils. In addressing the questions, “What is the soil like?” and “Where does it come from?” (i.e., provenance determination), we are involved in studies relating to characterizing and locating the sources of soils to make forensic comparisons. The sophistication and effectiveness of soil classification reflects the level of scientific maturity and an understanding of the particular area of study (Simonson 1959). A major aim of classification is to usefully summarize the natural variability of forms the entity takes and to enhance communication

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about that entity. However, soil classification may stimulate or may discourage scientists with an interest in soils. If a classification system proves to be relevant and user friendly, it stimulates and encourages further work because it is recognized for its inherent capacity to create order and to enhance the useful understanding of soils (e.g., USDA 2003). This approach has provided numerous international soil scientists with valuable conceptual understanding of soils in terms of textural differentiation of profiles, the relative development of diagnostic horizons (e.g., gypsic, calcic, natric, argillic, oxic), subsoil color, and mottle differences. Many of the concepts in soil classification also provided effective pedotransfer functionality, particularly in terms of soil water attributes (e.g., Bouma 1989). If a soil classification is not useful it hinders transfer of information— often because of the lack of distinct separation between classes, many soils were inconsistently classified and distinguished, leading to conceptual confusion and pointless argument of subtle differences. For example, many soil classification systems are significantly biased toward agricultural soils, the subject of study for most soil scientists. Consequently, many soils found in nonagricultural environments are not suitably categorized because they do not match the central classification concepts (Fitzpatrick et al. 2003). Soil classification systems are important tools within the context of the forensic comparison of soils. They are our attempts to bring conceptual order into the complex world of soils and to allow knowledge gained in one location to be used in another, given that we are transferring that knowledge to similar soil conditions with similar properties. The great variety of soils and climates makes classification a major task even if soils were changing. To appreciate the scale of the task we have to recognize that: soils are changing (e.g., due to erosion, salinization, disturbance, and oxidation of acid sulfate soils), their evolutionary history is only partially understood, and they are used for a range of purposes, all with unique requirements in relation to soil function and land use. The demands on soil classification are therefore so diverse that they cannot be satisfied by a single system at any point in time or for any part of the world. Changes in classification will be made with advances in data collection, storage, and processing, but their value depends on how easily class groups can be interpreted in relation to soil functions and processes. A sound basis for interpreting soils and their use in forensic soil comparisons resides in an improved understanding of soil processes and the interpretation of these from soil morphology in soil landscapes. Soil formation, or pedogenesis, is a major activity for pedologists. The origins of soil attributes, distinctive horizons, and profiles must be understood to develop conceptual models for soil evolution over both long and short time periods (e.g., Smeck, Runge, and MacKintosh 1983). Such models have intuitive, predictive power in the forensic comparison of soils.

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Factors of soil formation were proposed in the 1860s in the United States by E. W. Hilgard (Jenny 1961a) and in the 1880s in Russia by V. V. Dokuchaev (Krupenikov 1993) and have been developed in a semiquantitative fashion by Jenny (1941, 1961b, 1980), who formulated the now well-accepted Clorpt equation. Soil formation and the properties of the soil are the result of the following five key factors: 1. Parent material: The material from which the soil is formed. Soil parent material could be bedrock, organic material, an old soil surface, or a deposit from water, wind, glaciers, volcanoes, or material moving down a slope. 2. Climate: Heat, rain, ice, snow, wind, sunshine, and other environmental forces break down the parent material and affect how fast or slow soil processes proceed. 3. Organisms: All plants and animals living in or on the soil (including microorganisms and humans). The amount of water and nutrients plants need affects the way soil forms. Animals living in the soil affect decomposition of waste materials and how soil materials will be moved around in the soil profile. The dead remains of plants and animals (including human cadavers) become organic matter, which enriches soils. The way humans use soils influences soil formation. 4. Topography: The location of a soil in a landscape can affect how the climatic processes influence it. Soils at the bottom of a hill will get more water than soils on the slopes, and soils on the slopes that directly face the sun will be drier than soils on slopes that do not. 5. Time: All of these factors assert themselves over time, often over hundreds or thousands of years—but can even be hours (e.g., erosion; oxidation of pyrite to form sulfuric acid in acid sulfate soils). Simonson (1959) proposed a more general framework for soil formation based on four groups of processes: additions, transfers, transformations, and removals. A dynamic approach to pedogenesis, building on the perspectives offered by Jenny (1941) and Simonson (1959), can be used to provide a framework for the assessment of soil properties at different spatial and temporal scales. The way the five soil-forming factors interact differs from one place to another; accordingly, soils may differ greatly from each other. Each section of soil on a landscape has its own unique characteristics. The face of a soil, or the way it looks if one cuts a section of it out of the ground, is called a soil profile. Every soil profile is made up of layers called soil horizons. Soil horizons can be as thin as a few millimeters or thicker than a meter. Usually, each soil layer or horizon is given a pedogenic notation (e.g., A, B, C) indicating its position in the soil profile and drawing attention to

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Forensic Comparison of Soils



special features that may influence the use of the soil, such as surface layers or pans cemented by iron. Master pedogenic horizons or layers have the following features: A1: Surface horizon, usually with higher organic carbon content E: Paler subsurface horizon B: Subsurface horizon, usually with more clay and brighter colors C: Horizon with weathered rock or underlying sediment R: Indurated rock layer W: Water layers within or beneath the soil Soils are often also discussed in terms of topsoil and subsoil. These terms are not rigorously defined, but denote the following: • Topsoil: The surface zone, including the zone of accumulation of organic material (usually the A horizons). Topsoil can be modified by anthropogenic practices, such as road or foot traffic, plowing, and addition of fertilizers. • Subsoil: Underlying layers (B and C horizons), which cannot usually be modified except by deep excavation (e.g., graves) and drainage Soil descriptions follow strict conventions whereby a standard array of data is described in a sequence and each term is defined according to both the U.S. Department of Agriculture (USDA) Field book for describing and sampling soils, Version 2.0 (Schoeneberger et al. 2002) and national standard systems (e.g., McDonald et al. 1990). Soil morphological descriptors such as color, consistency, structure, texture, segregations/coarse fragments (charcoal, ironstone, or carbonates) and abundance of roots/pores are the most useful properties to aid the identification of soil materials (e.g., Fitzpatrick et al. 2003) and to assess practical soil conditions (e.g., Yaalon and Yaron 1966). Soil profiles and their horizons usually change across landscapes and also change as one digs deeper in the soil at one location. In fact, soil samples taken at the surface may have entirely different characteristics and appearances from soil dug deeper in the soil profile. One common reason that soil horizons are different as one digs deeper is the mixing of organic material in the upper horizons and weathering and leaching in lower horizons. Erosion, deposition, and other forms of disturbance might also affect the way a soil profile looks at a particular location. For example, soils on alluvial flats with regular flooding often have clear sedimentary layers. Various soil-forming processes create and destroy layers, and it is the balance between these competing processes that will determine how distinct layers are in a given soil. Some of the more common natural processes include the actions of soil fauna (e.g., worms, termites) and the depletion and accumulation and constituents

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10

Robert W. Fitzpatrick

including clay, organic matter, and calcium carbonate. In contrast, the anthropogenic soil-forming processes that destroy layers are excavation (e.g., plowing and grave digging) and fertilizer applications. In natural conditions the speed of pedogenesis is such that it is barely perceptible within human generation spans. In anthropogenic conditions the speed, and direction, of pedogenesis can be altered through engineered effects on the soil forming factors and processes. Microclimate is modified by irrigation, fallows, and mulches; organisms are reselected and controlled for agriculture and forestry; relief is altered by land forming as leveling or contour bank construction; parent material is augmented with fertilizers and mulches but deprived by crop and residue removal. These modified factors in turn influence the rates of the soil forming processes. Fertilizers, tillage (erosion), crop removal, and irrigation (leaching fraction) alter the balance of additions and removals, whereas irrigation affects transfers within the system, for example by mobilization of free CaCO3 in the upper part of the soil and precipitation deeper in the B horizon. Transformations such as humification/mineralization, mineral weathering, and clay degradation are all modified by the increased oxidation due to tillage, and hydrolysis due to changed moisture regimes. These human induced changes in pedogenesis have been referred to as metapedogenesis (Yaalon and Yaron 1966). In assessing soil for forensic comparisons or detecting buried objects (e.g., exhumations), metapedogenetic processes need to be clearly distinguished from the natural rate of pedogenesis.

1.5  Spatial Scale and Pedogenic Processes Dijkerman (1974) suggested an organizational hierarchy, based on size, of seven subsystems for soil studies and discussed the relationship of empirical scientific methodology to these different levels. Other researchers (e.g., Hoosbeek and Bryant 1992; Sposito and Reginato 1992) adopted a similar approach. The pedon (Soil Survey Staff 1988) is accepted as the basic threedimensional unit of soil encompassing the variations in horizon and profile features that would fully characterize the soil type under investigation. The pedon exists in the larger subsystem hierarchy of polypedon, toposequence, and catchment or region and contains smaller subsystems of horizons, peds, mineral organic complexes, and minerals. Investigation of soil at the pedon scale should always include details at the horizon scale and context at the toposequence or catchment scale (soil landscape). Description and quantification of attributes of subsystems, including their spatial variability, is advanced at all scales. Soil assessment can therefore be related at all scales of pedological interest. However, the temporal variability of soils is less well understood or

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documented. Assessment of soil features and properties that may change in anthropogenic time scales requires clarification of rates of change of pedological attributes in response to pedogenic processes. Hoosbeek and Bryant (1992) reviewed progress toward the quantitative modeling of soil processes. They characterized models with respect to their relative degree of computation (qualitative to quantitative), complexity (functional to mechanistic), and level of organization (microscopic to megascopic). They concluded that although qualitative models have aided in soil survey and understanding soils in landscapes, there is a need to devise more quantitative models to predict how soils will change in the future. This is particularly important if soil properties are to be used in making forensic comparisons.

1.6 Relationship between Soil Type and Scale: Regional and Global Typically, maps are used to provide pictorial representations of the distribution of soils, each map varying according to the specific soil classification scheme used. At levels above the soil profile scale in a soil landscape, or at small scales of investigation (1:250,000 and smaller) soil data become generalized, and soil comparisons for forensic purposes also become more generalized. Hence, map units at scales smaller than 1:50,000 cannot represent a single kind of soil. Reporting forensic soil comparisons using map data at these scales should only be used for intelligence (i.e., providing information for broad considerations) purposes and not for evidence. The reliability of such soil assessments depends on the density and quality of soil data collected at larger scales.

1.7 Most Favored Techniques Used by Forensic Soil Scientists The major question posed now is how can soils be used to make accurate forensic comparisons when we know that soils are highly complex and that there are thousands of different soil types in existence? For example, according to the USDA, which collects soil data at many different scales, there are more than 50,000 different varieties of soil in the United States alone. Parent material, climate, organisms, and the amount of time it takes for these properties to interact will vary worldwide. First of all, soil samples must be carefully collected and handled at the crime scene and then compared by a soil scientist with forensic science experience to ensure that the soil samples can be useful during an investigation.

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1.7.1  Theory of Making Comparisons between Soil Samples It is important to first define the word compare because no two physical objects can ever, in a theoretical sense, be the same (Murray and Tedrow 1991). Similarly, a sample of soil or any other earth material cannot be said, in the absolute sense, to have come from the same single place. However, according to Murray and Tedrow (1991) it is possible to establish “with a high degree of certainty that a sample is or is not associated with a given scene.” For example, a portion of the soil (or other earth material) could have been removed to another location during human activity. 1.7.2  A  pproaches and Methods for Making Comparisons between Soil Samples Forensic soil scientists must first determine if uncommon and unusual particles, or unusual combinations of particles, occur in the soil samples and compare them with similar soil in a known location. To do this properly the soil must be systematically described and characterized using standard soil testing methods to deduce whether a soil sample can be used as evidence (Figure 1.1). Methods for characterizing soils for a forensic comparison involve subdividing them into two major categories: descriptive (morphological) and analytical (Figure 1.1). Morphological soil indicators are arguably the most common and probably the simplest—and it is for this reason that all samples are characterized first using the four key morphological descriptors (Figure 1.1). In many respects, the soil resembles a sandwich with easily observed characteristics of thickness, color, consistency, texture, and structure, which convey the concept of different soil layers with different properties. In soil samples from crime scenes (polluted sites) and control sites in question where soil may have been transported—by vehicle, foot (e.g., Figure 1.2a), or shovel perhaps—and are suspect, these four visual properties are important indicators. The following checklist of six key soil morphological descriptors has been compiled from standard techniques used in soil science (e.g., Schoeneberger et al. 2002) for assessing the soil properties for forensic examinations. These are: (1) observations of depth changes in consistence, (2) color, (3) texture, (4) structure, (5) segregations/coarse fragments (carbonates and ironstone), and (6) abundance of roots in the different layers or horizons. Morphological descriptors are useful in assessing soil conditions for the following reasons: • They are rapid field and laboratory assessments. Other methods, such as mineralogy (see next section) and geochemistry, are complex and more costly to carry out.

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Figure 1.2  Contact traces of yellowish-gray soil on the suspect’s shoes (left) and the control soil specimen from the bank of a river (right), which comprises a mixture of 95% coarse gravel and rock fragments and only 5% clay and silt (< 50 µm fraction). (See color insert following p. 178.)

• They can be used in research to evaluate causes for variation in soil condition induced by anthropogenic activities, land management, hydrology, and weather conditions. 1.7.2.1  Soil Color In particular, soil color should be determined on dry and moist samples using Munsell Soil Color Charts (1994). Soil color is usually the first property recorded in a morphological description of soils (and may be the only feature of significance to a layperson) and provides an indicator of redox status because soil color relates to soil aeration and organic matter content (Fitzpatrick, McKenzie, and Maschmedt 1999). Soil color has been found to be extremely useful in forensic soil identification by Sugita and Marumo (1996). This more objective notion of soil color uses three coordinates: hue (shade), value (lightness), and chroma (intensity). Hue is the color frequency and in most soils ranges from red to yellow. Value or tone refers to lightness from white to black, and chroma defines the degree of color saturation or intensity of hue. Red soil matrices are generally described with hues 5 YR or redder (and chroma greater than 1), reddish with hues 7.5 YR (and chroma greater than 1), and yellow with hues 7.5 YR or yellower. Dark colors have low value (< 3) and low chroma (< 2). Training is recommended before consistent color matching is made (Post et al. 1993).

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Color of soils depends on the type of iron oxides and content of organic matter (Bigham and Ciolkosz 1993). Uniform high chroma red and yellow colors (hues) indicate oxidizing conditions, and uniform low chroma colors (dark gray and blue tints) indicate reducing, waterlogged, or aquic conditions. Mixtures of bright red or yellow soil matrices with blotches of dark gray or bluish form one type of mottle and indicate periodic conditions of water saturation (USDA 1998; Vepraskas 1992). Red soils are nearly always better drained than yellow soils. The content and type of iron oxide affects soil chemistry. Several workers (e.g., Scheinost and Schwertmann 1995) have shown that phosphate adsorption maxima increase from red (hematitic) to yellow (goethite-rich) soils. Consequently, because yellow soils in some regions are closely correlated to soil P sorption, soil color has been used to predict the likely need for phosphate applications. 1.7.2.2  Soil Consistence Soil consistence is a measure of the strength and coherence of a soil. Soil consistence or consistency is also called rupture resistance and is a very readily observed feature in the field. Consistence of a soil material can be measured in the field by simply manipulating a piece of soil in the hand and determining the magnitude of force needed to cause disruption or distortion. Consistence is expressed as loose, soft, firm, very hard, and rigid (USDA 1993). Terms used to describe consistence vary depending on the moisture content of the sample tested (e.g., soft when dry versus friable when moist). Changes in soil consistence with depth (cm) are recorded in the field using the field description checklist sheet (Table 1.1). The magnitude of force needed to cause disruption or distortion by manipulating a piece of block-like (25 mm to 30 mm on edge) soil in the hand or under foot. Stress is applied along the vertical in-plane axis of the block-like piece of soil by compressing it between extended thumb and forefinger, between both hands, or between foot and hard, flat surface. Obvious factors that influence consistency include soil texture, mechanical compaction, organic matter content, cementing agents, and water content. It is for this reason that consistency is best measured or assessed when the soil is either dry (i.e., standard moisture content) or moist (Table 1.1). If the piece of block-like soil is less than 25 mm to 30 mm on edge, then corrections should be made for class estimates given in Table 1.2 (i.e., 10 mm block will require about one third the force to rupture it). Changes in soil consistence are a useful surrogate measure for identifying restrictive layers because soil texture and structure are often difficult to measure consistently by inexperienced operators and because root abun-

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dance depends on other factors such as climate, vegetation type, soil fertility, and land management. 1.7.2.3  Soil Texture Field soil texture reflects the proportion of sand (2–0.02 mm), silt (0.02–0.002 mm) and clay (< 0.002 mm) in soil (Table 1.2). Field or hand soil texture is determined in the field by the following procedure: • Take a sample of soil sufficient to fit comfortably into the palm of the hand (separate out gravel and stones). Moisten soil with water, a little at a time, and work until it just sticks to your fingers and is not mushy. This is when its water content is approximately at field capacity. • Continue moistening and working until there is no apparent change in the ball (bolus) of soil. This usually takes one to two minutes. • Attempt to make a ribbon by progressively shearing the ball between thumb and forefinger. The behavior of the worked soil and the length of the ribbon produced by pressing out between thumb and forefinger characterizes ten selected soil texture grades as shown in Table 1.2. This surrogate is used to estimate the following: • Water and nutrient retention or leaching capacity: Coarse-grained sands have larger pores than those found in finer-textured soils. Consequently, coarse sands are typically drained rapidly and have a poor ability to hold water and nutrients. Loamy sands hold more water and nutrients, whereas the available water capacity and nutrient retention ability of clays are high. • Depth to restricting layers or subsurface compaction that may affect root growth or water movement (e.g., subsurface compaction, structure decline): Sandy soils are generally more prone to subsurface compaction than finer-textured soils. • Erodibility (e.g., sands are more easily eroded by wind): Grain size may also affect the susceptibility to erosion. Fine sand grains are easily transported by the wind. Coarser grains are heavier and require more force to be moved. Clay particles, though light and easy to transport, are often difficult to detach because they are bound together. Consequently, a trend in the change of texture down a profile is frequently used to classify soils (e.g., Isbell 1996; Northcote 1979) because of its importance for plant growth and water movement.

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Loose

Very weak to weak

Firm to very firm

1

2

3

Class Number

Firm

Soft

Loose

Dryb

Firm

Friable

Loose

Moistb

Consistence Classes Australiana/ U.S. Department of Agricultureb

Table 1.1  Interpreting Soil Consistence Consistence Test Inferred from Excavation Difficultyb

Environment Indication

Block-like piece not Can be excavated with a spade No restriction on root obtainable. Only individual using arm-applied pressure. growth for annuals and sand grains can be picked Neither application of impact perennials. No up between thumb and energy nor application of restriction on water forefinger. (0) pressure with the foot to a movement. spade is necessary. Fails (i.e., crumbles) under Arm-applied pressure to a Root growth of annuals slight force applied spade is insufficient. and perennials is between thumb and Excavation can be unrestricted. Slight forefinger. (< 8–20) accomplished quite easily by restriction on water application of impact energy movement; water is with spade or by foot usually available to pressure to spade. most crops and trees. Fails under moderate to Excavation with spade can be Water flow can be strong force applied accomplished, but with restricted contributing between thumb and difficulty. Excavation is easily to periodic forefinger. (20–80) possible with a full length waterlogging. pick using an over-the-head swing.

Rupture Resistance on a 25–30 mm Fragment of Dry Soil

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Rigid

5

Rigid

Very hard

Rigid

Very firm

Excavation with a full-length Root growth of most pick using an over-the-head species is restricted. swing is moderately to Water flow may be markedly difficult. restricted. Excavation is possible in a reasonable period of time with a backhoe mounted on a 40–60 KW (50–80 hp) tractor. Cannot be ruptured by blow Excavation is impossible with Root growth of most with hammer. (> 800) a full-length pick using an species is severely over-the-head arm swing or restricted. Water flow is with reasonable time period normally restricted. with a backhoe mounted on a 40–60 KW (50–80 hp) tractor.

Cannot be ruptured between thumb and forefinger but can be by applying full body weight under foot. (80–800)

Notes: Figures in parentheses are the force needed for failure in newtons. Two systems are used to describe soil consistence. Table 1.2 approximately correlates the two systems by using five classes with corresponding field tests and environmental indications. The force (expressed in newtons) required to fail a fragment of soil is calculated from the weight required to crush the fragment (expressed as in kg force) multiplied by 9.81, the gravitational factor. (a) The Australian system uses the concept of soil strength and is measured on a 20 mm piece of soil. The class names are the same for all moisture contents, but soil water status must be recorded. (b) The USDA system uses fragments of soil about 25–30 mm in size and has different class names for different moisture contents (dry and moist).

Strong to very strong

4

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S

LS

CS

SL

L

ZL

SCL

CL

Loamy sand

Clayey sand

Sandy loam

Loam

Silty Loam

Sandy clay loam

Clay loam

Code

Sand

Texture

40–50 mm

25–40 mm

25 mm

25 mm

15–25 mm

5–15 mm

5 mm

nil

Ribbon

coherent and plastic

Coherent and rather spongy Strongly coherent

Coherent and rather spongy

Coherence slight

Coherence nil to very slight Coherence very slight

Coherence nil to very slight

Ball

Sandy to touch; medium-size sand grains visible in finer matrix. Clay is 20–30%. Smooth to manipulate. Clay is 30–35%.

Smooth feel; may feel greasy if organic matter is present. Clay is about 25%. As above but more silky feel

Cannot be molded. Clay is 5–10%. Cannot be molded. Clay is 5–10%. Sandy to touch. Clay is 10–20%

Cannot be molded. Clay is < 5%.

Feel and Approximate Clay Content

Table 1.2  Interpreting Soil Texture from Behavior of a Moist Bolus (Ball)

As above

As above

Root growth of annuals and perennials is not restricted but has a high susceptibility to mechanical compaction. Very slight restriction on water movement; soil water is available to most crops and trees. Water drains from the soil readily but not rapidly. Root growth of annuals and perennials is not restricted with moderate susceptibility to mechanical compaction. As above

As above

Minimal physical restriction to root growth for annuals and perennials but has a moderate susceptibility to mechanical compaction. No restriction on water movement, but periodic soil moisture stress is common because water is drained very rapidly. As above

Interpretation

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MC

HC

Medium clay

Heavy clay

>75 mm

>75 mm

50–75 mm

smooth plastic

smooth plastic

plastic

Root growth of annuals and perennials is frequently restricted with moderate susceptibility to mechanical compaction. Soil water is available to most crops and trees. Water flow is restricted contributing to periodic waterlogging. Root growth of most species is moderately to severely restricted but with low susceptibility to mechanical compaction. Water drains very slowly. This does not apply to self mulching or subplastic clays. As above

sandy loam (SL) loam (L), sandy clay loam (SCL), silty loam (ZL) clay loam (CL) light clay (LC) medium clay (MC), heavy clay (HC)

2. The Sandy Loams 3. The Loams 4. The Clay loams 5. The Light Clays 6. The Medium-Heavy Clays

The Texture Groups according to Northcote (1979) sand (S), loamy sand (LS), clayey sand (CS) 1. The Sands

Can be molded into rods without fracture; has firm resistance to ribboning shear. Clay is > 55%

Can be molded into rods without fracture; has some resistance to ribboning shear. Clay is 45–55%.

Smooth to touch; slight to shearing between thumb and forefinger. Clay is 35–40%.

Source: Adapted from McDonald, R. C., Isbell, R. F., Speight, J. G., Walker, J., and Hopkins, M. S., Australian Soil and Land Survey: Field Handbook, 2nd ed., Melbourne, Australia: Inkata Press, 1990 (with permission).

Texture qualifiers: used as a prefix to refine texture description Coarse Coarse to touch; sand grains can be seen with the sandy naked eye Can be felt and often heard when bolus is Fine sandy manipulated; sand grains seen under hand lens of 10 times magnification Gritty More than 35% very coarse sand and very fine (1–3mm) gravel 35–70% of gravel by volume Gravely Stony 35–70% of stones by volume

LC

Light clay

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1.7.2.4  Soil Structure Soil structure relates to the way soil particles are arranged and bound together (Schoeneberger et al. 2002). Soil structure can easily be described from the visible appearance of in situ soil in a dry to slightly moist state by the presence or absence of the following: • Peds (granular, lenticular, platy, blocky, polyhedral, columnar, and prismatic) • Single grain or structureless • Massive • Slickensides (shiny, cracked, or grooved clay surfaces) The size, shape, and nature of soil aggregates, peds, or slickensides play a major role in determining profile hydrology and the ease of root penetration. Where soil particles are bound together in naturally formed aggregates (peds) separated by irregular spaces, the soil is described as having structure. The degree and nature of structural development is largely determined by clay mineralogy and organic matter content. Peds result from the natural subdivision of the soil by fine cracks to form either small (granular or polyhedral) or large blocks (columnar, prismatic, and platy). The cracks separating these peds do not usually have shiny slickensided surfaces, but ped size and development may range from weak to strong. Where peds are largely absent, the soil is described as being structureless. In a singlegrained material, two thirds of a soil is composed of individual particles, which are not bound together (loose and incoherent). In a massive material, two thirds of the soil occurs in one large block with the particles being bound together (coherent). Slickensides are easily observable shiny planes of weakness along which movement occurs in shrink–swell medium-to-heavy clay soils. These are shearing faults, which exist permanently in wet or dry expansive clays. They take the form of cracked, polished, or grooved surfaces, ranging from 10 mm to 200 mm across. Slickensides often run through the soil mass in many directions and may break the structure up into bowl-shaped blocks. They can move up to 25 mm per year. Hence, the frequency and size of slickensides present can quantify the potential capacity of the soil to shrink and swell (i.e., develop cracks when dry). Soils or soil layers with slickensides are highly impermeable to water movement, especially when they are moist and root growth is restricted. 1.7.2.5  Segregations and Coarse Fragments Segregations are accumulations of distinct mineral particles such as iron oxides, calcium carbonate, and gypsum that have formed in soil. They occur

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in a variety of sizes, shapes, and forms and can be either soft or hard. In many parts of the world, these segregations are common and can have a major influence on soil chemical and physical properties. Calcium carbonate commonly occurs either as masses and nodules. Hydrochloric acid (1M HCl) is commonly used in the field to confirm the presence of calcium carbonate in the soil (Schoeneberger et al. 2002). The reaction between acid and soil carbonate causes effervescence. This is known as the fizz test. Gypsum is often present as crystals, which glisten. They generally occur in lower rainfall areas and often indicate high electrolyte concentrations in soils. Coarse fragments include rock fragments, strongly cemented soil materials, and hard segregations, which are sized greater than 2 mm.

1.8 Petrographic and Other Advanced Techniques and Instruments The use of petrography is a major and often precise method of studying and screening soils for discrimination in forensics. For example, nearly fifty common minerals as well as several less common minerals can easily be seen by the naked eye (e.g., gypsum), but using a lens or low-power stereo-binocular microscope enables the forensic soil scientist to better detect mineral properties and to provide more accurate mineral identification. The petrographic microscope is also a common instrument used to study thin sections of soil samples (resin impregnated), minerals, and rocks. Thin sections of soil materials are mounted on a glass slide and are viewed with the petrographic microscope under different incident light conditions through its special attachments (e.g., Stoops 2003). A new rapid mid-infrared spectroscopic method called diffuse reflectance infrared Fourier transform spectra (DRIFTS), coupled with chemometrics, has been developed by Janik, Merry, and Skjemstad (1998) and routinely applied to rapidly screen and compare crime scene samples (Figure 1.1). Added to these rapid methods and techniques are the use of rapid mass and volume magnetic susceptibility methods, which should also always be used before moving to the more costly methods (Figure 1.1). Mineral magnetic techniques are a relatively recent development (post-1971) and have now become a very powerful and widely used research tool to characterize natural materials in landscapes (e.g., Thompson and Oldfield 1986). X-ray diffraction (XRD) methods are arguably the most significant for both qualitative and quantitative analyses of solid materials in forensic soil science. Extremely minute sample quantities or tiny sample areas as well as large quantities can be successfully analyzed using XRD. The critical

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advantage of XRD methods in forensic soil science is based on the unique character of the diffraction patterns of crystalline and even poorly crystalline soil minerals. Elements and their oxides, polymorphic forms, and mixed crystals can be distinguished by nondestructive examinations. Part of the comparison involves identification of as many of the crystalline components as possible, either by reference to the ICDD Powder Diffraction File (Faber, Fawcett, and Goehner 2005) or to a local collection of standard reference diffraction patterns (e.g., Rendle 2004). For analysis in a Debye-Scherrer powder camera, extremely small specimens (e.g., paint flakes) can be mounted on the end of glass fibers. Consequently, according to Kugler (2003), x-ray methods are often the only ones that will permit further differentiation of materials under laboratory conditions. Methods such as XRD, XRF, and DRIFTS are used, whose results overlap. These overlapping results confirm each other and give a secure result to the examination. Scanning electron microscopes (SEMs) and transmission electron microscopes (TEMs) are also frequently used to examine the morphology and chemical composition (via energy dispersive spectroscopy) of particles more than 100,000 times their original size, making them very useful. Soil minerals, fossils, and pollen spores that occur in soils and can be described and analyzed in detail by SEMs and TEMs and are very useful indicators when studying soil samples. All these techniques in combination achieve reliable, definite, and accurate results and provide additional information about the chemical and physical properties of the suspected material. For example, the following soil analyses methods were required in a burglary case (Fitzpatrick et al. 2007). The first step was to visually compare the suspect soil specimen (i.e., adhered soil scraped from the soles and sides of the running shoes shown in Figure 1.2a) and control specimen (i.e., soil shown in Figure 1.2b, which was obtained from the bank of the river where the suspect was seen to run through). This visual comparison was conducted by eye and by low-power stereo-binocular microscope light microscopy. From these detailed visual observations, it appeared that the fine fraction in the riverbank sample had a similar yellow color to the soil adhered to the shoe. Consequently, because the riverbank sample contained more than 95% coarse gravel and stones, a subsample was sieved using a 50 µm sieve to obtain a finer fraction (< 50 µm). The fine soil fraction from the riverbank and soil on the shoe had a remarkably similar color (Munsell color) and mass magnetic susceptibility. Hence, in accordance with the systematic approach outlined in Figure 1.1, the third step was to check their mineralogical and chemical composition by using XRD and DRIFTS analyses. The XRD diffraction patterns of the shoe (suspect) and riverbank (control) soil samples closely match each other—a technique that can be likened to fingerprint comparisons (Figure 1.3). However, what is the significance of this close match? If the two soil samples, for example, contain only one

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Comparison of LRJ-1(Red) with Clay from Sole of Shoe (Black) 24 22 Intensity (Counts) × 100

20 18 16 14

46-1045 9-466 31-966 36-426 6-263 29-701 5-586 41-1366 14-164

Quartz, SYN Albite, Ordered Orthoclase Dolomite Muscovite-2M1 Clinochlore-1MIIB, FE-RICI Calcite, SYN Actinolite Kaolinite-1A

12 10 8 6 4 2 10.00

20.00 30.00 2-Theta Angle (deg)

40.00

Figure 1.3  Comparisons between x-ray diffraction (XRD) patterns of soil samples from the shoe and riverbank (< 50 µm fraction) shown in Figure 1.2. The < 50 µm fraction was separated from the stony riverbank soil by sieving through a 50 µm sieve. Shoe and riverbank samples were both ground using an agate mortar and pestle before being lightly pressed into aluminum sample holders for XRD analysis. XRD patterns were recorded with a Philips PW1800 microprocessor-controlled diffractometer using Co Kα radiation, variable divergence slit, and graphite monochromator. (From Fitzpatrick, R. W., Raven, M. D., and Forrester, S. T., CSIRO Land and Water Client Report CAFSS_027, 2007. With permission.) (See color insert following p. 178.)

crystalline component such as quartz (i.e., silicon dioxide), which is very common in soils, the significance of the match and its evidential value will be low. If, however, the two soils contain four or five crystalline components, some of them unusual, then the significance of the match and its evidential value will be considered to be high. The mineralogical compositions of the two samples are summarized in Table 1.3 and are very similar; containing quartz, mica, albite, orthoclase, dolomite, chlorite, calcite, amphibole, and kaolin. Relative proportions of the minerals are slightly different, likely due to the different particle sizes of the samples. DRIFTS analyses, or Fourier Transform Infraredspectroscopy (FTIR), was conducted on the same samples used for XRD analyses (Fitzpatrick et al. 2007). Light energy in the mid-infrared range (8000–450 cm-1) is focused on the surface of the air-dried, finely ground soil samples. Some of the light beam penetrates a small distance into the sample and is reflected back into the spectrometer where the spectrum is collected. Although the two samples are spectrally similar (Figure 1.4) they do differ slightly in the amount of organic matter, which is reflected in some of broad peaks (i.e., because

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24

Table 1.3  Summary of Mineralogical Composition from X-Ray Diffraction Analysis Soil Samples River­banka Shoe

Quartz

Orthoclase

Mica

Albite

D

SDS

M

M

D

M

M

M

Dolomite

Chlorite

Calcite

Amphibole

Kaolin

M

T

T

T

T

T

T

T

T

T

Where < 50 µm fraction; D, dominant (> 60%); SD, subdominant (20–60%); M, minor (5–20%); T, trace (< 5%)%). Source: Adapted from Fitzpatrick, R. W., Raven, M. D., and Forrester, S. T., CSIRO Land and Water Client Report CAFSS_027, 2007 (with permission). a

2.0

1.5

1.0

0.5 499.99988 cm–1

Variables 1.37600e+03 cm–1 2.25200e+03 cm–1 3.12800e+03 cm–1

Figure 1.4  Comparison of diffuse reflectance infrared Fourier transform spectral (DRIFTS) patterns between the yellow-brown soil on the shoe (red) and the < 50 µm fraction in the stony soil from the riverbank (blue). Shoe and riverbank samples were both ground using an agate mortar and pestle. (From Fitzpatrick, R. W., Raven, M. D., and Forrester, S. T., CSIRO Land and Water Client Report CAFSS_027, 2007. With permission.) (See color insert following p. 178.)

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25

the shoe sample has slightly higher organic carbon content), indicating that the two samples are most likely to have been derived from the same general location. To conclude, sufficient soil morphological, mineralogical (XRD), and physicochemical (DRIFTS) data were acquired on the two samples to be able to determine if they compare or do not compare. The soil from the shoe is therefore most likely sourced from the stony/gravely soil on the riverbank.

1.9  Conclusions Qualitative soil morphological information should form an essential part of all investigations of crime scenes and burial site locations. Visual, morphological observations must be used by forensic soil scientists in the field and in the laboratory to recognize and characterize key soil features. The interpretation of these tests and methods is not equally applicable to all soils and should also be made in the context of the forensic soil examination (e.g., sieving of samples to obtain a more representative sample to make comparisons). Ideally, soil testing and soil interpretations should be carried out by experienced forensic soil scientists. Soil scientists, crime scene investigators, and forensic scientist groups must work closely to further test the proposed systematic approach to discriminate soils for forensic soil examinations using soil morphology (e.g., thickness, color, color, consistency, texture, structure), organic matter, mineralogy, geochemistry (e.g., spectroscopy, magnetic susceptibility analyses), geophysics, wet chemical techniques, and electron microscopy. This information is being used to develop and refine methodologies and approaches to develop a practical soil forensic manual with a soil kit for sampling, describing, and interpreting soils.

References Bingham J. M. and Ciolkosz E. J. (Eds.) (1993). Soil Color. Madison, WI: Soil Science Society of America Special Publication 31. Bouma, J. (1989). Using soil survey data for quantitative land evaluation. Adv. Soil Sci. 9, 177–213. Chisum, W. and Turvey, B. (2000). Evidence dynamics: Locard’s exchange principle and crime reconstruction. J. Behavioral Profiling 1, 1-15. Dent, B. B., Forbes, S. L., and Stuart, B. H. (2004). Review of human decomposition processes in soil. Environ. Geol. 45, 576–585. Dijkerman, J. C. (1974). Pedology as a science: The role of data, models and theories in the study of natural soil systems. Geoderma 11, 73–93.

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Faber, J., Fawcett, T., and Goehner, R. (2005). Powder Diffraction File (2003). Newtown Square, PA: The International Centre for Diffraction Data. http://journals.cambridge.org/downloadphpfile. Fitzpatrick R. W., McKenzie, N. J., and Maschmedt, D. (1999). Soil morphological indicators and their importance to soil fertility, in Soil Analysis: an Interpretation Manual (K. Peverell, L. A. Sparrow, and D. J. Reuter, Eds.). Melbourne, Australia: CSIRO Publishing, 55–69. Fitzpatrick, R. W., Powell, B., McKenzie, N. J., Maschmedt, D. J., Schoknecht, N., and Jacquier, D. W. (2003). Demands on soil classification in Australia, in Soil Classification: A Global Desk Reference (H. Eswaran, T. Rice, R. Ahrens, and B. A. Stewart, Eds.). Boca Raton, FL: CRC Press, 77–100. Fitzpatrick R. W., Raven M. D., and Forrester, S. T. (2007). Investigation to determine if shoes seized by South Australia Police contain soil materials that compare with a control soil sample from the bank of the Torrens River, Adelaide. CSIRO Land and Water Client Report CAFSS_027. Fitzpatrick R. W., Raven M., and McLaughlin M. J. (2006, May). Forensic soil science: An overview with reference to case investigations and challenges, in Proceedings of the First International Workshop on Criminal and Environmental Forensics (R. W. Fitzpatrick, Ed.). Perth. http://www.clw.csiro.au/cafss/. Garrison, E. C. (2003). Techniques in Archaeological Geology. Berlin: Springer-Verlag. Hoosbeek, M. R. and Bryant, R. B. (1992). Towards the quantitative modelling of pedogenesis a review. Geoderma 55, 183–210. Isbell, R. F. (1996). The Australian Soil Classification. Collingwood, Australia: CSIRO Publishing. Janik, L. J., Merry, R. H., and Skjemstad, J. O. (1998). Can mid-infrared diffuse reflectance analysis replace soil extractions? Aust. J. Exp. Agric. 38, 637–650. Jenny, H. (1941). Factors of Soil Formation: A System of Quantitative Pedology. New York: McGraw Hill. Jenny, H. (1961a). E. W. Hilgard and the Birth of Modern Soil Science. Pisa, Italy: Collana Della Rivista Agrochimica. Jenny, H. (1961b). Derivation of the state factor equations of soils and ecosystems. Soil Sci. Soc. Am. Proc. 25, 385–388. Jenny, H. (1980). The Soil Resource: Origin and Behaviour. New York: Springer Verlag, Ecological Studies 37. Krupenikov, I. A. (1993). History of Soil Science. Rotterdam, The Netherlands: Balkema. Kugler W. (2003). X-ray diffraction analysis in the forensic science: The last resort in many criminal cases. Adv. X Ray Anal. 46, 1–16. McDonald, R. C., Isbell, R. F., Speight, J. G., Walker, J., and Hopkins, M. S. (1990). Australian Soil and Land Survey: Field Handbook, 2d ed. Melbourne, Australia: Inkata Press. Munsell Soil Color Charts (1994). Munsell Color. New Windsor, NY: Macbeth Division of Kollinorgen Instruments Corporation. Murray, R. and Tedrow, J. C. F. (1975 [1986]). Forensic Geology: Earth Sciences and Criminal Investigation. New York: Rutgers University Press. Murray, R. C. (2004) Evidence from the Earth: Forensic Geology and Criminal Investigation. Missoula, MT: Mountain Press Publishing.

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Murray, R. C. and Tedrow, J. C. F. (1991). Forensic Geology. Englewood Cliffs, NJ: Prentice Hall. Northcote, K. H. (1979). A Factual Key for the Recognition of Australian Soils. Glenside, Australia: Rellim Technical Publications. Post, D. F., Bryant, R. B., Batchily, A. K., Heute, A. R., Levine, S. J., Mays, M. D., et al. (1993). Correlations between field and laboratory measurements of soil color, in Soil Color (J. M. Bigham, E. J. Ciolkosz, and R. J. Luxmoore, Eds.). Madison, WI: Soil Science Society of America Special Publication 31, 35–49. Rendle, D. F. (2004). Database use in forensic analysis. Crystallogr. Rev. 10, 23–28. Ruffell, A. and McKinley, J. (2004). Forensic geoscience: Applications of geology, geomorphology and geophysics to criminal investigations. Earth-Sci. Rev. 69, 235–247. Scheinost, A. C. and Schwertmann, U. (1995). Predicting phosphate adsorptiondesorption on a soilscape. Soil Sci. Soc. Am. J. 59, 1575–1580. Schoeneberger P. J., Wysocki, D. A., Benham, E. C., and Broderson W. D. (Eds.) (2002). Field Book for Describing and Sampling Soils, Version 2.0. Lincoln, NE: Natural Resources Conservation Service, National Soil Survey Center. Science and Art (1856). Curious use of the microscope. Scientific American 11, 240. Simonson, R. W. (1959). Outline of a generalized theory of soil genesis. Soil Sci. Soc. Am. Proc. 23, 152–156. Smeck, N. E., Runge, E. C. A., and MacKintosh, E. E. (1983). Dynamics and genetic modelling of soil systems, in Pedogenesis and Soil Taxonomy. 1. Concepts and Interactions (L. P. Wilding, N. E. Smeck, and G. F. Hall, Eds.). Amsterdam: Elsevier, 51–81. Spennemann, D. H. R. and Franke, B. (1995). Archaeological techniques for exhumations: A unique data source for crime scene investigations. Forensic Sci. Int. 74, 5–15. Sposito, G. and Reginato, R. J. (1992). Pedology: The science of soil development, in Opportunities in Basic Soil Science Research (G. Sposito and R. J. Reginato, Eds.). Madison, WI: Soil Science Society of America, 9–25. Stoops, G. (2003). Guidelines for Analysis and Description of Soil and Regolith Thin Sections. Madison, WI: Soil Science Society of America. Sugita, R. and Marumo, Y. (1996). Validity of color examination for forensic soil identification. For. Sci. Int. 83, 201–210. Thompson, R. and Oldfield, F. (1986). Environmental Magnetism. London: Allen and Unwin Ltd. USDA Soil Survey Division Staff (1993). Soil Survey Manual. Washington, DC: U.S. Department of Agriculture Handbook 18. USDA Soil Survey Staff (1988). Soil Taxonomy: A Basic System of Soil Classification for Making and Interpreting Soil Surveys, 2d ed. Malabar, FL: Krieger. USDA Soil Survey Staff (1998). Keys to Soil Taxonomy, 8th ed. Washington, DC: U.S. Department of Agriculture, Natural Resources Conservation Service. USDA Soil Survey Staff (2003). Keys to Soil Taxonomy, 9th ed. Washington, DC: U.S. Department of Agriculture, Natural Resources Conservation Service. Vepraskas, M. J. (1992). Redoximorphic features for identifying aquic conditions. Raleigh: North Carolina State University Technical Bulletin 301.

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Wilding, L. P. (1994). Factors of soil formation: Contributions to pedology, in Factors of Soil Formation: A Fiftieth Anniversary Retrospective (R. Amundson, J. Harden, and M. Singer, Eds.). Madison, WI: Soil Science Society of America, 15–30. Yaalon, D. and Yaron, B. (1966). Framework for man made soil changes: An outline of metapedogenesis. Soil Sci. 102, 272–277.

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Cadaver Decomposition and Soil: Processes David O. Carter and Mark Tibbett

2

Contents 2.1  Introduction................................................................................................. 29 2.2  Cadavers: Composition and Decomposition............................................31 2.3  The Formation of a Cadaver Decomposition Island.............................. 33 2.3.1  Fresh and Bloated Cadavers.......................................................... 35 2.3.2  Active Decay.................................................................................... 35 2.3.3  Advanced Decay, Dry, and Remains............................................ 36 2.4  Factors Influencing Cadaver Decomposition.......................................... 38 2.4.1  Aboveground Decomposition....................................................... 38 2.4.1.1  Temperature.................................................................... 38 2.4.1.2  Moisture........................................................................... 39 2.4.1.3  Trauma............................................................................. 40 2.4.1.4  Associated Materials...................................................... 40 2.4.2  Belowground Decomposition....................................................... 40 2.4.2.1  Temperature.................................................................... 40 2.4.2.2  Moisture and Soil Texture............................................ 41 2.4.2.3  Soil pH............................................................................. 42 2.4.2.4  Associated Materials...................................................... 43 2.4.2.5  Decomposer Adaptation............................................... 43 2.5  Concluding Remarks.................................................................................. 44 References............................................................................................................... 45

2.1  Introduction Forensic taphonomy is an applied science with clear aims: Use the processes associated with cadaver decomposition to estimate postmortem or postburial interval, determine cause and manner of death, locate clandestine graves, and identify the deceased (Haglund 2005; Haglund and Sorg 1997). Forensic taphonomy derives these aims from taphonomy, a branch of palaeontology (Efremov 1940). Taphonomy was developed to understand the ecology of a decomposition site, how site ecology changes on the introduction of plant or 29

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animal remains, and, in turn, how site ecology affects the decomposition of these materials (ibid.). Thus, the goals of forensic taphonomy are achieved through the ecology of cadaver decomposition. To date, several cadaver decomposition studies have been conducted in terrestrial ecosystems. In response to the myriad locations a cadaver can be deposited following death, these studies have been conducted inside structures (e.g., buildings, cars) (Galloway 1997; Galloway et al. 1989; Mann, Bass, and Meadows 1990), on the soil surface (Davis and Goff 2000; Rodriguez and Bass 1983; Vass et al. 1992), and following burial in soil (Mant 1950; Morovic-Budak 1965; Rodriguez and Bass 1985; VanLaerhoven and Anderson 1999). Thus far, the majority of these studies have focused on the activity of aboveground insects (Kocárek 2003; Motter 1898; Payne 1965; Reed 1958; VanLaerhoven and Anderson 1999) and scavengers (Berryman 2002; DeVault, Brisbin, and Rhodes 2003; DeVault, Rhodes, and Shivik 2004; Galdikas 1978; Haglund 1997; Willey and Snyder 1989) whereas less attention has been given to the processes that occur in soils associated with cadaver breakdown (gravesoils) (Carter and Tibbett 2003, 2006; Hopkins, Wiltshire, and Turner 2000; Putman 1978a; Sagara 1995; Tibbett et al. 2004; Vass et al. 1992). As a consequence, the relationship between cadaver decomposition and soil is poorly understood. The value of soil in a death investigation is most often as associative evidence. This reflects the traditional view of forensic science: Soil is a passive medium that can be defined by intrinsic biological, chemical, and physical properties (see Fitzpatrick, this volume). In reality, soil is a dynamic medium that can rapidly respond to environmental change such as pollution (Brookes 1995) and disturbance (Bongers 1990). Considering that the death of an animal in a terrestrial ecosystem is a natural disturbance (Putman 1983), potential exists for biophysicochemical characteristics of soil to be used as indices of criminal activity, such as the deposition of a body. To this end, soil biology and chemistry have been investigated as a means to estimate postmortem interval (PMI) (Carter and Tibbett 2003; Tibbett et al. 2004; Vass 2001; Vass et al. 1992) and to locate clandestine graves (Carter and Tibbett 2003; Rodriguez and Bass 1985). Although some of these approaches have proven successful in actual casework (Vass et al. 1992), the majority of them are in the early stages of development. This chapter reviews the processes in soils associated with cadaver decomposition (i.e., gravesoils). A portion of this review also concerns insects and scavengers, because the activity of these organisms can regulate the introduction of cadaver material to soil. However, these topics are dealt with in greater detail by Amendt, Krettek, and Zehner (2004) and DeVault, Brisbin, and Rhodes (2003). The hope is that this chapter will provide a greater understanding of the processes associated with cadaver decomposition and their potential for forensic application.

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2.2  Cadavers: Composition and Decomposition A cadaver is a complex resource that comes with a heavy microbial inoculum in the form of enteric and dermal microbial communities (Clark, Worrell, and Pless 1997; Hill 1995; Noble 1982; Wilson 2005; Yajima et al. 2001). A cadaver also comprises a large amount of water (60%–80%), a relatively high concentration of lipid and protein (Swift, Heal, and Anderson 1979; Tortora and Grabowski 2000) and a narrow C:N ratio (Table 2.1). These properties are characteristic of a high-quality resource; thus, the breakdown of a cadaver is usually rapid. This breakdown can broadly be described by three processes: autolysis, putrefaction, and decay. Following the cessation of the heart, internal aerobic microorganisms deplete the tissues of oxygen. This marks the onset of autolysis, which results in the destruction of cells (Gill-King 1997). Autolysis can begin within minutes of death (Vass et al. 2002) and is significantly affected by temperature and moisture (Gill-King 1997). Concomitantly, optimal conditions are created for anaerobic microorganisms (e.g., Clostridium, Bacteroides) originating from the gastrointestinal tract and respiratory system. Following the establishment of an anaerobic environment, carbohydrates, lipids and proteins are transformed into organic acids (e.g., propionic acid, lactic acid) and gases (e.g., methane, hydrogen sulphide, ammonia) that result in color change, odor, and bloating of the cadaver. This process is putrefaction. Putrefactive bloating can compromise the integrity of the skin and lead to ruptures that allow oxygen back into the cadaver. This reestablishes aerobic metabolism and designates the beginning of the decay process (Johnson 1975; Micozzi 1986). Decay typically represents the period of most rapid breakdown. Although a cadaver is subject to the intrinsic processes of autolysis and putrefaction, the majority of decomposition is due to the activity of noncadaveric organisms, particularly insects and scavengers. Insects can arrive at a cadaver within seconds of death (Mann, Bass, and Meadows 1990). Blowflies and flesh flies tend to dominate the early stages of cadaver decomposition in an attempt to find a suitable resource for the development of their offspring. The activity of these insects can have a significant effect on cadaver decomposition: Maggot activity can represent the primary driving force behind the removal of soft tissues. Thus, insect activity can also influence the success of scavengers if they can locate and consume a cadaver before a scavenger does so (DeVault et al. 2004). In addition, microorganisms can release repellent toxins (Janzen 1977). However, scavengers can consume from 35 to 75% of the cadavers in terrestrial ecosystems (DeVault, Brisbin, and Rhodes 2003), and, when insects and microbes are less active (such as during winter), scavenger success can approach 100% (Putman 1983).

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80

78

78

75

Pig (Sus scrofa L.) age: 56 days

Pig (Sus scrofa L.) age: 28 days

Rabbit age: 70 days

Rat (Rattus rattus L.) age: 70 days

—

—

—

7.7

5.8

C:N Ratio

3.2

29

29

26

32

N (g kg–1)

6.5

7.0

7.4

6.5

10

P (g kg–1)

3.5

3.2

2.7

2.9

4.0

K (g kg–1)

12

12

10

10

15

Ca (g kg–1)

0.5

—

0.4

0.4

1.0

Mg (g kg–1)

Spray and Widdowson (1950)

Spray and Widdowson (1950)

Manners and McCrea (1963)

Spray and Widdowson (1950); DeSutter and Ham (2005)

Tortora and Grabowski (2000)

References

Notes: Measurements of carbon (C), nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) are presented as grams per kilogram (g kg-1) cadaver mass (dry weight).

60

H2O (%)

Human (Homo sapiens L.) age: adult

Organic Resource

Table 2.1  Chemical Composition of Mammalian Cadavers during Life

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Cadaver mass apparently plays a critical role in scavenger activity and the formation of gravesoil. Small cadavers (i.e., infants, juveniles) can be consumed ex situ because they can be carried away in their entirety. Thus, the amount of cadaveric material entering the soil might be negligible (Putman 1983). Large cadavers tend to be consumed (at least partly) in situ, which allows cadaveric material to enter the soil (Coe 1978; Towne 2000) or be left on the soil surface (Putman 1983). Therefore, significant amounts of cadaveric material might only enter the soil when insects and microbes dominate cadaver decomposition or when a cadaver is too large to be carried away by a scavenger. This effect is characterized by a localized alteration of soil biology and chemistry. A fundamental understanding of this localized area, or cadaver decomposition island (CDI) (Carter, Yellowlees, and Tibbett 2007), is vital to the use of gravesoils in crime scene investigation.

2.3  The Formation of a Cadaver Decomposition Island A CDI is formed via an intense pulse of water, carbon (C), and nutrients (e.g., nitrogen, phosphorus [P]). This pulse, and the cadaver itself, can initially have a negative effect on surrounding vegetation observed as the death of underlying and nearby plants by leachate and smothering (Figure 2.1b) (Towne 2000). Though the dynamics of a CDI are poorly known, it is generally understood that the biophysicochemical characteristics of a CDI change over time (Towne 2000; Vass et al. 1992). These changes can be defined by a succession of insect (Kocárek 2003), plant (Towne 2000), and fungal (Tibbett and Carter 2003) communities as well as variation in the concentration of chemical compounds such as ammonium and nitrate (Towne 2000; Vass et al. 1992). In addition, the lateral (and probably vertical) extent of a CDI changes over time (Towne 2000) (Figure 2.1). These phenomena are likely related to the physicochemical composition of the cadaver—that is, the stage of cadaver decomposition. Several cadaver decomposition studies (Anderson and VanLaerhoven 1996; Carter 2005; Hewadikaram and Goff 1991; Kocárek 2003; Melis et al. 2004; Micozzi 1986; Payne 1965; Payne, King, and Beinhart 1968) have shown that cadaver breakdown follows a sigmoidal pattern (Figure 2.2). This pattern is probably due to the presence of skin, which will retain moisture, and the rate at which blowfly larvae consume cadaveric material (Putman 1977). Cadaver decomposition is also often associated with a number of stages (Bornemissza 1957; Coe 1978; Fuller 1934; Johnson 1975; Megyesi, Nawrocki, and Haskell 2005; Payne 1965; Payne and King 1968; Reed 1958). These stages are a subjective means to summarize physicochemical changes (Schoenly and Reid 1987). For consistency we refer to the six stages proposed by Payne (1965): fresh, bloated, active decay, advanced decay, dry, remains.

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34 a

b

Figure 2.1  Pig (Sus scrofa L.) cadaver in the bloated (a) and advanced decay (b) stage of decomposition on the soil surface of a pasture near Mead, Nebraska. Cadavers were 8 weeks old and approximately 40 kg at the time of death. Cadavers were placed on the soil surface within 30 minutes of death. Arrow indicates location and direction of maggot migration. (See color insert following p. 178.)

The progress of a cadaver through the decomposition stages is typically attributed to temperature. Accumulated degree days (ADDs, the sum of average daily temperature) can be used to compensate for differences in temperature (Megyesi et al. 2005; Vass et al. 1992). It is currently known that the advanced decay and remains stages associated with a 68 kg human cadaver occur at 400 and 1285 ADDs, respectively (Vass et al. 1992). Thus, an average summer daily temperature of 20°C would result in the onset of advanced decay after twenty days whereas an average daily winter temperature of 2°C would result in advanced decay after 200 days.

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Fresh Bloat 0

Mass Loss (%)

20

Active Decay

40 60 Advanced Decay

80 100

Remains Dry 0

5

10 15 Time (days)

20

25

Figure 2.2  Sigmoidal decomposition curves typically associated with cadaver decomposition on the soil surface (—) and following burial in soil (---). (Adapted from Carter, unpublished data. With permission).

2.3.1  Fresh and Bloated Cadavers The fresh and bloated stages of decomposition correspond to the time of death up to the rupture of the skin. During this time blowflies (Calliphoridae) and flesh flies (Sarcophagidae) arrive at a cadaver to find a suitable oviposition site. In addition, the activity of soil microbes (possibly zymogenous r-strategist bacteria) increases (Carter 2005; Putman 1978a). Thus, the CDI during the fresh stage of decomposition comprises the cadaver, the gravesoil, and ovipositing flies on moist areas of the cadaver (e.g., mouth, nose, anus). The bloated stage results from the accumulation of gases (hydrogen sulphide, carbon dioxide, methane) associated with anaerobic metabolism (putrefaction). During this stage, the pressure from these gases forces fluid to escape from natural cadaveric openings (mouth, nose, anus) and flow into the soil. The effect of this phenomenon on gravesoil ecology is currently not understood. Eventually, putrefactive bloating and maggot feeding activity cause ruptures in the skin. These openings allow oxygen back into the cadaver and expose more surface area for the development of fly larvae and aerobic microbial activity (Putman 1978a). This designates the beginning of active decay (Johnson 1975; Micozzi 1986). 2.3.2  Active Decay Active decay represents the period of greatest mass loss (Figure 2.2), which results from the release of cadaveric fluids into the soil. This flux can cause

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David O. Carter and Mark Tibbett

islands of purge fluids and, thus, can lead to the formation of a single CDI. The status of soil energy, nutrients, and microbial communities during active decay is currently unknown. Active decay will continue until maggot migration. This phenomenon represents the onset of advanced Decay. 2.3.3  Advanced Decay, Dry, and Remains The final three stages of cadaver decomposition, advanced decay, dry,t and remains, correspond to a second period of slow cadaver mass loss (Figure 2.2), probably due to the depletion of readily available cadaveric materials. The surface of gravesoil associated with maggot activity contains dead vegetation that can be visible for at least 100 days postmortem (which might vary due to initial cadaver mass) (Figure 2.1b). The cause of plant death is currently unknown. It might be due to decomposition fluids or the excretion of antibiotics by maggots (e.g., Thomas et al. 1999). Regardless, a CDI during advanced decay represents an area of increased soil carbon (Carter 2005; Putman 1978b; Vass et al. 1992), nutrients (Carter 2005; Towne 2000; Vass et al. 1992), and pH (Carter 2005; Vass et al. 1992). These changes are not surprising after considering that a cadaver comprises a large amount of water (50%–80%) and has a narrow C:N ratio (DeSutter and Ham 2005; Tortora and Grabowski 2000) (Table 2.1). These properties are characteristic of a high-quality resource that is associated with a significant input of energy and nutrients and a high level of microbial activity (Swift et al. 1979). Advanced decay is associated with a significant increase in the concentration of soil nitrogen. The decomposition of a 68 kg human cadaver resulted in an increase in approximately 525 µg ammonium g-1 soil (Vass et al. 1992). Cadaveric material contains several other nutrients, such as P, potassium (K), calcium (Ca), and magnesium (Mg) (Table 2.1), which will enter the soil upon decomposition. Soil (3–5 cm) beneath a 68 kg human cadaver in advanced decay also contained 300 µg K g-1 soil, 50 µg Ca g-1 soil, and approximately 10 µg Mg g-1 soil (Vass et al. 1992). It is difficult to determine when advanced decay ends and remains begins (Payne 1965). However, the increased growth of plants around the edge of the CDI (Figure 2.3) might act as an indicator of the progression into the remains stage. This provides evidence that the nutrient status of the gravesoil has not yet reached basal levels. The concentration of phosphorus (Towne 2000), ammonium, potassium, sulphate, calcium, chloride, and sodium (Vass et al. 1992) in soil (3–5 cm) associated with the decomposition of a 68 kg human cadaver can remain as high as 50–150 µg g-1 soil above basal levels during dry and remains stages. Soil texture and cadaver mass can affect the vertical extent of a CDI. For example, the CDI, as measured by soil moisture content, associated with an elephant (Loxodonta africana Blumenbach) cadaver (~1,629 kg) on sandy

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Figure 2.3  Pig (Sus scrofa L.) cadaver and cadaver decomposition island (CDI) on the soil surface of a pasture near Mead, Nebraska, at 70 days postmortem. Note area of dead plant material that defines the lateral extent of the CDI, which is bordered by plants that have undergone enhanced growth. (See color insert following p. 178.)

loam soil can extend to 40 cm below the cadaver, 35 cm at 1 m from the cadaver, and 8 cm at 2 m from the cadaver (Coe 1978). In contrast, the CDI associated with a 633 kg elephant cadaver on quartz gravel can extend to 1.5 m below the soil surface (Coe 1978). By comparison, the CDI associated with the decomposition of a 620 g guinea pig (Cavia porcellus L.) on sandy soil can extend to 14 cm below the cadaver (Bornemissza 1957). The majority of gravesoil research has been conducted during the advanced decay and remains stages. This is probably because most cadaver decomposition studies are empirical, and, thus, cadavers are not discovered until they have been exposed for an extended period of time. The latter stages of cadaver decomposition have been associated with a decreased abundance of Collembola (0–2 cm) and Acari (0–5 cm) (Bornemissza 1957). In addition, the formation of fungal fruiting bodies can occur during advanced decay (Figure 2.4). This chemoecological group of fungi, known as the postputrefaction fungi (Sagara 1995), fruit in a successional sequence that is believed to be in response to the form of N (Tibbett and Carter 2003). Early-phase fungi comprise zygomycetes, dueteromycetes, and ascomycetes that fruit in response to high concentrations of ammonia (Yamanaka, 1995a, 1995b) from one to ten months after N addition (Sagara 1992). Late-phase postputrefaction fungi fruit in response to organic N and high concentrations of ammonium and nitrate (ibid.) and can be present from one to four years after N addition (see Sagara et al., this volume). These findings show that a CDI is a long-lasting component of terrestrial death scenes.

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Figure 2.4  Putative postputrefaction fungi (Coprinus sp.) in association with a pig (Sus scrofa L.) cadaver in the advanced decay stage of decomposition 28 days after death.

2.4  Factors Influencing Cadaver Decomposition Several factors can influence the breakdown of a cadaver and the formation of a CDI. These include temperature, moisture, soil type, associated materials, decomposer adaptation, and trauma. Furthermore, these factors may be more or less influential depending on whether a cadaver has been placed on the soil surface (exposed) or buried in soil. The effect of these factors on both the decomposition of exposed and buried cadavers will be discussed (see Hopkins, this volume). 2.4.1  Aboveground Decomposition 2.4.1.1  Temperature It is well known that temperature has a significant effect on the decomposition of exposed cadavers (Mann, Bass, and Meadows 1990; Rodriguez and Bass 1983; Vass et al. 1992). Indeed, temperature is regarded as one of the most influential factors of decomposition (Gill-King 1997; Mann, Bass, and Meadows 1990). An increase in the rate of cadaver decomposition is associated with an increase in temperature. This is because an increase in temperature is typically associated with an increase in biological activity (Carter and Tibbett 2006) and chemical reaction rates (van’t Hoff 1898). Thus, temperature affects the processes of autolysis and putrefaction (Gill-King 1997) as well as adult (Rodriguez and Bass 1983; Turner and Wiltshire 1999) and larval (Higley and Haskell 2001) insect activity.

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It has been repeatedly observed that the rate of cadaver decomposition increases as temperature increases (Mann, Bass, and Meadows 1990; Rodriguez and Bass 1983; Vass et al. 1992). Reed (1958) stated that a cadaver placed in a cooler setting with large numbers of insects will not decompose significantly faster than a cadaver in a warmer setting with less insects. Rodriguez and Bass (1983) investigated the effect of seasonality on the decomposition of exposed cadavers in an open-wooded area of Knoxville, Tennessee. Temperature was observed to significantly influence decomposition during the spring and summer months because the colonization of cadavers by insects was greater. Predictably, cold temperature will slow the progression of a cadaver through the sigmoidal decomposition curve. In theory, decomposition should still proceed at 0 °C because of the concentration of salts in a cadaver. However, Micozzi (1997) observed a lack of putrefaction at temperatures below 4 °C. This phenomenon is believed to be the result of the simultaneous suppression of decomposer activity and promotion of desiccation (Janaway 1996). Interestingly, the freezing and thawing of a cadaver tends to promote aerobic decomposition rather than the anaerobic breakdown typically associated with putrefaction (Micozzi 1986). The reason for this is unknown. 2.4.1.2  Moisture Because cadavers comprise 60%–80% water their breakdown has been described as a “competition” between desiccation and decomposition (Aufderheide 1981). The relationship between these processes is important because rapid desiccation can inhibit decomposition and result in the natural preservation of a cadaver for thousands of years, such as the natural mummies observed in Egypt (Ruffer 1921) and Peru (Allison 1979). Sledzik and Micozzi (1997) distinguished three types of mummification: natural, intentional, and artificial. Dryness, heat, or absence of air may cause natural mummification. Intentional mummification is the result of exploitation or enhancement of natural mummification processes. Artificial mummification may be the result of evisceration, fire, or smoke curing and the application of embalming substances. Generally, extremely dry environments promote desiccation (Galloway 1997; Galloway et al. 1989) whereas extremely wet environments promote waterlogging and adipocere formation (see Forbes, this volume). Both of these process slow cadaver decomposition. Campobasso, Di Vella, and Introna (2001) noted that humid environments can slow decomposition by saturating the tissues with water; however, this is in contrast to the observation that humidity is positively correlated with insect activity (, Bass, and Meadows 1990). The latter finding is somewhat supported by the observation that rainfall has little to no effect on maggot activity (, Bass, and Meadows 1990; Reed 1958).

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2.4.1.3  Trauma An exposed body with traumas can decompose faster than an exposed body that has not encountered trauma (, Bass, and Meadows 1990). This is due to the attraction of insects to open wounds, where oviposition occurs. It is unknown how trauma affects the formation of CDI. However, it is likely that it results in a more rapid rate of CDI formation by allowing cadaveric fluids to enter the soil more readily. 2.4.1.4  Associated Materials The presence of clothing on an exposed body tends to increase the rate of cadaver decomposition because clothing provides a shaded area for maggots to feed (, Bass, and Meadows 1990). It is unknown how the presence of clothing affects the formation of a CDI, but it is likely that it would retard the flow of cadaveric moisture into the soil. The effect of clothing on cadaver decomposition is discussed in extensive detail by Janaway (this volume). 2.4.2  Belowground Decomposition A number of studies have been conducted to understand cadaver decomposition following burial in soil (Carter 2005; Carter and Tibbett 2006; Child 1995; DeGaetano, Kempton, and Rowe 1992; Fiedler, Schneckenberg, and Graw 2004; Forbes, Dent, and Stuart 2005; Forbes, Stuart, and Dent 2005a, 2005b; Hopkins et al. 2000; Lötterle, Schmierl, and Schellmann 1982; Lundt 1964; Mant 1950; Motter 1898; Payne et al. 1968; Rodriguez and Bass 1985; Sagara 1976; Spennemann and Franke 1995; VanLaerhoven and Anderson 1999; Weitzel 2005). It is generally accepted that the burial of a cadaver results in a decreased rate of decomposition (, Bass, and Meadows, 1990; Rodriguez, 1997; Fiedler and Graw, 2003). It has even been proposed that cadaver decomposition following burial proceeds at rate of eight times slower than aboveground decomposition (Rodriguez 1997), but little experimental evidence exists to support this proposition. The reduced rate of cadaver decomposition upon burial is attributed to a reduced presence of insects and scavengers (Rodriguez 1997; Turner and Wiltshire 1999). Thus, the decomposer organisms associated with cadaver decomposition in gravesoils are primarily comprised of soil microbes and animals (e.g., nematodes). The decomposition of a cadaver in soil also follows a sigmoidal pattern (Payne, King, and Beinhart 1968; VanLaerhoven and Anderson 1999) (Figure 2.2). 2.4.2.1  Temperature An increase in temperature has been repeatedly observed to result in an increase in the rate of the decomposition of buried cadavers. Mant (1950) conducted 150 exhumations in Germany and noted that cadavers buried in

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the summer experienced a greater rate of decomposition than those buried in winter. Morovic-Budak (1965) recognized a similar pattern while conducting human exhumations in Croatia and also observed that a seasonal effect was exceedingly noticeable on cadavers buried up to one year. In a continuation of the study by Rodriguez and Bass (1983), six unembalmed human cadavers were buried in separate unlined trenches at various depths (Rodriguez and Bass 1985) and exhumed after set time intervals (1, 2, 6, and 12 months). The decomposition of buried cadavers was slower than the exposed cadavers. This was attributed to the decrease in temperature and insect activity on burial. More recently, experimental work using skeletal muscle tissue (Ovis aries) buried in a sandy loam soil has provided insight into the relationships between temperature and decomposition (Carter and Tibbett 2006). This showed that, as observed in many studies, an approximate doubling of microbial activity occurs with an increase of 10°C up to 35°C–40°C (see Paul and Clark 1996). However, the relationship between temperature and decomposition was not linear, as the increase in muscle tissue between 2°C and 12°C was greater than between 12°C and 22°C. In addition, soil microbes were triggered into activity (observed as carbon dioxide respiration) within 24 hours of burial. The majority of this activity was observed to occur during the first fourteen days of tissue burial at 12°C and 22°C. Thus, these results show that temperature can regulate cadaveric decomposition and associated gravesoil microbial activity. Though this work does not represent the decomposition of a complete cadaver, it does demonstrate that cadaveric material can be readily utilized by the soil microbial biomass. 2.4.2.2  Moisture and Soil Texture Soil moisture can have a significant effect on decomposition (Swift et al. 1979). This is due, in part, to the fact that soil moisture can affect the metabolism of decomposer microorganisms. This effect can be modified by soil texture because bioavailable moisture is determined, in part, by the suction with which water is held between soil particles (matric potential). Thus, the calibration of soils to a known matric potential can lead to the assessment of the effect of bioavailability of moisture in soil (Hillel 1982) and allow for the comparison of process rates between soils at the same matric potential (Orchard and Cook 1983). It is generally accepted that coarse-textured (sandy) soil with a low moisture content frequently promotes desiccation (Fiedler and Graw 2003; Mant 1950; Santarsiero et al. 2000). This phenomenon is almost certainly related to the diffusion of gases through the soil matrix (see Tibbett et al. 2004). Coarsetextured soils are associated with a high rate of gas diffusivity (Moldrup et al. 1997), which allows gases and moisture to move relatively rapidly through the soil matrix. The ability of coarse-textured soil to rapidly lose moisture will also promote desiccation because hydrolytic enzymes associated with the

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cycling of carbon and nutrients are retarded by low moisture content (Skujins and McLaren 1967). Desiccation can inhibit decomposition and result in the natural preservation of a cadaver for thousands of years (Micozzi 1991). However, this phenomenon only occurs in a few extreme settings such as areas of Egypt (Dzierzykray-Rogalsky 1986; Ruffer 1921), Peru (Allison 1979), and Siberia (Lundin 1978). Alternatively, burial in coarse-textured soil with a high water content might result in the formation of pseudomorphs (shapes of human cadavers primarily in the form of sand), such as those observed at Sutton Hoo, England (Bethell and Carver 1987). These pseudomorphs were associated with an elevated concentration of calcium, phosphorus, and manganese, which is likely related to the breakdown of bone. Fine-textured (clayey) soil has been associated with an inhibition of cadaver breakdown (Hopkins, Wiltshire, and Turner 2000; Santarsiero et al. 2000; Turner and Wiltshire 1999). These soils have a low rate of gas diffusivity. The burial of a cadaver in a wet, fine-textured soil can result in decreased decomposition (Hopkins, Wiltshire, and Turner 2000; Turner and Wiltshire 1999) because the rate at which oxygen is exchanged with CO2 might not be sufficient to meet aerobic microbial demand (Carter 2005). Thus, reducing conditions are established whereby anaerobic microorganisms dominate decomposition. These organisms are less efficient decomposers than aerobes (Swift, Heal, and Anderson 1979). Reducing conditions can also promote the formation of adipocere (Fiedler and Graw 2003; Forbes, Stuart, and Dent 2004; Forbes, Stuart, and Dent 2005) around a cadaver or internal organs, which significantly slows cadaver decomposition (Dent, Forbes, and Stuart 2004; Fiedler, Schneckenberg, and Graw 2004; Froentjes 1965). However, many mammals (e.g., human, pig, sheep, cow, rabbit) contain sufficient moisture and fat to form adipocere in a moist, coarse-textured soil (Forbes, Stuart, and Dent 2005a). Gravesoil associated with adipocere formation has been observed to contain elevated levels of dissolved organic C, plant-available P, and total P (Fiedler, Schneckenberg, and Graw 2004) relative to soils without adipocere. It is also important to note that the formation of adipocere is not necessarily an endpoint (Evans 1963a; Froentjes 1965). On translocation to the soil surface or the establishment of an aerobic environment, adipocere can undergo decomposition (Evans 1963). This process is typically associated with the bacteria Bacillus spp., Cellulomonas spp., and Nocardia spp. (Pfeiffer, Milne, and Stevenson 1998). 2.4.2.3  Soil pH Little is known about the effect of soil pH on the decomposition of cadavers, but inferences may be drawn from other disciplines. In acid soils plants produce a greater number of tannins (Swift, Heal, and Anderson 1979). Tannins can combine with proteins and carbohydrates in organic matter, resulting in decreased microbial activity. Thus, acid soils might result in a slowing of

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cadaver decomposition. In turn, cadaver decomposition can have a significant effect on soil pH. It is generally understood that a buried body initially results in an alkaline environment (Carter 2005; Hopkins, Wiltshire, and Turner 2000; Rodriguez and Bass 1985), which is followed by the formation of an acidic environment (Gill-King 1997; Towne 2000). 2.4.2.4  Associated Materials Cadavers are commonly associated with clothing, plant material, and metal artefacts such as jewelry. Fully clothed cadavers buried directly in soil undergo a significant decrease in the rate of decomposition (Mant 1950). Clothing can inhibit the effects of the burial environment by partially preventing mesoand microorganisms from participating in cadaver decomposition. Clothed cadavers buried in moist, free-draining soils have been observed to undergo greater rates of adipocere formation, which tends to have a preservative effect on cadavers (Mant 1950, 1987). The relationship between clothing and cadaver decomposition is discussed by Janaway (this volume). A buried corpse surrounded by plant material (e.g., straw, pine branches) can display a more rapid rate of decomposition than a cadaver buried without these materials (Mant 1950). Mant (1950) believed that these plant materials introduced additional bacteria into the burial environment while providing a layer of air between the cadaver and the soil. Also, an increase in the rate of cadaver decomposition following the addition of plant material is due to the widening of the carbon to nitrogen ratio, which promotes microbial activity. In fact, this is the premise behind the composting of dead animals (Elwell, Moller, and Keener 1998). Concentrations of metal ions in the burial environment can lead to localized conditions of toxicity, which can prevent microbial activity (Janaway 1996). This phenomenon is typically associated with the preservation of associated grave materials such as textiles, leather, and wood (ibid.). However, an extensive collection of metallic artefacts usually contains insufficient concentrations of metal ions to result in significant retardation of decomposition (ibid.). 2.4.2.5  Decomposer Adaptation The rate of cadaver decomposition in soil can be affected by how often a particular site is subjected to cadaveric material. Microbial degradation is typically described as having three phases. The initial lag phase is defined by microbial or enzymatic enrichment. During the second phase the substrate is rapidly degraded. This is followed by a declining phase that results from a lack of readily available substrate or formation of humic substances (Ajwa and Tabatabai 1994). Forensic taphonomy holds that the burial of a number of cadavers in soil over time will result in an increased number of soil microorganisms (Janaway 1996). Experiments using controlled burial environment

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microcosms have demonstrated that soil microbiota can adapt to soft-tissue (Ovis aries) burial, resulting in an increased rate of decomposition without a significant change in microbial activity and biomass (Carter and Tibbett 2002). This adaptation may be due to enzyme or microbial specificity.

2.5  Concluding Remarks Clearly, our knowledge of the processes associated with cadaver decomposition in terrestrial ecosystems is limited, which is in direct contrast with the processes associated with the decomposition of other organic resources in and on the soil. This may be partly due to the reliance forensic taphonomy has placed on case studies, anecdotal evidence, and unreplicated experiments for data (Galloway et al. 1989; Mann, Bass, and Meadows 1990; Mant 1950; Micozzi 1986; Morovic-Budak 1965; Prieto, Magaña, and Ubelaker 2004; Rodriguez and Bass 1985; Sagara 1976). Techniques commonplace in the environmental sciences can be applied to studies on cadaver decomposition in soil to better understand the processes involved. A fundamental understanding of these processes should contribute to forensic taphonomy by designating biological and chemical markers with the potential to aid in the location and dating of clandestine graves (e.g., Carter and Tibbett 2003). Soils are a valuable but little exploited tool in forensic science generally and forensic taphonomy in particular. Soils are likely most valuable to forensic taphonomy following the onset of advanced decay. At this time fly larvae have migrated, which greatly decreases their value as a tool to estimate PMI. Interestingly, advanced decay is when soils are most affected by cadaver breakdown. Thus, soil science might be developed as the most accurate means to estimate the extended postmortem interval. Crucial to this development is the recognition that cadaver decomposition represents one of the most striking examples of how aboveground communities interact with belowground communities. Thus, an aboveground–belowground approach should be taken, which is in line with current thought in terrestrial ecology (Bardgett 2005). This approach should lend itself to collaboration among vertebrate ecologists, entomologists, and soil ecologists. As a consequence, several members of the cadaver decomposition food web should provide for more robust methods for estimating PMI and for locating clandestine graves. For example, an estimate of PMI based on insect larval development could be used in conjunction with the concentration of fatty acids in soils and soil bacterial community profiles. These measures should each provide a window in which criminal activity occurred. When used together, multiple aboveground and belowground measures will provide greater confidence to include or exclude individuals

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and to accept or reject alibis. This physical evidence is out there, and we are only just learning how to use it.

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Payne, J. A. and King, E. W. (1968). Coleoptera associated with pig carrion. Entomol. Month. Mag. 105, 224–232. Payne, J. A., King, E. W., and Beinhart, G. (1968). Arthropod succession and decomposition of buried pigs. Nature 219, 1180–1181. Pfeiffer, S., Milne, S., and Stevenson, R. M. (1998). The natural decomposition of adipocere. J. Forensic Sci. 43, 368–370. Prieto, J. L., Magaña, C., and Ubelaker, D. H. (2004). Interpretation of postmortem change in cadavers in Spain. J. Forensic Sci. 49, 918–923. Putman, R. J. (1977). Dynamics of the blowfly, Calliphora erythrocephala, within carrion. J. Anim. Ecol. 46, 853–866. Putman, R. J. (1978a). Patterns of carbon dioxide evolution from decaying carrion. Decomposition of small mammal carrion in temperate systems 1. Oikos 31, 47–57. Putman, R. J. (1978b). Flow of energy and organic matter from a carcase during decomposition: Decomposition of small mammal carrion in temperate systems 2. Oikos 31, 58–68. Putman, R. J. (1983). Carrion and Dung: The Decomposition of Animal Wastes: The Institute of Biology’s Studies in Biology 165. London: Edward Arnold Ltd. Reed, H. B. (1958). A study of dog carcass communities in Tennessee, with special reference to the insects. Am. Midl. Nat. 59, 213–245. Rodriguez, W. C. (1997). Decomposition of buried and submerged bodies, in Forensic Taphonomy: The Postmortem Fate of Human Remains (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 459–468. Rodriguez, W. C. and Bass, W. M. (1983). Insect activity and its relationship to decay rates of human cadavers in east Tennessee. J. Forensic Sci. 28, 423–432. Rodriguez, W. C. and Bass, W. M. (1985). Decomposition of buried bodies and methods that may aid in their location. J. Forensic Sci. 30, 836–852. Ruffer, M. A. (1921). Studies in the Paleopathology of Egypt. Chicago: University of Chicago Press. Sagara, N. (1976). Presence of buried mammalian carcass indicated by fungal fruiting bodies. Nature 262, 816. Sagara, N. (1992). Experimental disturbances and epigeous fungi, in The Fungal Community: Its Organisation and Role in the Ecosystem (G. C. Carroll and D. T. Wicklow, Eds.). New York: Marcel Dekker, Inc., 427–454. Sagara, N. (1995). Association of ectomycorrhizal fungi with decomposed animal wastes in forest habitats: A cleaning symbiosis? Can. J. Bot. 73, suppl. 1, S1423–S1433. Santarsiero, A., Minelli, L., Cutilli, D., and Cappielo, G. (2000). Hygienic aspects related to burial. Microchem. J. 67, 135–139. Schoenly, K. and Reid, W. (1987). Dynamics of heterotrophic succession in carrion arthropod assemblages: Discrete seres or a continuum of change. Oecologia 73, 192–202. Skujins, J. J. and McLaren, A. D. (1967). Enzyme reaction rates at limited water activities. Science 158, 1569–1570. Sledzik, P. S. and Micozzi, M. S. (1997). Autopsied, embalmed and preserved human remains: Distinguishing features in forensic and historic contexts, in Forensic Taphonomy: The Postmortem Fate of Human Remains (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 483–496.

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Spennemann, D. H. R. and Franke, B. (1995). Decomposition of buried human bodies and associated death scene materials on coral atolls in the tropical Pacific. J. Forensic Sci. 40, 356–367. Spray, C. M. and Widdowson, E. M. (1950). The effect of growth and development on the composition of mammals. Br. J. Nutr. 4, 332–353. Swift, M. J., Heal, O. W., and Anderson, J. M. (1979). Decomposition in Terrestrial Ecosystems. Oxford: Blackwell Scientific. Thomas, S., Andrews, A., Hay, P., and Bourgoise, S. (1999). The antimicrobial activity of maggot secretions: Results of a preliminary study. J. Tissue Viabil. 9, 127–132. Tibbett, M. and Carter, D. O. (2003). Mushrooms and taphonomy: The fungi that mark woodland graves. Mycologist 17, 20–24. Tibbett, M., Carter, D. O., Haslam, T., Major, R., and Haslam, R. (2004). A laboratory incubation method for determining the rate of microbiological degradation of skeletal muscle tissue in soil. J. Forensic Sci. 49, 560–565. Tortora, G. J. and Grabowski, S. R. (2000). Principles of Anatomy and Physiology, 9th ed. New York: John Wiley & Sons, Inc. Towne, E. G. (2000). Prairie vegetation and soil nutrient responses to ungulate carcasses. Oecologia 122, 232–239. Turner, B. D. and Wiltshire, P. E. J. (1999). Experimental validation of forensic evidence: A study of the decomposition of buried pigs in a heavy clay soil. Forensic Sci. Int. 101, 113–122. van’t Hoff, J. H. (1898). Lectures on Theoretical and Physical Chemistry: Part 1: Chemical Dynamics. London: Edward Arnold. VanLaerhoven, S. L. and Anderson, G. S. (1999). Insect succession on buried carrion in two biogeoclimatic zones of British Columbia. J. Forensic Sci. 44, 32–43. Vass, A. A. (2001). Beyond the grave-understanding human decomposition. Microbiology Today 28, 190–192. Vass, A. A., Barshick, S.-A., Sega, G., Caton, J., Skeen, J. T., Love, J. C., et al. (2002). Decomposition chemistry of human remains: a new methodology for determining the postmortem interval. J. Forensic Sci. 47, 542–553. Vass, A. A., Bass, W. M., Wolt, J. D., Foss, J. E., and Ammons, J. T. (1992). Time since death determinations of human cadavers using soil solution. J. Forensic Sci. 37, 1236–1253. Weitzel, M. A. (2005). A report of decomposition rates of a special burial type in Edmonton, Alberta from an experimental field study. J. Forensic Sci. 50, 641–647. Willey, P. and Snyder, L. M. (1989). Canid modification of human remains: implications for time-since-death estimations. J. Forensic Sci. 34, 894–901. Wilson, M. (2005). Microbial Inhabitants of Humans. Cambridge, UK: Cambridge University Press. Yajima, M., Nakayama, M., Hatano, S., Yamazaki, K., Aoyama, Y., Yajima, T., et al. (2001). Bacterial translocation in neonatal rats: the relation between intestinal flora, translocated bacteria and the influence of milk. J. Ped. Gastroenterol. Nutr. 37, 168–177.

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Yamanaka, T. (1995a). Changes in organic matter composition of forest soil treated with a large amount of urea to promote ammonia fungi and the abilities of these fungi to decompose organic matter. Mycoscience 36, 17–23. Yamanaka, T. (1995b). Nitrification in a Japanese red pine forest soil treated with a large amount of urea. J. Jap. For. Soc. 77, 232–238.

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3

The Role of Soil Organisms in Terrestrial Decomposition David W. Hopkins

Contents 3.1  Introduction................................................................................................. 53 3.2  Decomposition and Turnover.................................................................... 54 3.3  Factors Affecting Decomposition............................................................. 55 3.3.1  Resource Quality............................................................................. 56 3.3.2  Environmental Factors................................................................... 57 3.3.3  Presence and Activity of Organisms............................................ 61 3.4  Can Ecological Principles Be Applied to Forensic Investigations?...... 62 References............................................................................................................... 64

3.1  Introduction Decomposition is the progressive breakdown of organic matter ultimately into inorganic constituents. In soils, the decomposition of organic matter, such as plant, microbial, and animal remains and metabolic wastes, is mediated by decomposer organisms, which derive energy and nutrients from the organic matter. The majority of decomposer organisms exploit organic matter for the energy in the chemical bonds of the organic molecules as well as for the nutrients, such as carbon (C), nitrogen (N), phosphorus (P), and sulfur (S), they contain. The release of inorganic C from the organic matter as CO2 through the respiration of decomposer organisms is the major return route of C to the atmosphere as CO2, balancing the flux of CO2 from the atmosphere into biomass through photosynthesis. Since CO2 is relatively simple to detect, many studies of terrestrial decomposition use CO2 as the principal indicator of decomposition. However, mass loss of the original resource is also widely used, though it can often be difficult to separate mass loss from the original organic matter and the resynthesis of decomposer biomass and

53

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Volatile Organic Compounds

Rest In Peace

Decomposer Respiration

CO2

Leaf Litter, Faeces, Urine, Root Litter, Exudates

Dissolved Organic Matter

Autochthonous Decomposer Organisms

Uptake

Zymogenous Decomposer Organisms

Labile Stable Residues Residues Physically Protected Organic Matter

Chemically Protected Organic Matter

D E C O M P O S I T I O N Inert Organic Matter

Figure 3.1  Decomposition and carbon turnover in soil: A conceptual diagram summarizing the main elements of the initial Rothamsted carbon model (Jenkinson 1971). To this we have added other small, but potentially functionally important, compartments: the volatile organic carbon and the dissolved organic carbon derived during both decomposition of litter and exudation from plants. An inert organic matter pool is added as this appears in later versions of the Rothamsted model.

to detect mass loss in the terminal phase of decay when discrete remains have all but disappeared. In terrestrial ecosystems, the respiration by decomposer organisms usually exceeds the return of CO2 by respiration of plants and animals (Swift, Heal, and Anderson 1979). It therefore has major importance in ecological and biogeochemical investigations. However, because organic matter enters soil at different times, has mixed biochemical composition, and is derived from different sources, such as dead animals, leaf litter, and excreta, the decomposition process cannot be regarded as a single process with a discrete start and end points. Rather, organic residues are continually being broken down, replenished by new inputs, and modified by the decomposer organisms that synthesize new compounds and biomass as they grow and produce their own excretory products (Figure 3.1).

3.2  Decomposition and Turnover The closest we have come to a unified concept for summarizing the interaction of decomposing organic matter in soils and soil organisms is embod-

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Table 3.1  Size and Turnover Times of Different Soil Organic Matter Fractions Fraction Total Readily decomposable organic matter Resistant plant material Microbial (decomposer) biomass Physically protected organic matter Chemically protected organic matter

Size (t ha–1)

Turnover Time (Years)

29.2 0.1 0.6 0.3 13.6 14.6

1240 0.2 3.3 2.4 71 2900

ied in models of soil C turnover. The conceptual model summarized in Figure 3.1 owes much to the Rothamsted C model (Jenkinson and Rayner 1977). This model is based on compartments with functional relevance, and the figure therefore serves to illustrate the main processes of decomposition and turnover. Decomposing organic matter includes a wide range of compounds in complex mixtures (e.g., polysaccharides, proteins, lipids, and lignin) that arise from the bodily remains of plants, animals, and microorganisms, as well as their metabolic wastes. These, and the partial decomposition products that exist transiently, form a continuum from recently added and easily broken down residues, such as leaf litter and root exudates, to very stable, highly altered organic matter from which the easily exploited materials have been removed and that may be protected from further decomposition by interaction with mineral particles and soil colloids. Many components of fresh plant litter decompose very quickly, because they are rich in energy, readily accessible to decomposer organisms, and rapidly assimilated. Consequently, although leaf litter represents only a small fraction of C in soil, about half of the annual CO2 output from soil comes from decomposition of the leaf litter. At the other extreme, there is usually a very large amount stable organic matter that decomposes very slowly over centuries or millennia (Table 3.1).

3.3  Factors Affecting Decomposition In their now classic text on decomposition in terrestrial ecosystem, Swift et al. (1979) outlined three broad groups of factors that govern decomposition of organic residues in soils. The three groups of factors are the resource (or substrate) quality of the organic residue, the environmental factors, and the presence and activity of decomposer organisms.

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Amount of C

CO2

Residue Decomposer Biomass Time

Figure 3.2  Summary of decay or organic matter (residue) and accompanying synthesis of decomposer biomass and accumulation of carbon dioxide due to microbial respiration.

3.3.1  Resource Quality Resource quality factors include the organic residue’s biochemical composition and accessibility to decomposer organisms, and these have a major influence on the decomposition rate. Overall, the rate at which each of the different components of decomposition of organic matter is utilized by decomposer organisms determines the decomposition rate. The rate of decomposition of a discrete fraction of organic matter in soil generally follows first-order kinetics in which a constant fraction of the resource remaining is decomposed per unit of time: dS/dt = kS0, where dS is the substrate lost per unit of time dt, S0 is the initial amount of substrate, and k is the rate constant (i.e., the proportion of the remaining substrate in a particular fraction decomposes per unit of time), and each fraction, at least theoretically, has its own characteristic rate constant (Figure 3.2). For modeling purposes, it is assumed that each fraction decomposes according to a different first-order function, and the result is that decomposition is described by net result of multiple first-order decay functions. Though other mathematical functions may be fitted to decomposition functions on an empirical basis, it is usually difficult to justify anything other than multiple first-order decay functions. The turnover time of a decomposing resource is given by 1/k. Readily decomposable organic matter, such as glucose and cellulose, has a k value in the range 0.05–0.1 day-1 in soils. These k values correspond to relatively short turnover times of three to ten days for glucose and ten to twenty-five days for cellulose. By contrast, more resistant materials, such as lignin, have k values in the range 0.002–0.004 day-1, corresponding to turnover times of 250 to 500 days. The data summarized in Table 3.1 for the turnover times of the major soil organic matter fractions in the Jenkinson and Rayner (1977) model indicate even slower turnover of some fractions, with turnover times of centuries and millennia. This type of very slow turnover is the result of the combination of

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chemical stability of the resource as well as protection from decay. Although the evidence for the actual existence of some of the organic matter fractions in Figure 3.1 is indirect, many studies have shown that organic matter fractions decompose at different rates because of differences in their chemical structure (i.e., chemical protection) and that adsorption or association of organic matter with mineral soil particles reduces the decomposition rates (i.e., physical protection) (Jenkinson 1981). In many organic matter models, an inert or passive compartment is added to improve the performance of the model (Jenkinson et al. 1987; Parton et al. 1987). Clearly, there cannot be a completely inert soil C fraction in soil, and modeling simply points to the presence of a very stable organic matter fraction. Making the link between the presence of this inert soil C and its biological role remains elusive, but even soils that have been deprived of fresh organic matter inputs for prolonged periods (e.g., decades) are still biologically active, and activity must be being sustained by more degraded components of the soil organic matter (Lawson et al. 2000). 3.3.2  Environmental Factors The main environmental factors that influence decomposition are the water and oxygen availability, redox conditions, pH, temperature, and the degree of physical protection. In the context of decomposition in soils, the water content and oxygen availability often interact to influence decomposition. Without water biological processes cease. However, even under naturally arid conditions, this very rarely occurs because films of water persist over soil particles. Thus, decomposition slowly proceeds even in naturally dry soil because of the activity of those organisms able to access water. Griffin (1972) reported that fungi are more active in dry soils than bacteria. Because of the different range of water contents over which different components of the soil microbial community are active, there is a rather flat optimum soil water content for decomposition (Clement and Williams 1962). Fluctuations in microbial activity accompany rapid changes in moisture content; for example, Birch (1958) showed that rewetting a dry soil enhanced the short-term rate of organic matter decomposition and that this effect was reproduced following repeated drying and rewetting. These observations are consistent with the increased turnover of microbial biomass due to wet–dry cycles in the soil (Jenkinson and Ladd 1981). At the other extreme, biological activity in wet soils is usually limited by lack of O2 because of the low solubility and slow diffusion rate of O2 in water. However, even in the absence of O2, the wide physiological diversity among the bacteria means that facultatively and obligately anaerobic bacteria are able to use alternative electron acceptors to O2 (Table 3.2). The decomposition of plant material is also qualitatively

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Table 3.2  Alternative Electron Acceptors (redox couple) Used by Aerobic, Facultatively Anaerobic, and Obligately Anaerobic Bacteria at Neutral pH and the Associated Microbial Processes

Redox Couple

Microbial Process

Redox Potential (mV)

Organisms Involved

O2 → H2O

Aerobic respiration

+820

NO3– → N2O → N2 Mn4+ → Mn2+

Denitrification Manganese reduction Fermentation

+420 +410

Animals, plants roots, fungi, aerobic bacteria e.g., Pseudomonas spp e.g., Bacillus spp

+400

e.g., Clostridium spp

Iron reduction Dissimilatory nitrate reduction Sulphate reduction Methanogenesis

–180 –200

e.g., Pseudomonas spp. e.g., Acromobacter spp.

–220 –240

e.g., Desulfovibrio spp. e.g., Methanobacterium spp.

Oxidized organic compounds → reduced organic compounds such as organic acids Fe3+ → Fe2+ NO3– → NH4+ SO42– → H2S CO2 → CH4

different in waterlogged soils. For example, woody tissue in a riverbed persists for about 100,000 years (Alexander 1965), whereas in a well-drained soil even the slowest decomposing constituents persist for less than 3,000 years (Jenkinson 1981). This is because the breakdown of lignin in woody tissue is carried out by a restricted number of fungi that require O2 (Kirk 1984). Whether there is sufficient O2 in soil to support aerobic respiration can be strongly influenced by the availability of decomposable organic matter. This is particularly relevant in the context of grave soils, where there will be a large organic resource at a specific location in the soil. This will lead to the potential rate of O2 consumption during aerobic respiration exceeding the rate of O2 diffusion to the site of decomposition and thus will promote anoxic conditions and the prevalence of anaerobic microbial activities (Hopkins 2000; Janaway 1996). There are direct parallels between decomposing plant residues, with plant fragments acting as the foci for anaerobic microsites (Parkin 1987). In the context of grave soils, several of the products of anaerobic metabolism, such as volatile organic compounds arising from fermentation, sulfides, and CH4, may provide useful diagnostic indicators of the presence of a decomposing animal because of characteristic odor or color or because they can be detected by chemical analysis (Table 3.3) (Hopkins 2000; Vass et al. 2004).

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31 (0.7)

Control 115–30 cm

1.3 (0.13)

4.3 (0.39) 7.3 (0.63) 4.7 (0.23)

Total N (mg N g–1 soil)

11 (12.3)

130 (78.2) 700 (192) 3.9 (1.62)

[NH4+] (g N g–1 soil)

28 (0.71)

120 (78) 180 (51) 35 (11)

Amino Acid-N (g N g–1 soil)

3.5 (0.07)

4.5 (0.07) 5.6 (0.07) 3.5 (0.07)

pH

79 (20.5)

554 (231) 308 (15.6) 204 (12.7)

Microbial Biomass C (g C g–1 soil)

11 (3.5)

394 (96.2) 787 (86.3) 37 (9.9)

Basal Respiration Rate (nmol CO2 g–1 soil hour–1)

83 (45.3)

299 (13.4) 1990 (174) 102 (58.0)

N Mineral­ ization (g N g–1 soil day–1)

2.5Y 4/2

0.053*

2.5Y 5/3

10Y 5/1

0.81*

0.030 (0.0049)

5Y 5/2

Munsell Number 0.93*

[S2–] (pmol g–1 soil)

Dark grayish yellow Yellowish brown

Grayish olive Gray

Munsell Color

Notes: The values are the means of two replicate samples from a single grave followed by standard deviations in brackets, except where marked by *, for which there was only one replicated. In the original experiment (Hopkins, Wiltshire, and Turner 2000) there were three replicate graves, each containing a pig carcass and each with a separate control, but the data from only one of the graves are shown here.

57 (0.14) 162 (12.7) 89 (8.6)

Pig grave 0–15 cm Pig grave 15–30 cm Control 10–15 cm

Total C (mg C g–1 soil)

Table 3.3Selected Properties of Soils from an Experimental Pig Grave

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There are many examples of decomposition being slower in acidic compared with near neutral soils (e.g., Jenkinson 1971). This is in part because of the reduced overall microbial activity in acidic soils. As already mentioned, the absence of earthworms from low pH soils also indirectly affects the decomposition rate as well as the accumulation of partially decomposed organic matter in the upper part of the profile (Hopkins, Shiel, and O’Donnell 1988). Inorganic nutrients such as phosphorus, sulfur calcium, and potassium are rarely limiting to microbial activity in soil, but nitrogen may be. Nitrogen is released in inorganic (i.e., plant-available) forms—that is, mineralized— during organic matter decomposition when that organic matter provides a surplus of nitrogen in relation to the carbon supplied to the decomposer organisms. The converse of nitrogen mineralization, in which the decomposer organisms assimilate nitrogen from other sources in the soil, is referred to as nitrogen immobilization. The balance between nitrogen mineralization and immobilization depends on qualitative as well as quantitative aspects of the organic matter and the decomposer organisms (Jenkinson 1981; Swift et al. 1979), but in many cases a C-to-N ratio of around 20 is the threshold, above which nitrogen immobilization occurs and below which nitrogen mineralization occurs (Harmsen and van Schreven 1955). By comparison with plant biomass, animal biomass usually has a much lower C-to-N ratio because of the large proportion of structural proteins. As high C-to-N material decomposes, net N mineralization occurs and NH4+ accumulates. Even in acidic soils, the alkaline effect of NH4+ may lead to increased pH around decomposing animals (Table 3.3) (Carter and Tibbett 2006; Hopkins 2000). Like most biological processes, the rate of organic matter decomposition increases approximately in line with the van t’Hoff rule that the rate of reaction doubles for a 10°C temperature rise in the range 10–40°C (Jenkinson 1981). In temperate regions, the temperature of the upper surface of the soil may rise above 30°C, particularly if there is not dense plant cover, but at depths below about 10 cm the soil temperature is usually between 5°C and 20°C, with the size of the daily and seasonal fluctuations declining with increasing depth (Payne and Gregory 1988). This means that low temperature is one of the constraints on the decomposition of organic matter in soil. Floate (1970) showed that low temperature is an important factor leading to the accumulation of organic matter in upland soils, with virtually no decomposition at 0°C, and to a doubling in the decomposition rate between 5°C and 10°C (Carter and Tibbett 2006; Tibbett et al. 2004). At the macromolecular level, adsorption of clay particles to biological molecules such as proteins reduces their rate of decomposition by protecting them from microbial attack (Jenkinson 1981). On a larger scale, entrapment of organic matter in soil aggregates reduces the rate of decomposition at least while the aggregate persists. On a soil-profile scale, Shields and Paul (1973) showed that the rate of decay of organic matter on the soil surface was

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influenced to a far greater extent by environmental factors such as temperature and moisture than when incorporated into the soil. 3.3.3  Presence and Activity of Organisms The quantity of carbon from soil organic matter returned to the atmosphere as CO2 by the respiration of soil animals is usually small, but these organisms have an important functional role. Soil animals (e.g., earthworms, nematodes) incorporate organic matter from the surface into the soil, bring the organic matter into intimate association with soil microorganisms and inorganic nutrients in the soil, and comminute large fragments of organic matter, thereby increasing the surface area available for colonization by microorganisms and, by partially digesting the plant material, thereby biochemically and physically conditioning it. By contrast, the soil microorganisms are directly responsible for most of the CO2 returned to the atmosphere from soil organic matter. Microorganisms in the soil can be divided between two ecological strategies. Those that respond rapidly to addition of fresh substrate are referred to as the zymogenous component of the biomass (sensu Winogradsky 1924), whereas those that eke out an existence on the older, more stable organic matter are referred to as the autochthonous component of the biomass (sensu Winogradsky 1924). The soil community is probably not as sharply divided as Winogradsky’s definitions would imply, but the distinction is useful as it emphasizes extreme strategies and recognizes that at any one time a sizeable fraction of the microbial community may be dormant or relatively inactive because of a shortage of readily exploitable resources. Similarly, r-selected organisms (i.e., those showing rapid proliferation following addition of a pulse of substrate) and K-selected organisms (i.e., those maintaining a near constant but relatively inactive population) is probably not rigid. The zymogenous and autochthonous categories are approximately analogous to r-selected and K-selected organisms, respectively. Both concepts are useful for understanding soil C dynamics but cannot be related directly to particular taxa, which may switch strategies (Chapman and Gray 1981), or applied unreservedly in a complex environment such as soil where many factors other than C supply may affect biological activity and biomass. For many purposes, it has invariably been deemed adequate to treat the decomposer organisms as an undifferentiated entity in soil C turnover. This is clearly an oversimplification, and understanding of how or even whether the diversity of different types of decomposer organisms and the function of decomposition are causally related has in recent times become one of the most topical questions in ecology and in soil ecology in particular (Bardgett, Usher, and Hopkins 2005).

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It is often presumed by environmental microbiologists that all things are everywhere and that microorganisms are collectively infallible when it comes to degrading organic matter. From this it follows that given appropriate physical and chemical conditions it is possible to find any known microorganism and many unknown ones, so the presence or organic remains will lead to selection from the indigenous community of that particular environment of those capable of decomposing the residue. Neither of these pieces of environmental microbiological dogma is actually true, but both are sufficiently close to being true that they can be adopted as safe working principles under most circumstances when considering the decomposition of all natural and many synthetic organic residues. There is a related question about the diversity of the microbial decomposer community that is relevant. The conditions in a grave will select for a particular community of microorganisms because of, for example, the particular concentrations of nutrients and availability of water and O2. Those organisms favored by the environmental conditions will be selected for and will increase to the extent that they will become a dominant component of the microbial community, from perhaps having been present, but undetectable by current techniques, in the soil before the burial of the body or carcass. In addition to illustrating the diversity of the soil microbial community, the shift in composition toward a characteristic community may have some value as an indicator of grave sites (Tibbett and Carter 2003).

3.4  Can Ecological Principles Be Applied to Forensic Investigations? Although the organic residues with which Swift et al. (1979) were concerned were ecological materials mostly of plant or microbial origin, Hopkins (2000) argued that the same principles can be applied to any organic matter in soils, including archaeological and forensic remains. However, there have been few, if any, controlled and replicated studies of the decomposition of large animal remains under conditions taphonomically relevant conditions to test this proposal rigorously. Extensive observations have been made of the decomposition of both buried and unburied human cadavers (Mann, Bass, and Meadows 1990). Although there are difficulties interpreting some of the observations because of apparent lack of replication and experimental controls, Mann et al. (1990) ranked the factors affecting decay of human remains based on the considerable expertise of the scientists involved (Table 3.4). To the ranking of Mann et al. (1990), an assignment to one of the different factors (i.e., resource quality, environment conditions, and decomposer organisms) (Swift et al. 1979) has been added in Table 3.4. The observations

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Table 3.4  Factors Affecting the Decomposition of Animal Bodies

Factor

Score on Scale from 5 to 1, Where 5 Indicates the Greatest Effect

Type of Factor

5 5 5 4 4 4 3 3 3 2 1

Environment Organism Environment Organism Resource Environment Environment Resource Resource Resource Environment

Temperature Access by insects Burial depth Carnivores/rodents (scavengers) Trauma (penetration/crushing) Humidity/aridity Rainfall Body size and weight Embalming Clothing Substrate (little detailed information)

Source: Adapted from Mann, R. W., Bass, W. M., and Meadows, L., J. For. Sci. 35, 103–111, 1990 (with permission).

included both buried and unburied bodies. This accounts in part at least for the high ranking of temperature and depth, which are in any case linked factors since temperature decreases and becomes less variable with depth of burial. Similarly, it also accounts for the influence of organisms, specifically animals, on decay, because access by scavengers and sarcophagous insects is easier for bodies left at the surface. The different stages in cadaveric decay are summarized in Table 3.5. This table has been assembled with information from several sources, most Table 3.5  Stages of Cadaveric Decay Stage Fresh body Early decay Advanced decay Skeletonization

Characteristics Failure of metabolism and repair mechanisms Autolysis and bacterial decay characterized by methane, hydrogen sulphide, hydrogen, and carbon dioxide production Liquefaction of soft tissues, saponification of lipids, adipocere formation Bones remains, fluids disperse

Sources: Adapted from Gill-King, H., in Forensic Taphonomy—The Postmortem Fate of Human Remains (W. D. Haglund and M. H. Sorg, Eds.), Boca Raton, FL: CRC Press, 93–108, 1997; and Trick, J. K., Williams, G. M., Noy, D. J., Moore, Y., and Reeder, S., Paper presented at the British Society of Soil Science/Society for Environmental Geochemistry and Health joint meeting on Soil, Environment and Health at the University of Birmingham Medical School, April 6–7, 2000 (with permission).

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notably Gill-King (1997) and Trick et al. (2000). The time taken for the overall process to pass each stage is probably highly variable and will reflect many factors including those previously outlined. The microbial processes involved, together with intrinsic autolysis, during cadaveric decay are outlined by Janaway (1996). Microorganisms from the soil probably have a relatively minor role in cadaveric decay, at least at the outset or unless the body was dismembered or seriously mutilated. A succession from predominantly aerobic to anaerobic members of the enteric community, which colonize from the alimentary canal and gain access to the rest of the body via the lymphatic and artero-vascular system, is primarily responsible for the biodegradation of body tissue together with the activity of intrinsic enzymes that contribute to autolysis (ibid.). Fats are usually hydrolyzed as a result of both intrinsic lipases and those of microorganisms, which leads to the often rancid deposit, adipocere, containing a range of fatty acids, including stearic, palmitic, and oleic acids. The fats and fatty acids may be completely oxidized, but this is not usually observed because of the restricted oxygen supply. Similarly, proteolysis is catalyzed by both intrinsic enzymes and those of microorganisms (ibid.). The accumulation of microbially produced gases such as H2S and CH4 can lead to distension of the body cavity, which contributes to leakage, initially via the natural orifices and subsequently via ruptures in the skin as it decays, of a solution rich in the soluble products of decay (ibid.). The release of this liquid, which may be aided by percolation of water, leads to marked increases in the rate of microbial respiration and the size of the soil microbial community (Hopkins et al. 2000). For a greater understanding of the rate at which liquid and gaseous products of decay disperse from a corpse, knowledge of the physical conditions of the soil is important. It is probable that members of the soil microbial community have a major role in the biochemical processes of decay only in the later stages of decay. Although decay usually occurs under predominantly anaerobic conditions, because of the large amount of decomposable organic carbon in a body, invertebrates that burrow into corpses can increase the rate of gas transfer and possibly facilitate decomposition by mixing, secretion of enzymes, and the introduction of microorganisms from the soil (Janaway 1996).

References Alexander, M. (1965). Biodegradation: Problems of molecular recalcitrance and microbial falliblity. Adv. Appl. Micro. 7, 35–92. Bardgett, R. D., Usher, M. B., and Hopkins, D. W. (Eds.) (2005). Biological Diversity and Function in Soils. Cambridge, UK: Cambridge University Press, Cambridge. Birch, H. F. (1958). The effect of soil drying on humus decomposition and nitrogen availability. Plant Soil 10, 9–31.

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Carter, D. O. and Tibbett, M. (2006). Microbial decomposition of skeletal muscle (ovies aries) in a sandy loam soil at different temperatures. Soil Biol. Biochem. 38, 1139–1145. Chapman, S. J. and Gray, T. R. G. (1981). Endogenous metabolism and macromolecular composition of Arthrobacter globisformis. Soil Biol. Biochem. 13, 11–18. Clement, C. R. and Williams, T. E. (1962). An incubation technique for assessing the nitrogen status of soils newly ploughed from leys. J. Soil Sci. 13, 82–91. Floate, M. J. S. (1970). Decomposition of organic material from hill soils and pastures: III: The effect of temperature on the mineralization of carbon, nitrogen and phosphorus from plant materials and sheep faeces. Soil Biol. Biochem. 2, 187–197. Doyle, A. C. (1980). The Penguin Complete Sherlock Holmes. London, UK. Penguin Books. Gill-King, H. (1997). Chemical and ultrastructral aspects of decomposition, in Forensic Taphonomy: The Postmortem Fate of Human Remains (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 93–108. Griffin, D. M. (1972). Ecology of Soil Fungi. London: Chapman and Hall. Harmsen, D. A. and van Schreven, D. A. (1955). Mineralization of organic nitrogen in soil. Adv. Agron. 7, 299–398. Hopkins, D. W. (2000). Interfaces of soil biology with archaeological investigations, in Soil Biochemistry volume 10 (J.-M. Bollag and G. Stotzky, Eds.). New York: Marcel Dekker, 483–512. Hopkins, D. W., Shiel, R. S., and O’Donnell, A. G. (1988). The influence of sward species composition on the rate of organic matter decomposition in grassland soil. J. Soil Sci. 39, 385–392. Hopkins, D. W., Wiltshire, P. E. J., and Turner, B. D. (2000). Microbial characteristics of soils from graves: An investigation at the interface of soil microbiology and forensic science. Appl. Soil Ecol. 14, 283–288. Janaway, R. C. (1996). The decay of buried human remains and their associated materials, in Studies in Crime: An Introduction to Forensic Archaeology (J. Hunter, C. Roberts, and A. Martin, Eds.). London: B.T. Batsford Ltd., 58–85. Jenkinson, D. S. (1971). Studies of the decomposition of 14C labelled organic matter. Soil Sci. 111, 64–70. Jenkinson, D. S. (1981). The fate of plant and animal residues in soil, in The Chemistry of Soil Processes (D. J. Greenland and M. H. B. Hayes, Eds.). Chichester, UK: John Wiley and Sons, 505–561. Jenkinson, D. S. and Ladd, J. N. (1981). Microbial biomass in soil: measurement and turnover, in Soil Biochemistry volume 5, (E. A. Paul and J. N. Ladd, Eds.). New York: Marcel Dekker, 415–471. Jenkinson, D. S., Hart, P. B. S., Rayner, J. H., and Parry, L. C. (1987). Modelling the turnover of organic matter in long-term experiments at Rothamsted. Intecol. Bull. 15, 1–8. Jenkinson, D. S., and Rayner, J. H. (1977). The turnover of soil organic matter in some of the Rothamsted classical experiments. Soil Sci. 123, 298–305. Kirk, T. K. (1984). Degradation of lignin, in Lignin Biodegradation: Microbiology, Chemistry and Potential Applications (D. T. Gibson, Ed.). New York: Marcel Dekker, 399–437.

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Lawson, T., Hopkins, D. W., Chudek, J. A., Janaway, R. C., and Bell, M. G. (2000). Interactions of the soil organisms with materials buried for up to 33 years in the Wareham archaeological experimental earthwork. J. Arch. Sci. 27, 273–285. Mann, R. W., Bass, W. M., and Meadows, L. (1990). Time since death and decomposition of the human body: Variables and observations in case and experimental field studies. J. Foren. Sci. 35, 103–111. Parkin, T. B. (1987). Soil microsites as a source of denitrification variability. Soil Sci. Am. J. 51, 1194–1199. Parton, W. J., Schimel, D. S., Cole, C. V., and Ojima, D. S. (1987). Analysis of factors controlling soil organic matter in Great Plains grasslands. Soil Sci. Soc. Am. Proc. 53, 1173–1179. Payne, D. and Gregory, P. (1988). The temperature of the soil, in Russell’s Soil Conditions and Plant Growth (A. Wild, Ed.). Harlow, UK: Longman Scientific and Technical, 282–297. Shields, J. A. and Paul, E. A. (1973). Decomposition of 14C labelled plant material under field conditions. Can. J. Soil Sci. 53, 297–306. Swift, M. J., Heal, O. W., and Anderson, J. M. (1979). Decomposition in Terrestrial Ecosystems. Oxford: Blackwell Scientific Publications. Tibbett, M. and Carter, D. O. (2003). Mushrooms and taphonomy: The fungi that mark woodland graves. Mycologist 17, 20–24. Tibbett, M., Carter, D. O., Haslam, T., Major, R., and Haslam, R. (2004). A laboratory incubation method for determining the rate of microbiological degradation of skeletal muscle tissue in soil. J. Foren. Sci. 49, 560–565. Trick, J. K., Williams, G. M., Noy, D. J., Moore, Y., and Reeder, S. (2000, April 6–7). Pollution potential of cemeteries: Impact of the 19th century Carter Gate cemetery, Nottingham. Paper presented at the British Society of Soil Science/ Society for Environmental Geochemistry and Health joint meeting on Soil, Environment and Health at the University of Birmingham Medical School, Birmingham, AL. Vass, A. A., Smith, R. R., Thompson, C. V., Burnett, M. N., Wolf, D. A., Synstelien, J. A., et al. (2004). Decompositional odor analysis database. J. Foren. Sci. 49, 760–769. Winogradsky, S. (1924). Sur la microflore autochthone de la terre arable. Compte Rendu Acad. Sci. (Paris) 178, 1236–1239.

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Soil Fungi Associated with Graves and Latrines: Toward a Forensic Mycology

4

Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

Contents 4.1  Introduction................................................................................................. 68 4.2  Ammonia Fungi (AF)................................................................................. 69 4.2.1  Experimental Grouping of Fungi................................................. 69 4.2.2  Fungal Succession and Mycorrhizal Relations........................... 72 4.2.3  Environmental Conditions............................................................74 4.2.3.1  Initial Conditions............................................................74 4.2.3.2  Changes in the Soil..........................................................74 4.2.3.3  Responses of Other Organisms.................................... 75 4.3  Postputrefaction Fungi (PPF) and AF...................................................... 76 4.3.1  Fungal Growth Following Cadaver Decomposition on the Ground...................................................................................... 76 4.3.2  Fungal Growth Following Cadaver Decomposition Belowground................................................................................... 78 4.3.3  Fungal Growth Following Excreta Decomposition on the Ground...................................................................................... 80 4.3.4  Fungal Growth Following Excreta Decomposition Belowground................................................................................... 80 4.3.5  Habitat-Cleaning Symbiosis......................................................... 82 4.4  Physiology of the AF and PPF................................................................... 84 4.4.1  Spore Germination......................................................................... 84 4.4.2  Vegetative Growth.......................................................................... 84 4.4.2.1  Nitrogen Utilization....................................................... 84 4.4.2.2  Hydrogen Ion Concentration....................................... 86 4.4.2.3  Enzymatic Activity........................................................ 87 4.4.3  Formation of Reproductive Structures........................................ 87 4.4.4  Physiology and Succession............................................................ 89

67

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4.5  Possibilities for Forensic Application....................................................... 90 4.5.1  Human Behavior or Activities May Be Evidenced by Fungal Growth................................................................................ 90 4.5.2  Initial Conditions and the Postdeposition Interval May Be Assessed...................................................................................... 90 4.5.3  Simulation Experiments.................................................................91 4.5.4  Limitations....................................................................................... 93 Acknowledgments................................................................................................. 94 References............................................................................................................... 94 Appendix 4.1.......................................................................................................... 99 Appendix 4.2........................................................................................................ 102

4.1  Introduction The vertebrate body can be divided into soft and hard tissues: The former category is composed of muscles, internal organs, veins, and blood, and the latter is composed of bones, hair, horns, hooves, nails, feathers, and bills. Where a cadaver has been left to decompose on the ground and has not been scavenged before or during decomposition, the soft tissues can readily disappear, consumed mainly by invertebrates and bacteria, while the hard tissues endure. Probably for this reason, the hard tissues have long been known to support a specialized group of fungi adapted to this substratum: keratinophilic fungi (Hudson 1972). In contrast, the soft tissues have only relatively recently been shown to yield a particular group of fungi after their disappearance (Sagara 1976b). Similarly, feces, especially of herbivores, have long been known to bear coprophilous fungi (Webster 1970) as they endure, whereas urine and readily decomposing feces have only recently been shown to yield a specific group of fungi (Sagara 1975). These two new groups of fungi are in fact almost identical in species composition and successional development. The present chapter focuses on this neglected aspect of nature and its forensic potential. The prospective use of fungi in forensic taphonomy originated from studies on fungi that sporulate or fruit on (primarily) forest soils after experimental addition of urea (or nitrogenous compounds such as ammonia) (Sagara 1973, 1975; Sagara and Hamada 1965; see also Suzuki 2006). Observational studies under natural, or nonexperimental, conditions found the same fungi fruiting after the decomposition of cadavers and excreta and similar changes in the soil to the urea- or ammonia-treated soil. This process seemed sufficient to establish the sites of cadaver and excreta decomposition as new fungal habitats, giving a competitive advantage to a special group of

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fungi (Sagara 1975, 1992, 1995). Thus, the treatment of soil with urea (and other ammoniacal compounds) can simulate cadaver and excreta decomposition (Sagara 1975, 1992, 1995), at least from a mycological perspective. Since observations from nature are inevitably based on sporadic case studies, the chapter first discusses fungal responses to urea treatment as the fundamental basis for a forensic mycology. In this chapter, the fungi that form fruiting bodies on the soil after treatment with urea are termed ammonia fungi (AF), and those that fruit after cadaver or excreta decomposition under natural conditions are termed postputrefaction fungi (PPF). This definition of AF differs somewhat from its original description (Sagara 1975) but may be easier to understand and use. Both groups are fundamentally the same in species composition and fruiting successions. To clarify the nature of fungal trigger materials in the soil, the term excreta is used when both urine and feces are collectively referred to or when it is not known whether the waste in question was urine or feces, or both. The term latrine is used to denote a site of repeated excretal deposition, and the term feces is used to refer to solid waste.



(a)

(b)

Figure 4.1  Conidium production of Doratomyces putredinis, an early colonizer on urea plot. 160 g urea-N was spread to 45 × 90 cm plot in a Quercus forest, Derbyshire, United Kingdom, on May 21, 1986; photograph taken on July 14, 1986. Bar in (a) = 10 mm; bar in (b) = 1 mm.

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Figure 4.2  Flush of Lyophyllum tylicolor, marking an early phase of the fungus succession on urea plot. 1.6 kg urea-N was spread to 4 × 5 m plot in a PinusChamaecyparis forest, Kyoto, Japan, on May 2, 1971; photograph taken on May 21, 1971 (a part of the plot is shown).

4.2  Ammonia Fungi (AF) 4.2.1  Experimental Grouping of Fungi The application of urea or a related nitrogen compound to the soil encourages a unique group of fungi to form reproductive structures (conidia or fruit bodies) on the soil surface in a successional sequence (Figures 4.1–4.4). Fungal growth occurs without addition of any inocula and is accompanied by some characteristic changes in the soil (see Sections 4.2.3.2 and 4.2.3.3). The effective rate of fertilization is between 80 and 320 g of nitrogen (N) per m2, which is much greater than conventional forest fertilization practice. Ammonia, used in the aqueous form, is the simplest compound to cause these phenomena. A sudden and excess input of ammonia, which inevitably causes alkalinity in the soil, is the essential substance to trigger the phenomena in question. It is likely that urea treatment and ammonia treatment exert the same effect so far as the terrestrial (epigeous) fungi are concerned. Nonalkaline ammonium salts ((NH4)2SO4, NH4NO3, NH4Cl), nitrite (NO2-), nitrate (NO3-), carbohydrates, and so forth do not show such effects. Alkali and alkaline-earth materials are fairly effective; however, this may be

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Figure 4.3  Flush of Hebeloma radicosoides, marking a late phase of the fungus succession on urea plot. 160 g urea-N was spread to 0.5 × 1 m plot in a Quercus forest, Kyoto, Japan on February 2, 1994; photograph taken on October 12, 1994. The 1-m folding scale marked one edge of the plot. Amblyosporium botrytis Ascobolus denudatus Lyophyllum tylicolor Peziza urinophila Pseudombrophila petrakii Coprinus echinosporus Peziza moravecii Hebeloma vinosophyllum Hebeloma radicosoides Laccaria bicolor Collybia cookei Mycena pura 10 Apr. 89 0

1 Nov. 89 1 May 90 200

400

1 Nov. 90 1 May 91 600

800

1 Nov. 91 Date 1000 Days

Figure 4.4  An example of the fungal succession on urea plot. 327 g urea-N was spread to 0.5 × 2 m plot in a Pinus forest, Kyoto, Japan, on April 10, 1989, and observation continued until 1992. Occurrence of the reproductive structures is marked +. (Modified from Yamanaka, T., Bull. Jpn. Soc. Microbial Ecol. 10, 67– 72, 1995. With permission.)

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attributed to the liberation of ammonia from the soil itself. In northern forests where soil reserves of NH4+-N or organic N are larger, alkali treatment might replace nitrogen application by liberating a large amount of NH4+-N from the soil itself. This seems to have been shown with soil from Denmark (Petersen 1970). Those fungi that specifically respond to the treatment of soil with urea, aqueous ammonia, and related nitrogenous materials have been placed in a chemoecological group and termed AF (Sagara, 1975, 1992, 1995; see also Suzuki 2006). In this chapter, the group AF is taken to include only those responding to urea treatment. The number of species belonging to this group totals about 50 (Appendix 4.1). It should be noted that ammonia is primarily a trigger material and that many secondary effects and environmental conditions are important (see Sections 4.2.2 and 4.2.3). It is also to be noted that a single treatment is essential, as the repeated application of urea to the same soil shows different features in the growth of fungi as well as in the changes of soil (Sagara 1975). The fungal community in the urea-treated soil, as determined by isolation techniques (Furuya 1990), seems to be different from that seen on the soil surface. Although this chapter does not cover this aspect of AF, we do not exclude this approach in understanding the fungal community as a whole or for developing forensic tools. 4.2.2  Fungal Succession and Mycorrhizal Relations The succession of AF as seen by the sequential reproduction of fungi on soil in urea-treated plots roughly follows the following scheme: Deuteromycetes → Ascomycetes (mostly Discomycetes) → Basidiomycetes (mostly Agaricales)

Alternatively, the succession can be divided into an early phase or stage and a late phase or stage, at least when it takes place in central Japan (Figures 4.1–4.5) (Sagara 1975, 1992, 1995; Suzuki, Uchida, and Kita 2002; Yamanaka 1995a; see also Appendix 4.2). The early phase (EP) comprises molds (e.g., Amblyosporium botrytis, Doratomyces putredinis: Deuteromycetes), cup-fungi (e.g., Ascobolus denudatus, Peziza moravecii: Discomycetes), and smaller gill-fungi (e.g., Lyophyllum tylicolor, Coprinus spp.: Agaricales). The late phase (LP) comprises larger and smaller gill-fungi (e.g., Hebeloma spp., Laccaria spp., Lyophyllum ambustum: Basidiomycetes). The two phases usually occur discontinuously; see Figure 4.4, for example: The interruption occurred between day 142 (August 30) and 191 (October 18). The EP lasts for a period of 1 to 10 months and the LP for a period of one to several mushroom seasons (up to 4 years). This persistence depends on the season at treat-

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9

pH

7

5

3

Nitrogen Concentration (log 10)

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5 4 3

Ab Ad Lt

2

Pp

Pu Ce

1

Hr

0 19 Apr. 90

1 Nov. 90

0

1 Nov. 91

1 May 91

200

Hr

400

Date

600Days

Figure 4.5  Changes in the concentrations (µg N g -1 dry soil) of NH4+-N (▫) and NO3--N (), and in the pH value () in urea-treated soil. On April 19, 1990, 327 g urea-N was spread to 0.5 m × 2.0 m plot in a Pinus-Chamaecyparis forest, Kyoto, Japan. Occurrence of the reproductive structures of ammonia fungi is presented on the figure: Ab, Amblyosporium botrytis; Ad, Ascobolus denudatus; Lt, Lyophyllum tylicolor; Pu, Peziza urinophila; Pp, Pseudombrophila petrakii; Ce, Coprinus echinosporus; Hr, Hebeloma radicosoides. (From Yamanaka, T., Ph.D. diss., Kyoto University, 2002. With permission.)

pH11

pH10

pH9

pH8

pH7

pH6

pH5

pH4

pH3

Figure 4.5a  Colors of the leachate from the humus soaked in the buffer solutions with pH 11–3 at one-unit intervals (left to right), showing the importance of alkalinity in this color change and the sensitivity of the soil to treatment. The humus was collected from a Pinus-Quercus forest, Ibaraki, in April 1997. (From Yamanaka, T., Ph.D. diss., Kyoto University, 2002. With permission.) (See color insert following p. 178.)

ment, the amount of urea used, and the type of vegetation (Sagara 1976a). In the United Kingdom, a prolonged EP has been recorded, possibly due to the cooler climate (Sagara, unpublished). Species composition of the EP differs between forests and nonforested vegetation (e.g., weed communities) and between hills or mountains (i.e., nonpopulated areas) and level land (i.e., populated areas) (Sagara 1975).

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Embracing such differences, the EP species seem generally cosmopolitan (see Suzuki et al. 2003). This is probably because they are saprotrophic (Sagara 1975; Yamanaka 1995a; see also Section 4.4). From observations in the United Kingdom, Rhopalomyces elegans var. minor, a possibly nematophagous zygomycete, may now be included in the EP (Sagara, unpublished), as does Doratomyces putredinis (Figure 4.1). In Japan D. putredinis does not appear in field experiments but only in the laboratory (Sagara 1975). The LP fungi include many ectomycorrhizal species and therefore are strongly influenced by the ectomycorrhizal status of the vegetation (Sagara 1995). Ectomycorrhizas are a symbiosis between woody plants and certain species of higher fungi (see Smith and Read 1997). Consequently, the LP fungi tend to be more provincial and may vary among regions of the world. For further biogeographic discussions see Suzuki et al. (2003). When urea has been applied deep in the soil, the EP is not observed and only the deep-rooting Hebelomas (H. danicum and/or H. radicosoides) appear to fruit (Sagara 1995; Sagara et al. 2000). The LP also embraces the process of recovery from disturbance, and hence many fungi that are difficult to classify as AF may join this phase (Sagara 1995). For mechanisms of the AF succession, see Section 4.4.4. 4.2.3  Environmental Conditions 4.2.3.1  Initial Conditions The EP fungi generally require the presence of humus, the soil layer of organic matter termed Ao horizon or O horizon (Sagara 1975). Even for the LP deeprooted Hebelomas, the addition of humus deep in the soil (see Section 4.2.2) promotes their fruiting aboveground (see Figure 4.15, Sagara 1984; Sagara et al. 2000). The species composition of the AF community after a urea treatment differs with the time of year (i.e., season) and the application rate of the urea (Sagara 1975). It seems likely that this is an effect due to temperature and amount of N per unit area (or volume) of soil (Sagara 1976a). For instance, a combination of higher temperature (20–30°C) and higher rate of urea-N (20–40 mg g-1 dry humus) favors Cladorrhinum foecundissimum and Coprinus neolagopus to grow. A combination of moderate temperature (10–20°C) and medium rate of urea-N (10–20 mg g-1 dry humus) encourages Ascobolus denudatus, Lyophyllum tylicolor, and some others to grow. Exceptionally, Pseudombrophila petrakii, again an EP species, seems to require fresh leaf litter as a substrate (Sagara 1976c). The LP could also be affected by the phenology of plant root growth as a consequence of mycorrhizal relations. The

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appropriate time of treatment in a year for obtaining each ammonia fungus is shown in Appendix 4.1. 4.2.3.2  Changes in the Soil Urea added to the soil is decomposed to ammonia overwhelmingly in the O horizon (Imamura, Yumoto, and Yanai 2006). Under this condition, the humus becomes black, with a smell of ammonia and then of compost, holding higher water content (Sagara 1975). In the EP, the NH4+-N in the soil shows 500–2,000 times higher than that of the control, and the pH value increases to 8–9 (Figure 4.5). In the LP, NH4+-N decreases gradually but remains slightly higher than in the control. The NO3--N increases transiently to 160–400 times higher than control values, and, accordingly, the pH value decreases to 4–6 (Figure 4.5). In this phase, the fine roots of plants, which were once killed by the treatment, recover (Furuya 1990; Sagara 1975; Suzuki, Uchida, and Kita 2002; Yamanaka 1995a, 1995b; see also Section 4.2.3.3). This might be similar to the fatal effect that carcass decomposition has on prairie plants, which die and are ultimately replaced by pioneer species, thereby resetting plant succession (Towne 2000). Humus blackened by urea treatment produces water extract that is dark brown to almost black in color. In contrast, the water extract from the control (untreated) humus is light yellow (Sagara 1975; Yamanaka 1995c). The same color change takes place immediately after the treatment with aqueous ammonia—that is, far sooner than in the urea treatment. Aqueous ammonia adjusted to pH 4 or 6 with buffer solutions does not cause this change, whereas buffer solutions with pH 7 and greater do (Figure 4.5a) (Yamanaka 2002). This indicates that the color change is caused by the alkalinity that has been brought about by the excess of ammonia. The black color of the water extract may possibly represent, for example, humic acid or fulvic acid, which are known to be liberated under alkaline conditions (Yamanaka 2002). Organic matter extracted with 50% methanol increases during the EP (Yamanaka 1995a). This may be because of the increased solubility of the humus under the alkaline conditions as previously described. In contrast, water-soluble carbohydrates and phenolics decrease by the treatment. This may be explained, at least partly, by the damage of plant roots that are known to exude water-soluble carbohydrates to soil (Yamanaka 2002). 4.2.3.3  Responses of Other Organisms The urea treatment destroys the indigenous community of soil organisms. Heterotrophic bacteria as a whole increase approximately 1,000 times more than those in the untreated soil, following their slight decrease soon after the treatment (Yamanaka 1995b). Bacterial numbers then decrease gradually. Nitrifying bacteria increase transiently, in keeping with the increase of

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NO3--N (Yamanaka 1995b). Cellulose-decomposing bacteria decrease after the treatment and remain in much smaller numbers for a considerable period (He and Suzuki 2004). Nematodes that are possibly bacteriophagous also increase after the treatment (Yamanaka 1995a, 1995c), a possible reflection of increased bacterial numbers. This phenomenon has also been observed in association with decomposing pig carcasses (Carter et al., unpublished). The appearance of the possible nematode parasite Rhopalomyces elegans var. minor (see Section 4.2.2) creates a potential tripartite food web among bacteria, nematodes, and fungi, as hypothesized by Barron (1977). The soil arthropod fauna greatly changes following urea treatment. Certain species that are rarely found in the untreated soil, such as Atheta spp. (Coleoptera: Staphylinidae) and small dipterans, appear abundantly in the treated soil under the alkaline conditions of the EP. Conversely, some arthropods, which are often found in the untreated soil, disappear from the treated soil (Kohei Sawada, pers. comm.). The fine roots of plants are killed by the urea treatment, presumably due to ammonia toxicity, but recover vigorously and abundantly during the LP (Sagara 1975). This recovery of roots is accompanied by colonization of the hyphae of LP fungi and by formation of ectomycorrhizas if the associated plants are ectomycorrhizal (Sagara 1995).

4.3  Postputrefaction Fungi (PPF) and AF 4.3.1  Fungal Growth Following Cadaver Decomposition on the Ground Human or other animal cadavers abandoned on the ground decompose quickly. For example, in the warm season of Japan, where average temperature equals 25°C, a human body can undergo significant decomposition within 10 days, leaving primarily bones and hair. Immediately after this stage (decomposition approaching skeletonization—see Chapters 2 and 8), a zygomycete phase occurs: Mucor spp. and/or Rhopalomyces strangulatus appear on the remaining animal matter and on the surrounding soil (Figure 4.6; Appendix 4.2). These fungi do not appear after urea treatment (Sagara 1975) and hence are classified in the PPF but not in the AF (Sagara 1992, 1995). The appearance of R. strangulatus on animal remains is accompanied by a strong smell of the putrefying carcass (Sagara 1989). From this point onward, fungal succession seems to generally follow a comparable scheme to urea-treated plots, with a similar array of species fruiting (Figure 4.7) (Sagara 1975, 1995). However, some species that do not belong to AF (e.g., Scutellinia scutellata and Glomus pubescens) may join the succession, as observed after the experi-

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b

d

c

Figure 4.6a–4.6d  A carrion crow (Corvus corone) carcass and Rhopalomyces strangulatus, an early colonizer on such a substratum (further information in Appendix 4.2). (a) The carcass lying on the ground. (b) Rhopalomyces strangulatus sporulating on part of the carcass; bar = 10 mm. (c) Photomicrograph of a R. strangulatus sporophore and spore (arrowhead); bar = 200 µm. (d) Photomicrograph of one of the R. strangulatus spores that are characteristically large; bar = 10 µm.

a

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78 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

Figure 4.7  Hebeloma vinosophyllum (arrowhead) fruiting beside the skull of an abandoned domestic cat body. The wrapped body seemed to have initially been buried 80–90 cm away but to have been dug out and moved by some animals. Place and date: Quercus forest, Hyogo, Japan, September 20, 1989. The folded scale is 51.5 cm long.

mental application of fish to the forest floor (Sagara 1975). The changes to the soil at sites where cadavers have decomposed are similar to those of ureatreated plots (Sagara 1975). The color change to black in the humus is often mistaken for oil or fat but it is in fact due to alkalinity of ammonia or amines (see Section 4.2.3.2). 4.3.2  Fungal Growth Following Cadaver Decomposition Belowground Examples here (Appendix 4.2) are not drawn from formal graves and coffin burials, but see Weimann (1940), Mueller (1953), Bonnet (1967), and Dent et al. (2004). The EP of AF succession is not observed unless burial has been very shallow. This is because the EP fungi require humus as a substrate (see Section 4.2.3.1) and because they do not develop pseudorhizas (i.e., prostipes in Sagara 1999) to grow out of deep soil. The shallow burial of a mammalian cadaver, with a soil cover of, for example, less than 10 cm, gives rise to most LP AF (Figure 4.8) (Sagara 1976b, 1981; Takayama and Sagara 1981). Under a deeper burial, maybe more than 20 cm, only those species that can respond to resources (including plant roots) deeper in the soil and develop pseudorhizas may fruit (e.g., Hebeloma radicosoides or H. danicum) (Kuroyanagi et al. 1982; Sagara, unpublished data). This phenomenon has taken root in popular culture (Sagara 1984), and in North America, H. syrjense (which may be conspecific with H. danicum; Appendix 4.1) has become known as

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H

U

S

d

c

R

F

Figure 4.8a–4.8d  Fruiting of Laccaria bicolor and detection of a buried mammal carcass from under the fungus (Appendix 4.2). (a) Decaying fruit bodies of L. bicolor with sticks as markers; the folded scale is 20 cm long. This spotted, unusual appearance of the ammonia fungus on the superficially undisturbed ground suggested that something that liberated ammonia had been buried there. (b) Excavation revealed remains of a mammal buried under the fruit bodies: (S) skull (broken), (U) upper arm bone still half-buried in the soil, (H) decaying fur and hair. (c) Luxuriant colonization of tree roots (R) and decaying fat tissue (adipocere, F) observed in the soil under the fruit bodies (arrowheads). (d) Bones collected from there; this array of bones showed that one domestic cat had been buried. (From Sagara, N., Trans. Mycol. Soc. Japan 22, 271–275, 1981. With permission.)

b

a

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80 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

the corpse finder (see Lincoff 1981). There seems to be no other report of fungal growth on a buried human corpse, although there is dated literature that cannot be clearly related to modern biology (Sagara 1977, 1984). In nonectomycorrhizal vegetation, the LP is rarely observed, because nonectomycorrhizal species are rare among the LP fungi. Adipose tissue sometimes remains as adipocere in the soil (Figure 4.8c) due to oxygen deficiency (Forbes Stuart, and Dent 2005, Ch. 8), which probably acts as a barrier to invertebrate and microbe activity. 4.3.3  Fungal Growth Following Excreta Decomposition on the Ground From what little data are available (Appendix 4.2), it is clear that human excreta (urine or feces) yield some AF. The fungal succession seems to follow the same sequence as that which occurs on urea plots, although the number of species found at one particular site is lower (Sagara 1975, 1995). Raccoon dogs (Nyctereutes procyonoides) make latrines in the forest occupying an area of 0.5 m2 or smaller. Here they repeatedly urinate and defecate over a number of years. Such a site also yields AF but usually lacks the EP observed on urea plots (Kasuya 2002; Sagara 1989). This may be because the excreta deposition is repeated. Instead, a few non-AF (e.g., Mucor spp. and Ascobolus sp.) may appear on the feces themselves (Sagara 1989). Furthermore, some ectomycorrhizal non-AF seem to often add to the fungal assemblage at such sites (Kasuya 2002; Sagara, unpublished data). The location of sites in which excreta have been deposited and the identification of animal species that have excreted are difficult. Typically, by the time fungi fruit at a site, little remains to indicate an excretion event in the past (Figures 4.9, 4.9a), although a color change in soil as described earlier may be seen (Figure 4.9a). Because of this difficulty, many records of fruiting of the AF from nonexperimental sites have been ignored in this chapter and omitted from Appendix 4.2, although they certainly indicate antecedent excretion. 4.3.4  Fungal Growth Following Excreta Decomposition Belowground Information on belowground excrement is disparate with some data available on the buried human latrine, on the vespine (wasp) nest, on the mole latrine, on the shrew latrine, and on the wood-mouse latrine (Appendix 4.2). Similarly with buried cadavers, these sites are characterized by the growth of deep-rooting ectomycorrhizal fungi from the genus Hebeloma (Figure 4.10). They also lack the EP of AF succession. The major source of ammonia in the case of the wasp (Vespula flaviceps) nest is uric acid that is contained in the

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Figure 4.9  Humaria velenovskyi fruiting on the ground after decomposition and disappearance of dumped night soil (Appendix 4.2). Apothecia 7–8 mm across.

Figure 4.9a  Coprinus tuberosus (arrows) fruiting on the ground after decomposition and disappearance of human feces (Appendix 4.2). Note the black color of the humus that indicates the flow-in of ammonia in the past. For scale see pine needles. (See color insert following p. 178.)

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82 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

L

Figure 4.10  Soil profile showing Hebeloma radicosoides fruiting out of a human latrine buried together with fallen leaves (Appendix 4.2). The folded scale is 37 cm long. The fruiting of this ammonia fungus at the nonexperimental site prompted the excavation to identify the source of ammonia. No trace of the excreta remained at this moment.

faecal pellets excreted by the wasp larvae (see Figure 4.15). These pellets are deposited on the ceiling of each cell of the combs (Sagara et al. 1985). Hebeloma radicosum—which grows exclusively on the mole (Talpidae) latrine, shrew-mole (Talpidae) latrine, and wood-mouse (Muridae: Apodemus) latrine under the ground (Figure 4.11) (Sagara 1999; Sagara et al. 1988, 2006)—is a non-AF. This fungus has never been found on other substrates or after any experimental treatment of soil (Sagara 1995; Sagara et al. 2000) (This has been another reason to propose the term PPF). Hebeloma danicum, an AF, sometimes joins H. radicosum on those small mammal latrines (Sagara 1999). 4.3.5  Habitat-Cleaning Symbiosis The fungal growth and accompanying soil-biological events described in Sections 4.3.1 through 4.3.4 can be regarded as the process by which the habitats of those animals concerned are cleaned (Sagara 1995). Decomposition

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N

83

L L

Figure 4.11  Soil profile showing Hebeloma radicosum (fallen) fruiting out of the deserted latrines (L) near the nest (N) of a mole; stick indicates occurrence a little before of another fruit body. The folded scale is 51.5 cm long. Place and date: Quercus forest, Hiroshima, Japan, November 11, 2000. Mole species concerned: possibly Mogera imaizumii. There is no other way than using this fungus to locate a mole’s nest unless radio-tracking techniques are used.

of a cadaver or excreta seems to be carried out largely by bacteria and invertebrates. Products from this process may be first immobilized by the EP postputrefaction organisms including fungi. The products of these organisms may then be absorbed by both LP PPF and plants, often via ectomycorrhizas (Figure 4.12). The absorbed materials may be transformed and translocated above ground, partly by the fruiting of the fungi and partly by the growth of the trees. This concept of tripartite association among plants, animals, and fungi has been termed habitat-cleaning symbiosis (Sagara 1999). The stage of cadaver or excreta decomposition by bacteria and invertebrates and the EP of fungal succession following it are not represented in Figure 4.12 but should be considered as being inevitably involved in that symbiosis as the prerequisites. Furthermore, bacteria, nematodes, and other organisms as discussed earlier (Section 4.2.3.3) may also need to be explicitly included in that symbiosis (Yamanaka 2002). The previously known tripartite associations among plants, animals, and fungi are based on mycophagy (i.e., the consumption of fungi) and mycorrhizal symbiosis (for bibliography see Sagara 1995, 2000). The newly hypoth-

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84 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett Hebeloma vinosophyllum Laccaria bicolor

Trees Hebeloma danicum Hebeloma radicosoides

Ground surface

Fagaceae betulaceae pinaceae

Animal matter

Ectomycorrhizas

Root stock

5 cm

Figure 4.12  Diagram showing the relationship among the abandoned cadaver or excreta, the fungi growing after its decomposition, and the trees hosting the fungi in mycorrhizal symbiosis. This relationship forms part of the habitatcleaning symbiosis (see text). The cadaver here is shown buried, but “buried” or “unburied” does not matter for the establishment of this symbiosis. The possible early phase of this symbiosis is not shown here.

esized association is based on death or excretion and mycorrhizal symbiosis (Sagara 2000). Thus, the proposal of the habitat-cleaning symbiosis implies to establish death and excretion as the bases of symbiotic associations, in addition to ingestion.

4.4  Physiology of the AF and PPF 4.4.1  Spore Germination Basidiospore germination in the AF, both of EP species (Coprinus spp.) and LP species (Hebeloma vinosophyllum), is stimulated by a sufficient concentration of NH4+-N under alkaline to neutral conditions (Suzuki 1978; Suzuki et al. 1982). This suggests that they might start growth only after a sudden and abundant release of ammonia and that they might exist latently in the normal (i.e., untreated) soil in the form of basidiospores (Yamanaka 2002).

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Table 4.1 Growth of the Ammonia Fungi (mg Dry Weight of Mycelium 40 ml–1 Medium) in Liquid Media Containing Different Nitrogen Sources Nitrogen Sources Species Early phase species   Amblyosporium botrytis I   Amblyosporium botrytis II   Peziza urinophila   Pseudombrophila petrakii   Lyophyllum tyliocolor I   Lyophyllum tyliocolor II   Coprinus echinosporus I   Coprinus echinosporus II   Coprinus phlyctidosporus I   Coprinus phlyctidosporus II Late phase species   Laccaria bicolor   Hebeloma vinosophyllum I   Hebeloma vinosophyllum II   Hebeloma radicosoides I   Hebeloma radicosoides II

NH4+

NO3–

GL

AS

ET

PU

UR

BSA

18 18 2 5 22 26 22 23 38 19

0 1 2 4 1 0 0 2 29 20

2 1 5 2 74 24 19 1 4 9

38 61 3 2 52 10 96 97 35 35

0 0 0 0 0 0 0 0 0 0

0 0 1 0 0 0 1 1 0 0

99 91 3 1 47 69 56 78 14 26

0 0 3 8 64 84 24 10 6 11

44 44 37 37 53

15 149 124 162 56

3 76 36 57 61

4 170 223 136 159

1 1 0 0 2

1 1 0 1 2

69 163 163 235 114

1 196 215 171 109

Note: NH4, ammonium chloride; NO3, potassium nitrate; GL, glycine; AS, asparagine; ET, ethylenediamine; PU, putrescine; UR, urea; BSA, bovine serum albumin. Source: Data from Yamanaka (1999).

4.4.2  Vegetative Growth 4.4.2.1  Nitrogen Utilization Generally, the EP species seem to utilize NH4+-N, amino acids, urea and bovine serum albumin but not NO3--N, whereas the LP species utilize NO3--N, in addition to NH4+-N and organic nitrogen utilized by the EP species (Table 4.1). The EP species are primarily saprotrophic whereas LP species are mostly ectomycorrhizal (see Section 4.2.2). Saprotrophic fungi are generally known to grow better on NH4+-N or amino acids than on NO3--N, whereas ectomycorrhizal species can grow on NO3--N as well (Finlay, Frostegård, and Sonnerfeldt 1992; Fries 1955; Hacskaylo, Lilly, and Barnett 1954; Keller 1996). Thus, nitrogen utilization in the AF seems similar to that in non-AF, so far as the form of nitrogen is concerned. However, there may be differential sensitivities to NH4+-N and/or to NO3--N between the AF and non-AF, where the AF may prefer or endure higher concentrations.

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86 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett Table 4.2 Growth of the Ammonia Fungi (mg Dry Weight of Mycelium 40 ml-1 medium) in Liquid Media at Different pH. Initial pH Species Early phase species   Amblyosporium botrytis I   Amblyosporium botrytis II   Peziza urinophila   Pseudombrophila petrakii   Lyophyllum tyliocolor I   Lyophyllum tyliocolor II   Coprinus echinosporus I   Coprinus echinosporus II   Coprinus phlyctidosporus I   Coprinus phlyctidosporus II Late phase species   Laccaria bicolor   Hebeloma vinosophyllum I   Hebeloma vinosophyllum II   Hebeloma radicosoides I   Hebeloma radicosoides II

3

4

5

6

7

8

9

0 0 0 0 2 1 0 0 0 0

0 1 9 0 6 5 2 2 0 0

9 8 29 1 9 16 38 85 0 3

23 29 36 9 61 64 111 171 10 51

89 42 47 29 55 64 148 172 2 69

57 51 100 18 35 77 135 162 0 24

52 34 81 1 1 0 21 37 0 2

3 1 0 1 1

6 18 3 9 9

10 90 69 87 32

12 102 113 71 45

7 62 108 36 30

2 24 17 13 4

0 0 0 0 0

Source: Data from Yamanaka (2003)

Some ectomycorrhizal AF utilize amino acids (glycine, asparagine) (Table 4.1). This might allow these fungi to survive in ectomycorrhizal symbioses in untreated soils, utilizing amino acids exudated from plant roots and other sources of nutrients in the soil. Furthermore, nonectomycorrhizal EP AF also might survive in the form of vegetative hyphae in the rhizosphere or rhizoplane, since they also utilize amino acids as mentioned already (Yamanaka 2002). Amines are the particular products of putrefaction. None of the AF tested utilized amines (putrescine, ethylenediamine) as the sole source of nitrogen in culture (Table 4.1), although some amines (ethylenediamine, trimethylamine) are as effective as urea in the field (Sagara 1975, 1992). This would show that AF do not directly utilize amines but utilize them only after decomposition to ammonia. Thus, the word postputrefaction in the term PPF may seem inappropriate from the fungal physiological point of view (Yamanaka 2002), but this word indicates the characteristic feature of cadaver and fecal decomposition. It also describes the major cause for growth of the AF in nature.

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4.4.2.2  Hydrogen Ion Concentration The EP species sporulate or fruit under neutral to slightly alkaline conditions in the treated soil and the optimum pH for their vegetative growth in culture is 7 to 9. In contrast, LP species fruit under slightly acidic conditions and have their optimum pH at 5 to 7 (Figure 4.5, Table 4.2). Thus, the optimum pH for growth of the AF in culture has relevance to the pH in ureatreated soil in which the AF grow (Yamanaka 2003; see also Soponsathien 1998; Suzuki 2006). In culture (Table 4.2), EP species grow even at pH 4, a similar value to that of untreated soils, and this suggests that the EP fungi might survive in untreated soils in the form of vegetative hyphae. Similarly, the growth of LP species at pH 8 suggests that they might commence vegetative growth during the EP (Yamanaka 2002). 4.4.2.3  Enzymatic Activity Water-soluble carbohydrates in soil that are to be readily utilized by fungi as carbon sources decrease after urea treatment (Yamanaka 1995a). Under such a condition, the AF would have to utilize high molecular weight organic matter through degradation. Accordingly, EP species (Amblyosporium botrytis, Ascobolus denudatus, Peziza urinophila, Pseudombrophila perakii, Lyophyllum tylicolor, and Coprinus echinosporus) show strong abilities to decompose cellulose, lignin, chitin, protein and lipid (Yamanaka 1995a). In contrast, LP species (Laccaria bicolor and Hebeloma spp.) have a poor capacity to degrade these compounds. As mycorrhizal fungi, their carbon supply is probably from the host plants. EP species (L. tylicolor and Coprinus spp.) possess cellulases that are most active at pH 7–9, whereas LP species (L. bicolor and Hebeloma spp.) have an activity optimum of pH 6–7 (Enokibara et al. 1993). Thus, the optimum pH for enzymatic activity in some AF seems to reflect the pH of soil in which they grow. 4.4.3  Formation of Reproductive Structures The AF and PPF can be detected in the field only by the occurrence of their reproductive structures. The reproduction in these fungi is a secondary phenomenon in their response to urea, cadaver, or excreta decomposition. However, it may still be directly related with such an event. Coprinus tuberosus (Morimoto, Suda, and Sagara 1982), C. phlyctidosporus (He and Suzuki 2003), and Lyophyllum tylicolor (Yamanaka 1994) develop fruit bodies on nitrogenrich media. Coprinus cinereus fruits in the dark by adding ammonia to the medium (Morimoto, Suda, and Sagara 1981). Hebeloma vinosophyllum forms small fruit bodies in synthetic media containing NH4+-N and urea as a sole nitrogen source (Suzuki 2006). The sizes of fruit bodies can vary enormously.

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88 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

Figure 4.13  Lyophyllum tylicolor fruit bodies collected from urea plot (left) and from human urination sites (right), showing size variation due probably to the rate of nitrogen deposited. Collections from a Pinus-Chamaecyparis forest, Kyoto, Japan, on May 23, 1966. Bar = 1 cm. a

b

Figure 4.14  Lyophyllum tylicolor in slide culture, showing viability of the fungus. (a) The mycelium having spread on the slide glass out of the disc of nutrient agar (arrow) and now exhibiting a powdery appearance wherein basidia and basidiospores have developed; bar = 5 mm. (b) Photomicrograph of the basidia and basidiospores formed there; arrows indicate the spores formed on the basidia; bar = 100 µm. (From Yamanaka, T. and Sagara, N., Mycol. Res. 94, 847–850, 1990. With permission.)

In the case of L. tylicolor shown in Figure 4.13, the size variation possibly corresponded to the amount of nitrogen added to soil. Such correspondence between the size or amount of fruit bodies and the intensity of treatment is often observed in the field (Sagara 1976a, 1992).

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Figure 4.15  Hebeloma danicum (arrowhead) and H. radicosoides (the rest) fruiting on buried uric acid, which simulates their growth on abandoned nests of wasps underground (see Section 3.4). Twenty-five g of uric acid was buried together with humus (arrow) at the bottom (20 × 20 cm) of a 20 cm deep hole on December 8, 1984, in a Pinus-Quercus forest, Shiga, Japan; photograph taken on October 17, 1985.

Lyophyllum tylicolor forms basidia and basidiospores on the mycelium that has been cultured on a small amount of nutrient agar, without forming normal fruit bodies (Figure 4.14). It also forms basidia and basidiospores on the tops of young, undeveloped fruit bodies. These are observed not only in the laboratory studies but also in the field experiment with urea under certain conditions. This fungus may well reproduce sexually even by spotted and minute flow-in of nitrogen that would not be enough for the formation of normal fruit bodies. Such nitrogen may be expected from decomposition of resources such as a dead worm or a bird’s dropping (Yamanaka and Sagara 1990). It has been a question as to how the long-rooting (deep-colonizing) mushrooms can grow up to the ground surface and form fruit bodies there. Hebeloma radicosum, a PPF representing such mushrooms (Figure 4.11), forms fruit bodies under experimental conditions in the laboratory nonphototropically but negatively gravitropically from early development, unlike mushrooms in general. In the dark, the fruit bodies grow upward, forming pseudorhizas but remaining immature; they mature only in the light (Kaneko

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90 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

and Sagara 2002). These characteristics well explain that fruiting habit of the fungus and would also apply to H. radicosoides and other long-rooting AF (Figures 4.10 and 4.15). 4.4.4  Physiology and Succession Based on the observations from the field (see Sections 4.2.3.2 and 4.2.3.3) and from culture (see Sections 4.4.1 and 4.4.2), the successional change from EP to LP in urea plot may be explained as follows (Yamanaka 2002). The EP species grow soon after the treatment as they prefer NH4+-N and alkaline conditions and are able to obtain carbon sources saprotrophically from soil organic matter in the absence of living tree roots. They are then less suited to the soil as NO3--N increases and acidic conditions prevail. The EP species are thus replaced by the LP species as the latter utilize NO3--N as well as NH4+-N, favor acidic conditions, and meet ectomycorrhizal tree roots from which they can obtain carbon sources. The successional change within EP may result from the combination of sequential colonization by the fungi concerned and time required for each fungus to produce reproductive structures. Here, the sequence of colonization, which is accomplished by spore germination or mycelial growth, may result from the preference or tolerance of each fungus to higher concentrations of NH4+-N under neutral to alkaline conditions (Suzuki, Uchida, and Kita 2002).

4.5  Possibilities for Forensic Application 4.5.1  Human Behavior or Activities May Be Evidenced by Fungal Growth Examples of fungal growth described earlier (see Section 4.3) have already shown that, for example, abandoning or burying animal cadavers, urination, and defecation can be detected by the occurrence of reproductive structures of particular fungi. Another excellent example is given here where considerable interpretation of human activities may be made through mycological evidence (Figure 4.16). At this site, there is no discernible trace of remaining excreta. The presence of paper residue on the humus behind the bush might give cause to suggest that human excretion has taken place. The occurrence of fruit bodies of two AF—Hebeloma danicum and Laccaria bicolor—confirms this is almost certainly true. Moreover, the distribution of the paper residue and fruit bodies indicates that a person squatted facing to the right in the figure and urinated and defecated. Assuming the paper should have been dropped near the feces, H. danicum is located at the presumed site of defecation whereas L. bicolor at the site of urination.

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Figure 4.16  Hebeloma danicum (arrows) and Laccaria bicolor (arrowheads) fruiting beside remains of paper, which indicates not only antecedent human excreta but also a squatting pose (see text). Photograph was taken in a Pinus forest, Shiga, Japan, on October 20, 1981. (See color insert following p. 178.)

4.5.2  Initial Conditions and the Postdeposition Interval May Be Assessed Observations on the stage of fungal succession and other features in situ may allow a rough assessment of the time since a cadaver was abandoned or an excretion deposited. The example given here is of a cat carcass (Figure 4.8). In this case, the fruiting of Laccaria bicolor in May–June (early summer) seemed to be the first flush of the LP in the known AF succession. The humus was still black, the fatty tissue remained as adipocere, earthworms were still active, and the growth of new roots was prominent. These details help us establish that the cat body had been buried not too long ago. However, the period during which the carcass decomposition and fungal colonization took place had to include the season of lower temperatures. Therefore, the burial must have occurred sometime during the preceding winter (November–March). 4.5.3  Simulation Experiments The soil can rapidly respond to disturbance. Generally, the assemblage of fungal species that respond to any chemical treatment of soil is specific and reflects the characteristics of the chemical used (Sagara 1992). For instance, urea and calcium hydroxide yield AF and burnt-ground fungi, respectively, whereas calcium cyanamide, which is hydrolyzed to urea and calcium hydroxide in soil, yields both AF and burnt-ground fungi on the same soil

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92 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

Figure 4.17  Growth of Lyophyllum tylicolor under certain defined conditions using humus. The humus was collected from a Pinus densiflora forest in the suburbs of Kyoto and brought to the laboratory at Kyoto University. As a pretreatment, the humus was dressed with 0, 5, 10, and 20 mg of urea per 1 gram of fresh material (from left to right) and incubated at 20˚C. After 5 days, it was dried at 40˚C, sterilized by gamma-irradiation, put in the plastic cup (23 mm in inner diam and 47 mm in depth), inoculated with hyphal suspension of this fungus, and incubated again at 20˚C. Photograph was taken after further 10 days. (From Yamanaka, T., Ph.D. diss., Kyoto University, 2002. With permission.)

(Sagara 1975, 1992) (such a fact may also have forensic potential). Similarly, amino acids, peptone, and proteins that are more complex than urea may yield additional species to the AF (Sagara 1975, 1992). So, material to be used for simulation here may vary from animal waste itself to pure chemicals (Figure 4.15) (Sagara 1975, 1992). The EP of the AF succession can be reproduced in laboratory pot experiments using humus collected from the forest (Figure 4.17) (Sagara 1975, 1976a, 1976c, 1992; Suzuki 2006; Yamanaka 2002). The humus is not heavy in weight and can easily be transported to the laboratory. Mixing L, F, and H layers as a pretreatment does not retard the fungal growth (Sagara 1976c).

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93

These may enable a simulation to confirm the observations from the field or to unveil the facts that could have been overlooked in the field. Most AF and PPF can be reared in pure culture, and even some ectomycorrhizal species cultured to the point of fruiting (Kaneko and Sagara 2002; Ohta 1998; Suzuki 2006). Therefore, they may be subjected to any tests under axenic conditions (see Suzuki 2006). 4.5.4  Limitations If the soil lacks the layer of humus, the EP of the PPF succession will be poorly observed (see Section 4.1, Section 4.3.2). Even in these soils, Mucor spp. and Rhopalomyces strangulatus may grow, as they can colonize the animal remains themselves (see Section 4.3.1). In a case where the deposition of a cadaver or excreta has been made in nonectomycorrhizal vegetation, the LP will scarcely be observed, since nonectomycorrhizal species are rather rare among the LP fungi (see Section 4.2.2; Appendix 4.2). This should be particularly true when the cadaver or excreta has been buried, since the PPF that colonize at depth are ectomycorrhizal. In such cases, there should be little possibility of detecting a buried carcass or excreta through the fungal growth. To what depth AF or PPF can grow is not precisely known. This is likely related to the availability of oxygen and activity of organisms other than the fungi, especially of plant roots to act as hosts in mycorrhizal symbioses. The growth of Hebeloma radicosoides and H. danicum is likely to a depth of up to 30 cm (see Figures 4.10 and 4.15; see also Sagara 1995; Sagara et al. 2000). Hebeloma radicosum may colonize deeper than 50 cm (see Figure 4.11; see also Sagara 1999). Anyhow, burying deeper will reduce the chance of fungal growth. Excessive addition of any material to soil may retard the fungal growth (see Sections 4.2.1 and 4.3.1). How an adult human body, which may hold excessive resources for soil organisms, affects the fungal community is not precisely known, especially when it has been buried. Some LP PPF (e.g., Laccaria bicolor, Lactarius chrysorrheus, Suillus bovinus) grow not only on nitrogen-added soil but also on burnt ground and some other disturbed sites (Sagara 1992 and unpublished date). Namely, the growth of these species does not necessarily indicate a deposition in the past of animal matter at the site. The most important barrier to forensic application lies within the investigators themselves. First, it is hard for beginners to detect fungi in the field, since fungal reproductive structures are often minute and ephemeral (Figures 4.1, 4.2, 4.9, 4.9a, 4.13, 4.14) (see Bunyard 2004; Tibbett and Carter 2004). When one wishes to find fungi in the field, the investigator should specifically look for their occurrence. Unless intentional mycological observations are made, an investigator is likely to overlook fungal reproductive structures. Second, it is difficult to recognize species and to identify them,

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94 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

because the size and form of fruit bodies vary considerably (Figure 4.13) and because new species or unreported species may be involved. Therefore, an experienced field mycologist is desirable. The advocacy for forensic potential of fungi has recently been revived (Carter and Tibbett 2003; Tibbett and Carter 2003). However, there have been no major developments in this field for thirty years (see Sagara 1976b). We hope mycologists and forensic scientists will collaborate to take the concept of forensic mycology forward into an accomplished and effective forensic science tool.

Acknowledgments We are indebted to Dr. Joop van Brummelen, Nationaal Herbarium Nederland, Leiden, The Netherlands, for confirming the identification of Pseudombrohila petrakii, to Dr. Kouhei Sawada, Osaka, Japan, for allowing the use of his unpublished observations on arthropods in the urea plot, to Ms. Kyoko Kobayashi, Hyogo, Japan, for communicating the fruiting of Hebeloma vinosophyllum beside a carcass (Fig. 4.7), and to Ms. Mayumi Araki and Mr. Yukio Morinaga, Hiroshima, Japan, for offering the opportunity to excavate the Hebeloma radicosum site (Fig. 4.11). All those who have been previously acknowledged by the senior author (N.S.) for communicating the fruiting of the postputrefaction fungi in the field are thanked again. Mr. Taiga Kasuya, Tsukuba University, Ibaraki, Japan, kindly read the manuscripts, for which we are grateful.

References Barron, G. L. (1977). The Nematode-Destroying Fungi. Guelph, ON: Canadian Biological Publications. Bonnet, E. F. P. (1967). Medicina Legal. Buenos Aires: Lopez Liberos Ed. Breitenbach, J. (1979). Untersuchung einer aspektbildenden Pilzsukzession auf Vogeldung. Z. Mykol. 45, 15–34. Bunyard, B. A. (2004). Commentary on: Carter DO, Tibbett M. Taphonomic mycota: Fungi with forensic potential. J. Forensic Sci. 49, 1134. Carter, D. O. and Tibbett, M. (2003). Taphonomic mycota: Fungi with forensic potential. J. Forensic Sci. 48, 1–4. Dent, B. B., Forbes, S. L., and Stuart, B. H. (2004). Review of human decomposition process in soil. Environm. Geol. 45, 576–585. Ellis, M. B. and Ellis, J. P. (1997). Microfungi on Landplants. Slough, UK: Richmond Publishers. Enokibara, S., Suzuki, A., Fujita, C., Kashiwagi, M., Mori, N., and Kitamoto, Y. (1993). Diversity of pH spectra of cellulolytic enzymes in Basidiomycetes. Trans. Mycol. Soc. Japan 34, 221–228 (in Japanese with English summary).

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Finlay, R. D., Frostegård, Å., and Sonnerfeldt, A.-M. (1992). Utilization of organic and inorganic nitrogen sources by ectomycorrhizal fungi in pure culture and in symbiosis with Pinus contorta Dougl. ex Loud. New Phytol. 120, 105–115. Forbes, S. L., Stuart, B. H., and Dent, B. B. (2005). The effect of the burial environment on adipocere formation. Forensic Sci. Int. 154, 24–34. Fries, L. (1955). Studies in the physiology of Coprinus: I. Growth substance, nitrogen and carbon requirements. Svensk Bot. Tidskr. 49, 475–535. Fukiharu, T. and Hongo, T. (1995). Ammonia fungi of Iriomote Island in the southern Ryukyus, Japan and a new ammonia fungus, Hebeloma luchuense. Mycosciense 36, 425–430. Fukiharu, T., Yokoyama, G., and Oba, T. (2000). Occurrence of Hebeloma vinosophyllum on the forest ground after decomposition of crow carcass. Mycoscience 41, 401–402. Furuya, K. (1990). Coprophilous fungi as microbial resources. Sankyo Kenkyusho Nempo 42, 1–31. Hacskaylo, J., Lilly, V. G., and Barnett, H. L. (1954). Growth of fungi on three sources of nitrogen. Mycologia 46, 691–701. Hansen, L. and Knudsen, H. (Eds.). (1992). Nordic Macromycetes vol. 2. Copenhagen: Nordsvamp. Hansen, L. and Knudsen, H. (Eds.). (2000). Nordic Macromycetes vol. 1. Copenhagen: Nordsvamp. Harmaja, H. (1986). Studies on the Pezizales. Karstenia 26, 41–48. Harmaja, H. (2002). Alciphila vulgaris, a new genus and species of Deuteromycetes. Karstenia 42, 33–38. He, X. and Suzuki, A. (2003). Effect of nitrogen sources and pH on growth and fruit body formation of Coprinopsis phlyctidospora. Fungal Diversity 12, 35–44. He, X. and Suzuki, A. (2004). Effect of urea treatment on litter decomposition in Pasania edulis forest soil. J. Wood Sci. 50, 266—270. Hilton, R. N. (1978). The ghoul fungus, Hebeloma sp. ined. Trans. Mycol. Soc. Japan 19, 418. Hudson, H. J. (1972). Fungal Saprophytism. London: Edward Arnold. Imamura, A. (2001). Report on Laccaria amethystina, newly confirmed as an ammonia fungus. Mycoscience 42, 623–625. Imamura, A., Yumoto, T., and Yanai, J. (2006). Urease activity in soil as a factor affecting the succession of ammonia fungi. J. Forest Res. 11, 131–135. Kaneko, A. and Sagara, N. (2002). Responses of Hebeloma radicosum fruit-bodies to light and gravity: negatively gravitropic and nonphototropic growth. Mycoscience 43, 7–13. Kasuya, T. (2002). Reports on fruiting of ammonia fungi on the ground after decomposition of animal wastes. Nippon Kingakukai Kaiho 43, 99–104 (in Japanese with English summary). Keller, G. (1996). Utilization of inorganic and organic nitrogen sources by highsubalpine ectomycorrhizal fungi of Pinus cembra in pure culture. Mycol. Res. 100, 989–998. Kuroyanagi, E., Honda, S., Yoshimi, S., and Sagara, N. (1982). The appearance of Hebeloma radicosum from a buried cat carcass. Trans. Myc. Soc. Japan 23, 485–488 (in Japanese with English summary).

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96 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett Lehmann, P. F. (1976). Unusual fungi on pine leaf litter induced by urea and urine. Trans. Br. Mycol. Soc. 67, 251–253. Lewis, D. P. and McGraw, J. L., Jr. (1984). Studies on Big Thicket Agaricales. Southwest. Naturalist 29, 257–264. Lincoff, G. H. (1981). The Audubon Society Field Guide to North American Mushrooms. New York: Alfred A. Knopf. Mäkinen, Y. and Pohjola, A. (1969). Three discomycetous genera new to Finland. Karstenia 9, 5–8. Miller, O. K., Jr. and Hilton, R. N. (1986). New and interesting agarics from Western Australia. Sydowia, Ann. Mycol., 39, 126–137. Morimoto, N., Suda, S., and Sagara, N. (1981). Effect of ammonia on fruit-body induction of Coprinus cinereus in darkness. Plant Cell Physiol. 22, 247–254. Morimoto, N., Suda, S., and Sagara, N. (1982). The effects of urea on the vegetative and reproductive growth of Coprinus stercorarius in pure culture. Trans. Mycol. Soc. Japan 23, 79–83. Mueller, B. (1953). Gerichtliche Medizin. Berlin: Springer. Nagao, H., Udagawa, S., Bougher, N. L., Suzuki, A., and Tommerup, I. C. (2003). The genus Thecotheus (Pezizales) in Australia: T. urinamans sp. nov. from ureatreated jarrah (Eucalyptus marginata) forest. Mycologia 95, 688–693. Neda, H. and Doi, Y. (1998). Notes on agarics in Kyushu District. Mem. Natn. Sci. Mus. Tokyo 31, 89–95. Ohta, A. (1998). Fruit-body production of two ectomycorrhizal fungi in the genus Hebeloma in pure culture. Mycoscience 39, 15–19. Paulsen, M. D. and Dissing, H. (1979). The genus Ascobolus in Denmark. Bot. Tidsskr. 74, 67–78. Petersen, P. M. (1970). Changes of the fungus flora after treatment with various chemicals. Bot. Tdsskr. 65, 264–280. Pirozynski, K. A. (1969). Reassessment of the genus Amblyosporium. Can. J. Bot. 47, 325–334. Rea, C. (1922). British Basidiomycetae. Cambridge, UK: Cambridge University Press. Sagara, N. (1973). Proteophilous fungi and fireplace fungi (A preliminary report). Trans. Myc. Soc. Japan 14, 41–46. Sagara, N. (1975). Ammonia fungi—A chemoecological grouping of terrestrial fungi. Contr. Biol. Lab. Kyoto Univ. 24, 205–276. Sagara, N. (1976a). Growth and reproduction of the ammonia fungi, in Ecology of Microorganisms (3) (Biseibutsu-seitai Kenkyukai, Eds.). Tokyo: University of Tokyo Press, 153–178 (in Japanese). Sagara, N. (1976b). Presence of a buried mammalian carcass indicated by fungal fruiting bodies. Nature 262, 816. Sagara, N. (1976c). Supplement to the studies of ammonia fungi (1). Trans. Mycol. Soc. Japan 17, 418–428 (in Japanese with English summary). Sagara, N. (1977). Detecting dead bodies in 1718 and 1976. Nature 269, 284. Sagara, N. (1978). The occurrence of fungi in association with wood mouse nests. Trans. Mycol. Soc. Japan 19, 201–214. Sagara, N. (1980). Not mouse but mole. Trans. Mycol. Soc. Japan 21, 519. Sagara, N. (1981). Occurrence of Laccaria proxima in the grave site of a cat. Trans. Mycol. Soc. Japan 22, 271–275. Sagara, N. (1984). On “Corpse Finder.” McIlvainea 6, no. 2, 7–9.

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Sagara, N. (1989). Mushrooms and Animals. Tokyo: Tsukiji-shokan (in Japanese). Sagara, N. (1992). Experimental disturbances and epigeous fungi, in The Fungal Community, 2nd ed. (G. C. Carroll and D. T. Wicklow, Eds.). New York: Marcel Dekker, 427–454. Sagara, N. (1995). Association of ectomycorrhizal fungi with decomposed animal wastes in forest habitats: a cleaning symbiosis? Can. J. Bot. 73, suppl. 1, S1423–S1433. Sagara, N. (1999). Mycological approach to the natural history of talpid moles—A review with new data and proposal of “habitat-cleaning symbiosis,” in Recent Advances in the Biology of Japanese Insectivora (Y. Yokohata and S. Nakamura, Eds.). Shobara, Hiroshima: Hiba Society of Natural History, 33–55. Sagara, N. (2000). Symbioses between animals and fungi, in Mushroom Handbook (K. Kinugawa and M. Ogawa, Eds.). Tokyo: Asakura-shoten, 284–289 (in Japanese). Sagara, N. and Hamada, M. (1965). Responses of higher fungi to some chemical treatments of forest ground. Trans. Mycol. Soc. Japan 6, 72–74. Sagara, N., Honda, S., Kuroyanagi, E., and Takayama, S. (1981). The occurrence of Hebeloma spoliatum and Hebeloma radicosum on the dung-deposited burrows of Urotrichus talpoides (shrew mole). Trans. Mycol. Soc. Japan 22, 441–455. Sagara, N., Hongo, T., Murakami, Y., Hashimoto, T., Nagamasu, H., Fukiharu, T., and Asakawa, Y. (2000). Hebeloma radicosoides sp. nov., an agaric belonging to the chemoecological group ammonia fungi. Mycol. Res. 104, 1017–1024. Sagara, N., Kitamoto, Y., Nishio, R., and Yoshimi, S. (1985). Association of two Hebeloma species with decomposed nests of vespine wasps. Trans. Br. Mycol. Soc. 84, 349–352. Sagara, N. and Kobayashi, T. (1979). Hebeloma spoliatum appeared from abandoned nest-chambers of Vespula lewisi, a ground wasp. Trans. Mycol. Soc. Japan 20, 266–267 (in Japanese). Sagara, N., Matsuda, I., Kareki, K., and Ako, S. (1978). Habitats of Hebeloma spoliatum. Trans. Mycol. Soc. Japan 19, 90. Sagara, N., Murakami, Y., and Clémençon, H. (1988). Association of Hebeloma radicosum with a nest of the wood mouse Apodemus. Mycol. Helvet. 3, 27–35. Sagara, N., Okabe, H., and Kikuchi, J. (1993). Occurrence of an agaric fungus Hebeloma on the underground nest of wood mouse. Trans. Mycol. Soc. Japan 34, 315–322. Sagara, N., Senn-Irlet, B., and Marstad, P. (2006). Establishment of the case of Hebeloma radicosum growth on the latrine of the wood mouse. Mycoscience 47, 263–268. Sagara, N. and Takayama, S. (1982). Hebeloma radicosum appearing on the old latrine of a boy-scouts’(?) camping site. Nature Study 28, 99–100 (in Japanese). Smith, S. E. and Read, D. J. (1997). Mycorrhizal Symbiosis, 2nd ed. San Diego: Academic Press. Soponsathien, S. (1998). Some characteristics of ammonia fungi. 1. In relation to their ligninolytic enzyme activities. J. Gen. Appl. Microbiol. 44, 337–345. Suzuki, A. (1978). Basidiospore germination by aqua ammonia in Hebeloma vinosophyllum. Trans. Mycol. Soc. Japan 19, 362. Suzuki, A. (2006). Experimental and physiological ecology of ammonia fungi: studies using natural substrates and artificial media. Mycoscience 47, 3–17.

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98 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett Suzuki, A., Motoyoshi, N., and Sagara, N. (1982). Effects of ammonia, ammonium salts, urea, and potassium salts on basidiospore germination in Coprinus cinereus and Coprinus phlyctidosporus. Trans. Mycol. Soc. Japan 23, 217–224. Suzuki, A., Tanaka, C., Bougher, N. L., Tommerup, I. C., Buchanan, P. K., Fukiharu, T., et al. (2002). ITS rDNA variation of the Coprinopsis phlyctidospora (syn.: Coprinus phlyctidosporus) complex in the Northern and Southern Hemispheres. Mycoscience 43, 229–238. Suzuki, A., Uchida, M., and Kita, Y. (2002). Experimental analyses of successive occurrence of ammonia fungi in the field. Fungal Diversity 10, 141–165. Suzuki, A., Fukiharu, T., Tanaka, C., Ohono, T., and Buchanan, P. K. (2003). Saprobic and ectomycorrhizal ammonia fungi in the Southern Hemisphere. New Zeal. J. Bot. 41, 391–406. Svrček, M. (1968). Galactinia moravecii sp. nov., eine neue Art aus der Tschechoslowakei. Česká Mykol. 22, 90–92. Svrček, M. (1988). New or less known Discomycetes: XVIII. Česká Mykol. 42, 137–148. Takayama, S. and Sagara, N. (1981). The occurrence of Hebeloma vinosophyllum on soil after decomposition of the corpse of domestic rabbit. Trans. Mycol. Soc. Japan 22, 475–477 (in Japanese with English figure legends). Thaxter, R. (1891). On certain new or peculiar North American Hyphomycetes. I. Oedocephalum, Rhopalomyces and Sigmoideomyces n. g. Bot. Gaz. 16, 14–36. Tibbett, M. and Carter, D. O. (2003). Mushrooms and taphonomy: the fungi that mark woodland graves. Mycologist 17, 20–24. Tibbett, M. and Carter, D. O. (2004). Commentary on: Carter DO, Tibbett M. Taphonomic mycota: Fungi with forensic potential. J. Forens. Sci. 2003, 48:168–171. Author’s response. J. Forens. Sci. 49, 1135–1136. Towne, E. G. (2000). Prairie vegetation and soil nutrient responses to ungulate carcasses. Oecologia 122, 232–239. Webster, J. (1970). Coprophilous fungi. Trans. Br. Mycol. Soc. 54, 161–180. Weimann, W. (1940). Leichenflora, in Handwörterbuch der Gerichtlichen Medizin und Naturwissenshaftlichen Kriminalistik (F. von Neureiter, F. Pietrusky, and E. Schütt, Eds.). Berlin: Julius Springer, 444. Yamanaka, T. (1994). Fruiting of Lyophyllum tylicolor in plate culture on Soytoneglucose agar and urea-treated soil extract agar. Mycoscience 35, 187–189. Yamanaka, T. (1995a). Changes in organic matter composition of forest soil treated with a large amount of urea to promote ammonia fungi and the abilities of these fungi to decompose organic matter. Mycoscience 36, 17–23. Yamanaka, T. (1995b). Nitrification in a Japanese red pine forest soil treated with a large amount of urea. J. Jpn. Forestry Soc. 77, 232–238. Yamanaka, T. (1995c). Changes in soil conditions following treatment with a large amount of urea to enhance fungal fruiting-body production in a Japanese red pine forest. Bull. Jpn. Soc. Microbial Ecol. 10, 67–72. Yamanaka, T. (1999). Utilization of inorganic and organic nitrogen in pure cultures by saprotrophic and ectomycorrhizal fungi producing sporophores on ureatreated forest floor. Mycol. Res. 103, 811–816. Yamanaka, T. (2002). Growth mechanism of the ammonia fungi—Experimental mycological studies on cleaning processes in the sites after decomposition of deadbodies and excrement of animals. Ph.D. diss., Kyoto University (in Japanese).

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Yamanaka, T. (2003). The effect of pH on the growth of saprotrophic and ectomycorrhizal ammonia fungi in vitro. Mycologia 95, 584–589. Yamanaka, T. and Sagara, N. (1990). Development of basidia and basidiospores from slide-cultured mycelia in Lyophyllum tylicolor (Agaricales). Mycol. Res. 94, 847–850. Yang, Z. L., Matheny, P. B., Ge, Z-W., Slot, J. C., and Hibbett, D. S. (2005). New Asian species of the genus Anamika (euagarics, hebelomatoid clade) based on morphology and ribosomal DNA sequences. Mycol. Res. 109, 1259–1267.

Appendix 4.1  List of the Ammonia Fungi and Postputrefaction Fungi Species concept and nomenclature follow Ellis and Ellis (1977) for Deuteromycetes, Hansen and Knudsen (2000) for Ascomycetes, and Hansen and Knudsen (1992) for Basidiomycetes. For those species not included in these publications see Sagara (1975, 1992) or the note added to each species. Only the first report and important ones among those published in this concern are cited. For further bibliography see Suzuki et al. (2003). Some of the fungi now seem uncertain to be included here but remain for their potentiality. The letter E or L following the reference citation denotes the early phase or late phase, respectively, in the succession at which the fungus appears (reproduces); E-L means that the fungus appears at a transitional phase from E to L, while E~L continuously from E to L. The letter W or S following this characterization denotes winter or summer as the necessary or good season to trig the growth of the fungus, and the letters WS indifference to those seasons, as seen from the urea treatment experiments in Kyoto, Japan (Sagara 1975 and unpublished data).

A. Zygomycetes Mucor spp. (Sagara 1973, 1975), E Rhopalomyces elegans Corda (Thaxter 1891) Rhopalomyces elegans Corda var. minor (Rayss) J. Ellis (see Sagara 1992), E Rhopalomyces strangulatus Thaxt. (Thaxter 1891; Sagara 1973, 1975; Figure 4.6), E

B. Deuteromycetes Alciphila vulgaris Harmaja (Harmaja 2002), E Amblyosporium botrytis Fresen. (Pirozynski 1968; Sagara 1973, 1975), E, WS Cladorrhinum foecundissimum Sacc. et March. (Sagara 1973, 1975), E, S Doratomyces microsporus (Sacc.) Morton et Smith (Sagara 1975 as D. purpureofuscus), E Doratomyces putredinis (Corda) Morton et Smith (Sagara 1975; Figure 4.1), E Oidiodendron truncatum Barron (Sagara 1975) Penicillium lividum Westling (Sagara 1973, 1975), E C. Ascomycetes Ascobolus denudatus Fr. (Lehmann 1976; Sagara 1973, 1975; Sagara and Hamada 1965), E, W Ascobolus hansenii Mar. Paulsen et Dissing (Sagara 1973, 1975 as Ascobolus sp. no. 2), E Byssonectria terrestris (Alb. et. Schwein.:Fr.) Pfister (Mäkinen and Pohjola 1969 as Octospora aggregata; Sagara, 1973, 1975 as Byssonectria aggregate; Sagara 1992 as Inermisia fusispora), E Chaetomium globosum Kunze (Sagara 1975), E Humaria velenovskyi (Vacek) Korf et Sagara (Sagara 1973, 1975; Figure 4.9), E~L Iodophanus carneus (Pers.:Fr.) Korf (Sagara 1975), E Melastiza sp. (Sagara 1973, 1975), E

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100 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett Nannfeldtiella guldeniae (Svrček) Svrcek (Mäkinen and Pohjola 1969 as N. aggregata), E? Peziza moravecii (Svrček) Donadini (Svrček 1968 as Galactinia moravecii; Sagara 1973, 1975 as Peziza sp. no.1), E, W Peziza perpara Harmaja (Harmaja 1986), E? Peziza urinophila Y.-Z. Wang et Sagara (Sagara 1973, 1975 as Gelatinodiscus sp.), E, S Pseudombrophila merdaria (Fr.) Brumm. (Lehmann 1976 as P. deerata; identification needing confirmation; maybe P. petrakii), E Pseudombrophila petrakii (Sacc.) Brumm. (Sagara 1973, 1975 as Fimaria? sp.; Breitenbach 1979 as P. deerata; Harmaja 1986 as P. obliquerimosa; Svrček 1988 as P. fellneri), E, W Thecotheus urinamans Nagao, Udagawa et Bougher (Nagao et al. 2003) Trichophaea gregaria (Rehm) Boud. (Sagara 1975), L Scutellinia scutellata (L. :Fr.) Lambotte (Sagara 1975), E-L

D. Basidiomycetes Alnicola lactariolens Clémençon et Hongo (Fukiharu and Hongo 1995; confused with Hebeloma vinosophyllum in Sagara 1973, 1975), L Calocybe constricta (Fr.) Kühn. (Rea 1922 as Lepista constricta; Sagara 1973, 1975 as Lyophyllum constrictum or L. leucocephalum?), L, W Cantharellus omphalinoides Corner? (Sagara 1973, 1975 as C. minor?), L Collybia cookei (Bres.) J. D. Arnold (Sagara 1975), L Collybia? sp. (Sagara 1973, 1975; maybe Collybia biformis (Peck) Singer), L Coprinopsis sp. (Suzuki et al. 2002), E Coprinus cinereus (Schaeff.:Fr.) S. F. Gray (Sagara 1973, 1975 as C. lagopus), E Coprinus echinosporus Buller (Sagara 1973 as C. insignis?; Lehmann 1976; Sagara 1975), E, W Coprinus laanii Kits van Wav. (Sagara 1992), E(~L?) Coprinus narcoticus (Batsch:Fr.) Fr. (Sagara 1975; once treated as a misidentification of C. laanii (Sagara, 1992) but remain here for confirmation), E(~L?) Coprinus neolagopus Hongo et Sagara (Sagara 1973, 1975), E, S Coprinus phlyctidosporus Romagn. (Sagara 1973, 1975), E, S Coprinus tuberosus Quél. (Sagara 1973, 1975 as C. stercorarius; Fig. 4.9a), L Coprinus sp. (Sagara 1973, 1975 as Coprinus sp. no. 7), L, W? Coprinus sp. (Sagara 1975 as Coprinus sp. no. 8), L? Crucispora rhombisperma (Hongo) Horak (Sagara 1973 as Panaeolina? sp. no. 2; Sagara 1975 as Panaeolina rhombisperma), E, S? Entoloma lampropus (Fr.:Fr.) Hesler (Sagara 1973, 1975 as Rhodophyllus lampropus), L Hebeloma aminophilum Hilton et Miller (Hilton 1978; Miller and Hilton 1986; Suzuki et al. 2003), L Hebeloma danicum Gröger (Sagara 1973, 1975 as H. spoliatum; Figures 4.15, 4.16), L, W Hebeloma luchuense Fukiharu et Hongo (Fukiharu and Hongo 1995), L Hebeloma radicosoides Sagara, Hongo et Y. Murak. (Sagara 1973, 1975 as H. radicosum; Sagara et al., 2000; Figures 4.3, 4.10, 4.15), L, WS Hebeloma radicosum (Bull:Fr.) Rick. (Sagara 1978, 1980; Sagara, Murakami, and Clémençon 1988; Figure 4.11), L Hebeloma syrjense Karst. (Lewis and McGraw 1984; Lincoff 1981; most probably a misidentification of H. danicum (Sagara 1984)) Hebeloma vinosopyllum Hongo (Sagara 1973, 1975; Figure 4.7), L, S Hebeloma sp. (from Norway; Per Marstad, personal communication with the senior author, 2000)

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Laccaria amethystina Cooke (Imamura 2001; Kasuya 2002; Sagara 1995), L Laccaria bicolor (Maire) Orton (Sagara 1973, 1975, 1981 as L. proxima; Kasuya 2002; Figs. 8, 16), L, WS Laccaria laccata (Scop.:Fr.) Berk. et Br. (Kasuya 2002), L Laccaria spp. (Sagara, 1989, 1995; Kasuya 2002), L Lactarius chrysorrheus Fr. (Sagara 1973, 1975), L Lactarius lividatus Berk. et Curt. (Sagara 1989; nomenclature after Neda and Doi 1998), L Lepista nuda (Bull.:Fr.) Cooke (Sagara 1975, maybe a misidentification of L. sordida), L Lepista sordida (Schum.:Fr.) Sing. (Sagara 1973, 1975 as L. subnuda), L Lyophyllum ambustum (Fr.) Sing. (Sagara 1973, 1975 as L. gibberosum; Sagara 1992 as Tephrocybe ambusta), L, W Lyophyllum mephiticum (Fr.) Sing. (Sagara unpublished Data from England; referred to in Sagara 1992 as Tephrocybe mephitica), E Lyophyllum tylicolor (Fr.:Fr.) M. Lange et Siverts. (Sagara and Hamada 1965 as L. tylicolor f. typicum; Lehmann 1976; Sagara 1973, 1975 as Tephrocybe tesquorum; Figures 4.2, 4.13, 4.14, 4.17), E, W Mitrula sp. (Sagara, 1995), L Panaeolina sagarae Hongo (Sagara 1973 as Panaeolina? sp. no. 1; Sagara 1975), E Panaeolina? sp. (Sagara 1975 as Panaeolina? sp. no. 3), E-L? Rhodophyllus japonicus (Hongo) Hongo (Sagara 1975 as R. babingtonii f. japonicus), L Suillus bovinus (L.:Fr.) Roussel (Sagara 1989, 1995; Sagara et al. 1978), L Suillus luteus (L.:Fr.) Roussel (Kasuya 2002; Sagara 1989, 1995; Sagara et al. 1978), L

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69918.indb 102

Trigger Material

Ascobolus hansenii

Ascobolus denudatus

Amblyosporium botrytis

Kyoto, Japan Kyoto, Japan

Kyoto, Japan Kyoto, Japan Kyoto, Japan Kyoto, Japan

Pinus Pinus Pinus Castanopsis

Human feces Human body Domestic cat body Domestic dog body Human feces Domestic dog body Pinus Castanopsis

Kyoto, Japan

Pinus

Kyoto Japan

Fagus

Mole excreta

Human urine

Connecticut, USA

Kyoto, Japan

Cryptomeria Not stated

Kyoto, Japan

Location

Aphananthe

Vegetation

Vertebrate body

Early Successional Phase Rhopalomyces Human body strangulatus Crow body

Fungal Species

21.iv.1968 20?viii.1970

6.x.1968 10?ix.1968 ii?1966 20?viii.1970

16.iv.1966

A little before

Not stated

vi.1979

22?viii.1968

Deposition

6.vi.1968 20.ix.1970

14.xii.1968 3.x.1968 30.v.1966 20. ix.1970

4.v.1966

12.x.1984

1888–1889

9.vii.1979

11.ix.1968

Fruiting

Sagara (1975) Sagara (1975)

Sagara (1975) Sagara (1975) Sagara (1975) Sagara (1975)

Sagara (unpublished data) Sagara (1975)

Sagara (unpublished data); Figure 4.6 Pirozynski (1969)

Sagara (1975)

Reference

The fungal species are listed very roughly in order of their successional appearance. In principle, only the first report for each trigger material is cited. The data include those omitted from Sagara’s previous publications, and, in some instances, the data (date) of “deposition” are approximately given with the question mark.

Appendix 4.2  Major Postputrefaction Fungi and Data of Their Appearance that May Have Forensic Implications

102 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

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Coprinus echinosporus

Coprinus neolagopus

Peziza urinophila

Peziza moravecii

Pseudombrophila petrakii

Lyophyllum tylicolor

Domestic dog body Fowl body Brambling finch excreta

Brambling finch excreta Human urine Human feces Cow excreta Dog urine Human urine Human feces Human body Domestic dog body Fowl body Domestic cat body

Domestic dog body Fowl body Elk urine

Wood-pigeon excreta Human urine Kyoto, Japan Saitama, Japan Varsinais-Suomi, Finland Obwalden, Switzerland Kyoto, Japan Kyoto, Japan Bohemia, Czech Bohemia, Czech Kyoto, Japan Kyoto, Japan Kyoto, Japan Kyoto, Japan Saitama, Japan Kyoto, Japan Kyoto, Japan Saitama, Japan Obwalden, Switzerland

Castanopsis Quercus Woods

Pinus Pinus Not stated Not stated Pinus Pinus Cryptomeria Castanopsis Quercus Phyllostachys Castanopsis Quercus Picea

Picea

Kyoto, Japan

Jylland, Denmark

Pinus

Conifers

Winter 1999–2000 Winter 1977–1978

20?viii.1968

Winter 1999–2000 10?ix.1968

19.ii.1967 18.xii.1967 Not stated Not stated 16.iv.1966 1.viii.1966 12?viii.1968 20?viii.1970

Winter 1977–1978

Winter 1999–2000 Not stated

20?viii.1970

16.iv.1966

Not stated

22.v.2000 vi–vii.1978

20.ix.1968

22.v.2000 7.x.1968

10.iv.1967 23.iii.1968 11.iv.1966 10.iv.1967 23.v.1966 11.ix.1966 13.ix.1968 20.ix.1968

iv–vi.1978

22.v.2000 18.vii.1978

20.ix.1970

23.v.1966

24.v.1975

(continued)

Kasuya (2002) Breitenbach (1979)

Sagara (1975)

Kasuya (2002) Sagara (1975)

Sagara (1975) Sagara (1975) Svrček (1968) Svrček (1968) Sagara (1975) Sagara (1975) Sagara (1975) Sagara (1975)

Breitenbach (1979)

Kasuya (2002) Harmaja (1986)

Sagara (1975

Paulsen and Dissing (1979) Sagara (1975)

Soil Fungi Associated with Graves and Latrines 103

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69918.indb 104

Domestic dog body Domestic pig feces

Kyoto, Japan Shiga, Japan Kyoto, Japan Shiga, Japan Hiroshima, Japan

Castanopsis Pinus Pinus Quercus Pinus

Shiga, Japan

Pinus

Late Successional Phase Hebeloma danicum Human feces Night soil Domestic cat body

Crusispora rhombisperma Humaria velenovskyi

Night soil

Saitama, Japan

Castanea Shiga, Japan

Saitama, Japan Kyoto, Japan

Quercus Pinus

Pinus

Saitama, Japan

Castanea

Domestic dog urine Night soil

Kyoto, Japan

Pinus

Domestic dog body Domestic dog urine Fowl body Human excreta

Coprinus tuberosus

Saitama, Japan

Location

Quercus

Human urine

Coprinus phlyctidosporus

Vegetation

Trigger Material

Fungal Species

xi.1975

xi?1966

11.xi.1967 Not known ii?1966

Not known

Not known

Not stated

Winter 1999–2000 25.iv.1968

Not stated

v?1984

12.v.1999, vi.1999

Deposition

viii–xi.1976

26.x.1967

7.x.1968 26.x.1967 26.viii.1966

26.x.1967

26.x.1967

28.vi.1999

22.v.2000 5.x.1968

28.vi.1999

10.vii.1984

12.ix.1999

Fruiting

Appendix 4.2 (continued)  Major Postputrefaction Fungi and Data of Their Appearance that May Have Forensic Implications

Sagara et al. (1978)

Sagara (1975)

Sagara (1975) Sagara (1975) Sagara (1975)

Sagara (1975); Figure 4.9

Sagara (1975)

Kasuya (2002) Sagara (1975); Figure 4.9 Kasuya (2002)

Kasuya (2002)

Sagara (1995)

Kasuya (2002)

Reference

104 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

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69918.indb 105

Hebeloma vinosophyllum

Hebeloma syrjense (see Appendix 1)

Kyoto, Japan Kyoto, Japan Kyoto, Japan Kyoto, Japan

Quercus Pinus Quercus Pinus

Raccoon dog body

Raccoon dog excreta Mole excreta

Vespine wasp nest

E. N. America Saitama, Japan Shiga, Japan Kyoto, Japan Kyoto, Japan Saitama, Japan Saitama, Japan

Thicket Deciduous wood Quercus Pinus Pinus Castanopsis Quercus Quercus

Mammalian body

Human body Human urine

Night soil Domestic cat body Domestic dog body Fowl body Crow body

Texas, USA

Kyoto, Japan

Pinus

Sika deer body

Not known vi?1975 Winter 1967–1968? Winter 1999–2000 Late 1997

Not stated 2.ix.1999

Not stated

xi.1977

Not known

Intermittently

6.x.1983

Winter 2003–2004

19.ix.2000 ix–x.1998

26.x.1967 8.x.1975 24.viii.1968

ix–x 22.v.2000

4.xii.1976

15.x.1978

26.ix.1976

15.x.1979

28.ix.1984

9.ix.2004

Kasuya (2002) Fukiharu, Yokoyama, and Oba (2000) (continued)

Sagara (1975) Sagara (1976) Sagara (1975)

Sagara (1978, 1980) Sagara and Kobayashi (1979) Lewis and McGraw (1984) Lincoff (1981) Kasuya (2002)

Sagara (unpublished data) Sagara (unpublished data) Sagara (1989)

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Kalamunda, W. Australia

Eucalyptus

Kangaroo body

Laccaria bicolor

Hebeloma radicosoides

Manjimap, W. Australia

Eucalyptus

Snake body

Kyoto, Japan Aichi, Japan Kyoto, Japan Ibaraki, Japan Kyoto, Japan Kyoto, Japan Kyoto, Japan Shiga, Japan

Quercus Castanopsis Pinus Fagus Pinus Pinus Pinus Pinus

Human excreta

Domestic cat body

Raccoon dog excreta Mouse excreta

Vespine wasp nest Human urine Human feces Night soil

Kyoto, Japan

Location

Hebeloma aminophilum

Castanopsis

Kingfisher excreta

Alnicola lactariolens

Vegetation

Trigger Material

Fungal Species

x.1979 Not known 10.vi.1967 Not known

Not known

Some day between viii.1979 and iv.1980 Intermittently

Early 1981

Some months previously

Not stated

v–ix?1993

Deposition

25.vi.1981 1966 21.x.1967 13.x.1968

11.x.1989

14.x.1983

26.x.1980

1.xi.1981

22.vi.1977

21.v.1976

27.vi.1994

Fruiting

Appendix 4.2 (continued)  Major Postputrefaction Fungi and Data of Their Appearance that May Have Forensic Implications

Sagara, Okabe, and Kikuchi (1993) Sagara et al. (1985) Sagara (1975) Sagara (1975) Sagara (1975)

Sagara (1989)

Sagara (unpublished data) Hilton (1978); Miller and Hilton (1986) Hilton (1978); Miller and Hilton (1986) Sagara and Takayama (1982); Figure 4.10 Kuroyanagi et al. (1982)

Reference

106 Naohiko Sagara, Takashi Yamanaka, and Mark Tibbett

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Lactarius chrysorrheus

Laccaria amethystina

Kyoto, Japan Kyoto, Japan Kyoto, Japan Kyoto, Japan

Pinus Pinus Pinus Pinus

Human urine

Human feces Domestic cat body Raccoon dog excreta

Saitama, Japan Saitama, Japan

Pinus Quercus

Kyoto, Japan

Pinus

Raccoon dog body Raccoon dog excreta

Kyoto, Japan

Pinus

Raccoon dog excreta Giant hornet wastes Domestic cat body Kyoto, Japan

Kyoto, Japan

Pinus

Sika deer body

Quercus

Kyoto, Japan

Pinus

Domestic cat body

i.viii.1966 ii?1966 Intermittently

Not stated Intermittently for more than three years 14.vi.1966

Summer–autumn 1982 v?1983

Intermittently

Winter 2003–2004

6 months or more before

6.x.1966 16.xi.1967 17.x.1980

6.x.1966

26.ix.1999 ix–xi.1998

2.x.1983

16.x.1983

17.x.1980

9.ix.2004

6.vi.1980

Sagara (1975) Sagara (1975) Sagara (1989)

Sagara (1975)

Kasuya (2002) Kasuya (2002)

Sagara (1995)

Sagara (1995)

Sagara (unpublished data) Sagara (1989)

Sagara (1981); Figure 4.8

Soil Fungi Associated with Graves and Latrines 107

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The Role of Invertebrates in Terrestrial Decomposition: Forensic Applications

5

Ian R. Dadour and Michelle L. Harvey

Contents 5.1  Introduction............................................................................................... 109 5.2  The Invertebrates........................................................................................110 5.3  Forensic Entomology.................................................................................111 5.3.1  Succession.......................................................................................111 5.3.2  Current Research.......................................................................... 112 5.4  The Soil–Corpse Interface.........................................................................113 5.4.1  Class Arachnida.............................................................................114 5.4.1.1  Mites (Acari)...................................................................114 5.4.1.2  Spiders (Araneae)...........................................................115 5.4.1.3  Millipedes.......................................................................115 5.4.2  Class Insecta...................................................................................115 5.4.2.1  Springtails (Collembola)...............................................115 5.4.2.2  Silverfish (Thysanura)...................................................116 5.4.2.3  Cockroaches (Blattodea)..............................................116 5.4.2.4  Ants (Hymenoptera).....................................................116 5.4.2.5  Earwigs (Dermaptera)..................................................116 5.4.2.6  Flies (Diptera)................................................................116 5.4.2.7  Beetles (Coleoptera)......................................................117 5.5  Deeper Down: Invertebrates on Buried Bodies.....................................118 5.6  Conclusions................................................................................................ 120 References............................................................................................................. 120

5.1  Introduction The decomposing corpse provides an ephemeral yet nutrient-rich substrate that can be inhabited by a wide variety of organisms. As the bacteria begin the processes of cell breakdown, fermentation, and putrefaction, at the other end of the spectrum the large scavenging animals begin to play a significant 109

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110 Ian R. Dadour and Michelle L. Harvey

role in the consumption of the soft tissues of the corpse (Smith 1986). This in association with the smaller, more abundant invertebrate fauna is the major factor responsible for a large part of the decompositional process of the exposed corpse. Carrion itself forms a lucrative substrate for invertebrate colonization; however, in outdoor situations the interface between this substrate and the underlying soil, and the soil itself, also form attractive environments for certain organisms. The seepage of nutrient-rich fluids into the soil beneath the corpse significantly alters the microenvironment, affecting the inhabitant fauna. The arthropod assemblage may thus be considered to be affected by and reflective of the decomposition of the corpse and may therefore have some potential in contribution to forensic investigation (Bornemissza 1957). The invertebrate assemblage associated with carrion is composed mainly of insects (Smith 1986). The role of insects in the decomposition process has become a well-recognized area of forensic investigation, with forensic entomology commonly employed in homicide investigations (Morris and Dadour 2005). However, the use of invertebrates other than carrion-dwelling insects, particularly the soil fauna, has received little attention in a forensic context. Goff and Catts (1990) state that a wide variety of arthropods may be found associated with a corpse and may be indicative of the stage of decomposition, but not all are equally useful in estimation of postmortem interval (PMI). This may be attributed to the lack of research on groups other than the most abundant carrion inhabitants: the flies and beetles. Those invertebrates found in the soil beneath a corpse and how they might contribute to forensic investigations is considered in this chapter.

5.2  The Invertebrates The invertebrates are a diverse group of organisms classified by the absence of a backbone in all taxa. The invertebrates include sponges, molluscs, a variety of worm groups, and arthropods, and their enormous variability in morphology is reflected by their diversity in general biology and habitat. Certainly the most diverse invertebrate group is the phylum Arthropoda. This phylum contains at least 750,000 species, greater than three times the number of species found in all other described animal taxa (Ruppert and Barnes 1994). This group includes the crustaceans, insects, spiders, mites, scorpions, centipedes, and millipedes and includes species with both terrestrial and aquatic affinities. From a forensic perspective, the insects have been the main focus of invertebrate studies and applications. A wide variety of insects are attracted to carrion and are responsible for the consumption of much of the soft tissues of a corpse and are thus the most frequently encountered organisms on

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carrion. The ability of this group to locate and colonize carrion is exploited in the field of forensic entomology

5.3  Forensic Entomology Forensic entomology is broadly defined as the interaction of insects and other arthropods with legal matters. The field includes a wide variety of applications, encompassing any situation that may involve an interaction between insects and other arthropods and the law. The applications are generally categorized as urban, stored-product, and medicolegal forensic entomology (Lord and Stevenson 1986). The urban aspect generally deals with the effect of insects on manmade structures or other facets of human society. This may include the infestation of buildings by arthropod pests (Hall 1990) and the breeding of flies in livestock facilities (Hall 2001). The stored-product facet concerns the infestation of stored commodities by insect pests or domestic invasion of kitchen products by insects. This also encompasses the infestation of food sold by retailers to the public (ibid.). Undoubtedly the most widely recognized aspect of forensic entomology is the medicolegal aspect. Frequently featured as the main forensic investigative application of entomology in fiction, television, and film, this is the area toward which the majority of research is directed. Applications of this facet are wide-ranging and include the investigation of cases involving neglect by caregivers, generally in the elderly or very young (Benecke 2001), investigation of unexplained cases of death, or the cause of traffic accidents as a result of insect involvement (Hall 2001). The most recognized applications of medicolegal entomology, however, and perhaps forensic entomology as a whole, involve the estimate of PMI and circumstances surrounding a death. 5.3.1  Succession The estimation of PMI is based on knowledge of the locality-specific succession of insects occurring on a corpse following death. This predictable succession may be used, in conjunction with data describing the temperaturedependent developmental data for carrion-frequenting insects, to estimate PMI based on the minimum amount of time required for insect development. The use of successional data in estimation of PMI assumes that following death, an orderly and predictable succession of insect species occurs on a corpse. Insects are generally the first organisms to locate a body following death. The insects may be classified as adventive or incidental species, simply visiting by chance and having little forensic relevance (Goff and Catts 1990), predators that may feed on other species that have already colonized

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112 Ian R. Dadour and Michelle L. Harvey

the body, or necrophages that feed from the body itself and are most useful in estimation of PMI. As the body progresses through the stages of decomposition—flesh, bloat, decay, dry, and skeletal—the odors emitted by the corpse change (Anderson 2001), reflecting the physical changes in the body. The odors vary in attractiveness to different insects, and as the body decomposes and various resources are depleted, new insect types will colonize, being more suited to the current decompositional stage. These insect taxa reflect the physical changes in the body and are therefore predictable and useful in estimation of PMI. Flies (Diptera) and beetles (Coleoptera) are the insects most frequently collected from corpses (Lord 1990), and these are consequently the focus of most forensic invertebrate research and applications. The blowflies (Diptera: Calliphoridae) are usually the first insects to arrive following death. Female flies will deposit eggs or live larvae around orifices or wound sites on the corpse, and larvae will secrete enzymes and bacteria, facilitating consumption of the soft tissues of the corpse. Larvae will feed through three stages of growth (instars), each punctuated by the moulting of their size-restricting cuticle, enabling further growth. At the cessation of feeding, larvae will pupate in soil, in clothing, or beneath surrounding objects, and, following a period of metamorphosis, the adult fly emerges. The empty pupal casings may persist in soil for many years. The arrival of blowflies, and subsequently their larvae, is followed quickly by the arrival of the flesh flies (Diptera: Sarcophagidae), other carrion flies (Diptera: Muscidae), and predaceous beetle species such as rove beetles (Coleoptera: Staphylinidae), carrion beetles (Silphidae), clown beetles (Histeridae), skin beetles (Dermestidae), and checkered beetles (Cleridae). A variety of other fly families may be found in association with the body, and hide beetles (Trogidae) and larvae of some of the aforementioned beetle groups may feed on carrion itself, often on remains of hair, skin, and clothing in late decomposition (Smith 1986). 5.3.2  Current Research Current research focuses largely on aboveground successional studies, developmental rate studies, and other factors that impact on PMI estimation. DNA-based studies are becoming more common, facilitating the use of DNA for identification of insects and increased accuracy and efficiency (Harvey et al. 2003; Wallman, Leys, and Hogendoorn 2005). Studies have also concerned the possible characterization of human DNA ingested by blowfly larvae (Carvalho et al. 2005; Wells and Sperling 2001). Deaths occur in a variety of situations; clothed or unclothed, the corpse may be wrapped following death (Goff 1992), covered with vegetation, burned

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(Avila and Goff 1998), submersed in water, hanged, or buried. Each of these circumstances alters the decompositional environment in some way and defines the environment within which carrion-related organisms will interact. Though the body itself forms the primary decompositional site, the soil beneath it may be equally important. Corpses located in outdoor environments on a terrestrial surface create an interface within which soil fauna and carrion-dwelling organisms interact. The interactions in this zone are affected by soil type, vegetation, decomposition of the corpse, and a variety of environmental factors. Apart from the work by Bornemissza (1957) and Lundt (1964) the succession of insects in this interface, and within the soil itself, has been largely overlooked in the literature, and the forensic implications have yet to be considered.

5.4  The Soil–Corpse Interface The decomposing corpse in a terrestrial environment alters the substrate beneath it. This initiates a series of changes in vegetation and fauna, beginning a succession of arthropods affected by the decomposing carrion above. Bornemissza (1957) observed the greatest effect of the decomposing corpse on the soil beneath to occur during the black putrefaction and butyric fermentation stages. Fluid seepage contributes to development of a crust of hair, plant matter, and the uppermost soil layer beneath the body. During fermentation, the decomposition fluids released from the body, along with the waste products excreted by the insects feeding on the body, combine to kill the plants beneath the body and the soil fauna, altering the microenvironment. Anderson and VanLaerhoven (1996) found that vegetation under and around the body for 20–30 cm was killed by fluids released during active and advanced decay stages, and the number of arthropod species in the soil was reduced from thirty species before carrion placement to two species by fifteen days following placement in British Columbia. During the decay stage the soil beneath the carrion may become disturbed to a depth of approximately 1 in by the action of arthropods, particularly dipteran larvae, burrowing (Reed 1958). Decomposition fluids and associated arthropods are reported to affect the soil to a depth of 14 cm, with most effect in the upper soil layers. The area directly beneath the body, the carrion zone, serves as a decompositional zone occupied by carrion dwellers, distinct from a surrounding area of approximately 10 cm, which provides an intermediate zone of both carrion and regular soil-dwelling invertebrates (Bornemissza 1957). At a distance of 10–20 cm away from the body, the soil fauna is typical of general litter dwelling fauna, but perhaps the size of these zones may be dependent on the size of the carrion, as the work of

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114 Ian R. Dadour and Michelle L. Harvey

Bornemissza (1957) was based on guinea pigs and human decomposition may produce greater amounts of fluid. Payne (1965) indicated that in South Carolina, once carrion reached the dry stage of decomposition, there began an overlap of carrion and soil-dwelling insects on and around the corpse. Bornemissza (1957) recorded that one year following the placement of carrion, the soil arthropod assemblage had not yet returned to its predecompositional state. The soil–surface and litter-dwelling arthropods took longer to recolonize as compared with subterranean taxa. The crust formed by hair, vegetation, and fluids persisted long after placement and was most pronounced beneath oral and anal regions where seepage was greatest. Restoration of the soil community occurred only following heavy rains. Anderson and VanLaerhoven (1996) similarly observed that 271 days following death, the vegetation and soil fauna had not returned to normal. This alteration to the soil may perhaps be informative in PMI estimation for an extended period of time following death. A variety of arthropods associated with soil beneath a corpse are discussed herein. Many have forensic potential in that they are useful as broad markers for estimating time frames; however, in most cases research is scant in relation to their association with cadavers. The presence of many of these invertebrates is recorded for a variety of locations, but the timing of their arrival and departure varies markedly. This may be attributed to edaphic and climatic factors or simply to the particular species involved at a location. The discussion is centered around each type of organism in a more generalized sense, and variation between geographic localities is inevitable. 5.4.1  Class Arachnida 5.4.1.1  Mites (Acari) Mites are commonly associated with soil under the corpse in late decomposition stages (Goff and Catts 1990; Johnson 1975; Payne 1965; Smith 1986). Anderson and VanLaerhoven (1996) recorded mites as the most abundant invertebrates beneath surface carrion. Goff and Catts (1990) indicate several mite groups of significance. The Macrochelidae are phoretic on beetles, including the sylphid Necrophorus species, they attach themselves to flies and are transported to the body in early decomposition. They leave the beetle and may be found feeding mostly on fly eggs (Gibbs and Stanton 2001). Anderson and VanLaerhoven (1996) observed macrochelid abundance in soil to increase over time in the presence of carrion, and Reed (1958) similarly observed a great abundance around carrion. The soil-dwelling mites of the Cunaxidae are predatory mites living in the soil beneath remains, feeding on insects and their larvae associated with the remains. The Winterschmidtiidae and Acaridae may be found toward

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The Role of Invertebrates in Terrestrial Decomposition

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the end of decay, feeding on fungus and detritus (Goff and Catts 1990). Bornemissza (1957) also recorded the activity in late decay of tyroglyphid mites consuming any remaining skin and completely skeletonizing remains. The presence of particular mite groups varies markedly with location. In Perth, Australia, Bornemissza (1957) found gamasid mites during active decay and, as the remains began to dry, tyroglyphid mites following in the dry stage. He cites Kühnelt (1950), however, for the presence of mites only in the final stages of decomposition in Europe. This may be indicative of geographical behavioral variation, caused by climatic or edaphic factors. 5.4.1.2  Spiders (Araneae) Spiders may be collected from remains where they may be predaceous on other corpse-inhabiting arthropods (Goff and Catts 1990; Reed 1958; Smith 1986). They may be encountered beneath carrion capturing prey but as general predators with unpredictable arrival time at carrion are unlikely to be useful in estimation of PMI. 5.4.1.3  Millipedes Millipedes are commonly found in the moist environment beneath a body (Byrd and Castner 2001; Goff and Catts 1990). They are generally recognized as being plant feeders but have been reported to frequent carrion in many studies, generally in the dry stage of decomposition. Smith (1986) cited several studies as reporting the presence of Cambala annulata (Say) during active decay. 5.4.2  Class Insecta 5.4.2.1  Springtails (Collembola) Springtails are small insects commonly recognized by their springing behavior when disturbed in leaf litter or soil. They are considered to be mainly plant feeding with some species consuming decomposing animal material (Goff and Catts 1990) in damp soil and leaf litter habitats. The environment created by the seepage of fluids from the decomposing corpse provides springtails with a damp and nutrient-rich habitat. During active decay, however, the seepage of large amounts of decomposition products into the soil may alter springtail numbers. In Perth, Australia, Bornemissza (1957) recorded significantly lower numbers during active decay when fluids were being released, and during the formation of a crust beneath the body the subterranean springtails were entirely absent. Anderson and VanLaerhoven (1996) reported the return of springtails to the soil beneath carrion ninety-four days following placement of the body in British

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116 Ian R. Dadour and Michelle L. Harvey

Columbia, with populations increasing to higher abundances by day 111 than prior to placement of the carrion. Springtails have also been recorded from graves (Motter 1898) and surface-exposed carrion in other localities but are often herbivorous species that appear to have little relevance in estimation of PMI. 5.4.2.2  Silverfish (Thysanura) The silverfish, commonly found as household and stored-product pests, may be found in association with remains in the drier stages of late decomposition (Goff and Catts 1990). 5.4.2.3  Cockroaches (Blattodea) Cockroaches may be found feeding on the decomposing remains (Goff and Catts 1990; Payne 1965). 5.4.2.4  Ants (Hymenoptera) Ants are frequently associated with carrion, but their significance is difficult to determine. They are often located living within the soil beneath or close to surface carrion, making it unclear whether they are simply adventive or may actually be predictable members of the succession beneath carrion. Bornemissza (1957) recorded ants from soil beneath the carrion feeding on the carrion itself. Similarly, Payne (1957) observed ants feeding around body orifices and recorded their greatest activity to occur during nighttime hours. Ants are often responsible for the removal of immature insects from carrion, particularly fly larvae (Johnson 1975). It is suggested by Fuller (1934), however, that the presence of ants is not predictable, as carrion equally proximate to ant nests in her study were not always colonized. Ants are therefore likely to be adventive, and though seasonality and nesting sites may be informative in PMI estimation, their arrival at carrion may not be overly useful in estimation of PMI. 5.4.2.5  Earwigs (Dermaptera) Earwigs, inhabiting the soil beneath carrion, have been reported to feed on carrion (Bornemissza 1957). 5.4.2.6  Flies (Diptera) Following the cessation of feeding in the third larval stage, fly larvae will leave the body and pupate in soil, in clothing, or under nearby objects. Bornemissza (1957) noted that the soil structure beneath a corpse was significantly altered by the activity of the burrowing larvae, as well as other carrion-related insects. The discovery of larvae, pupae, or empty puparia may be important in the estimation of PMI, given relevant sets of developmental data for significant dipteran species. The piophilid, or cheese-skipper species

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Piophila casei, is reported to pupate in soil around carrion after cessation of feeding (Fuller 1934) and thus may be encountered. 5.4.2.7  Beetles (Coleoptera) Beetles are important members of the carrion assemblage, useful in estimation of PMI. They are often voracious predators of dipteran larvae on carrion and arrive following colonization by the larvae during the fresh stage with numbers increasing during bloat (Johnson 1975). Once the initial necrophages have left the corpse, PMI estimation may require use of other taxa that persist beyond soft-tissue consumption at a crime scene, such as the Coleoptera (Kulshrestha and Satpathy 2001). A number of studies have specifically focused on the carrion-frequenting Coleoptera (e.g., Easton 1965; Kaufmann 1937a, 1937b, 1937c; Shubeck 1970). The Silphidae, or carrion beetles, possess a variety of life histories, from development in decomposing vegetation to predation, and a number of species have been observed to feed on decomposing animal material (Byrd and Castner 2001; Leblanc and Strongman 2002). The burying beetles of the genus Necrophorus capture and bury small animals, on which they subsequently lay their eggs. Steele (1927) reported Necrophorus orbicollis, N. tomentosus, Silpha Americana, and S. novaboracensis all to prey on dipteran larvae on carrion. Other species will make impressions in decomposing flesh and lay their eggs in these impressions; however, it is the Necrophorus species that will primarily be located within the soil. They may be encountered in leaf litter beneath and around the body. Putman (1978) observed Necrophorus spp. frequenting rodent carcasses in England, with the greatest number during summer and autumn. The author notes, however, that the carrion was rarely buried by the beetles, with burials attempted but not completed. Adults were found on carcasses as early as 24 hours following placement, presumably preying on blowfly larvae. Reed (1958) similarly observed no burials by Necrophorus spp. but did encounter them beneath carrion in Tennessee. The Staphylinidae, or rove beetles, may be found in the surface layer of soil. They are voracious maggot predators, actively chasing maggots below the soil surface (Smith 1986). These beetles may arrive shortly following colonization of the corpse by blowfly larvae, with peak numbers under and around the corpse observed just prior to and at the time of larval migration from the corpse (Putman 1978). Clown beetles, of the Histeridae, are predators in both adult and larval stages, preying predominantly on fly larvae. They may be found in upper soil layers and are found as predators on the body between bloat to dry stages of decomposition (Byrd and Castner 2001). Payne and King (1969) record the occurrence of these beetles in the soil–carrion interface during daylight hours, with feeding activity occurring at night. The Cleridae (checkered

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beetles) are similarly predators in larval and adult stages and may be associated with upper soil layers. The Scarabaeidae (scarab or dung beetles) of the subfamily Scarabaeinae and also the Geotrupidae (also called dung beetles) frequent dung or carrion, which is generally rolled into a ball with eggs laid in the ball prior to burial (Byrd and Castner 2001). The egg hatches, and larvae develop on this nutrient-rich ball and may therefore be found in the soil beneath a body. The scarab and dung beetles construct tunnels in soil beneath carrion (Payne and King 1969; Smith 1986). Payne (1965) observed adult scarab beetles arriving as early as the bloat stage in South Carolina. During nocturnal hours they emerged from soil and fed on carrion, remaining in tunnels they constructed beneath carrion during the daylight hours. A variety of other beetle groups may be associated with carrion. Payne and King (1969) recorded the occurrence of Hydrophilidae underneath surface carrion. In addition, many beetle groups usually found dwelling in leaf litter or other decomposing organic matter have been collected from surface carrion. Leiodid beetles, usually found in decaying vegetative matter, were found on carrion, as were the fungus-feeding orthoperids and the vegetation-dwelling anthicids (Payne and King 1969). Reed (1958) also recorded the presence of Leptodiridae species, frequently recovered from decomposing vegetative matter, gathering around body openings, underneath carrion, or in nearby leaf litter. In the same study, Trogidae species, both adult and immature, were observed in soil under carrion.

5.5  Deeper Down: Invertebrates on Buried Bodies The burial of a body alters the decomposition process significantly from surface decomposition, particularly with relation to scavenging, temperature, and insect colonization (Fiedler and Graw 2003). The insects generally colonizing a body laid on the surface are inhibited, as are the airborne bacteria (Smith 1986). The depth, soil type and nature of the burial are obviously important factors (Lundt 1964). The most important effect of burial on decomposition is the increase in time required for biomass reduction, relative to exposed carrion (Smith 1986). Smith (1986) suggested that blowflies, which are responsible for the majority of biomass reduction on carrion, are excluded from the corpse at a depth of just 2.5 cm. However, Simpson and Strongman (2002) reported the occurrence of the blowfly Cynomyopsis cadaverina on carrion buried at a depth of 30 cm. Rodriguez and Bass (1985) also observed Sarcophagidae (flesh fly) and blowfly larvae on burials at a depth of 1 ft, as did VanLaerhoven and Anderson (1999). In the latter study, adult flies were observed attempting to

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move through surface soil cracks and reach the body, particularly following heavy rain. Eggs were also laid on the surface following rain, and on hatching, larvae moved down the soil cracks. Blowflies would therefore be unexpected at depths greater than this, and presence of larvae at greater depths may suggest burial occurred some time following death, allowing flies access to the body. In cases where exhumation occurs a considerable time after burial, the activity of blowflies on carrion may be indicated only by the presence of empty puparia in soil. Puparia may persist for hundreds of years following death (Gilbert and Bass 1967). Certain fly species are seasonal, and the presence of their puparia may be a useful indicator as to the seasonality of a death or burial (ibid.). Burial does not preclude invertebrates from accessing the corpse. In a burial situation, certain insects such as the flies Muscina spp. (family Muscidae) and Morpholeria kerteszi (family Heliomyzidae) will lay eggs on the soil surface, and following hatching, larvae will burrow through the soil to the carrion (Smith 1986). Other adult insects will burrow down through the soil to oviposit directly onto carrion, such as the Staphylinidae (Coleoptera) and Phoridae (Diptera). Adults of the phorid Conicera tibialis will burrow up to 2 m below the surface to oviposit on a body (Bourel at al. 2004). Cheeseskipper larvae (Piophilidae) have been recorded on buried carrion in rural areas of Nova Scotia (Simpson and Strongman 2002), and Sphaeroceridae (Diptera) may also be abundant on buried carrion (Bourel et al. 2004). Beetles may also be found on buried carrion. Payne and King (1969) discovered a carabid beetle species Anillinus fortis Horn, associated with buried carrion. Simpson and Strongman (2002) also reported the sylphid Necrophila americana and some histerid, carabid, and staphylinid species on buried carrion in rural areas of Nova Scotia but not in urban areas. VanLaerhoven and Anderson (1999) report the occurrence of many beetle species, including silphids, on buried carrion, but suggest these beetles to be unreliable in a successional sense as they are generalized predators and their arrival is not predictive. A thorough knowledge of the succession occurring on buried carrion is required if it is to be employed in PMI estimation. As with surface carrion, a predictable succession may be used to estimate PMI, and in cases where dipteran larvae are still present, the use of temperature dependent developmental data may be applied. The use of such data obviously requires consideration of season and temperature. VanLaerhoven and Anderson (1999) determined soil temperature to be a better predictor of internal temperature of buried carrion than ambient temperature and suggested use of soil temperature for estimation of insect development. VanLaerhoven and Anderson (1999) recorded the occurrence of some species on buried carrion to be predictable and suggested that burial successions can be used in estimation of PMI. This obviously requires further

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studies in each specific locality where such data are to be employed. The variability in species present and differences in behavior and developmental rates make the development of a generalized successional database impractical, as with the current successional data for aboveground carrion.

5.6  Conclusions The type of soil is an important factor in decomposition (Bornemissza 1957). Edaphic conditions affect the type of vegetation found in an area and, consequently, the invertebrate fauna. The properties of a soil and its potential for drainage would be assumed to also affect the faunal assemblage found beneath carrion. Soil type is particularly important in the decomposition of buried carrion (Simpson and Strongman 2002), as affected by numerous variables such as climate, vegetation, soil temperature, and moisture (Motter 1898) and drainage (Rodriguez and Bass 1985). Anderson and VanLaerhoven (1996) concluded from their studies that soil fauna and its succession may be very useful in estimation of PMI, being useful for a longer time following death than the surface carrion assemblage alone. This review has shown that there has been limited study of carrion-related soil invertebrates. Studies in the relevant literature indicate the presence of a few groups to occur in soil beneath carrion at distinct and possibly predictable stages of decomposition, but there are numerous invertebrate groups still neglected. The use of the soil succession in PMI estimation will require development of successional databases for locations where it is to be applied. This will involve consideration of all invertebrate groups associated with carrion and selection of groups that are truly predictable in their arrival at a corpse in relation to decompositional stage. Numerous studies have identified groups both beneath the body and associated with burials that may be useful in PMI estimation, but the successional data required to apply such observations are still lacking. Invertebrates play an important role in terrestrial decomposition, and further study will provide a new method for estimation of PMI and open new frontiers in forensic entomology.

References Anderson, G. S. (2001). Insect succession on carrion and its relationship to determining time of death, in Forensic Entomology: The Utility of Arthropods in Legal Investigations (J. H. Byrd and J. L. Castner, Eds.). Boca Raton, FL: CRC Press, 143–175. Anderson, G. S. and VanLaerhoven, S. L. (1996). Initial studies on insect succession on carrion in Southwestern British Columbia. J Forensic Sci. 41, 617–625.

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Avila, F. W. and Goff, M. L. (1998). Arthropod succession onto burnt carrion in two contrasting habitats in the Hawaiian Islands. J Forensic Sci. 43, 581–586. Benecke, M. (2001). Child neglect and forensic entomology. Forensic Sci. Int. 120, 155–159. Bornemissza, G. F. (1957). An analysis of arthropod succession in carrion and the effect of its decomposition on the soil fauna. Aust. J. Zool. 5, 1–12. Bourel, B., Tournel, G., Hedouin, V., and Gosset, D. (2004). Entomofauna of buried bodies in northern France. Int. J. Legal Med. 118, 215–220. Byrd, J. H. and Castner, J. L. (2001). Insects of forensic importance, in Forensic Entomology: The Utility of Arthropods in Legal Investigations (J. H. Byrd and J. L. Castner, Eds.). Boca Raton, FL: CRC Press, 43–79. Carvalho, F., Dadour, I. R., Groth, D. M., and Harvey, M. L. (2005). Isolation and detection of ingested DNA from the immature stages of Calliphora dubia (Diptera: Calliphoridae). Forensic Sci. Med. Pathol. 1, 261–265. Easton, A. M. (1965). The Coleoptera of a dead fox (Vulpes vulpes (L.)); including two species new to Britain. Entomol. Mon. Mag. 102, 205–210. Fiedler, S. and Graw, M. (2003). Decomposition of buried corpses, with special reference to the formation of adipocere. Naturwissenschaften 90, 291–300. Fuller, M. E. (1934). The insect inhabitants of carrion: a study in animal ecology. Common. Aust. Council Sci. Ind. Res. Bull. 82, 1–62. Gibbs, J. P. and Stanton, E. J. (2001). Habitat fragmentation and arthropod community change: Carrion beetles, phoretic mites, and flies. Ecol. Appl. 11, 79–85. Gilbert, B. M. and Bass, W. M. (1967). Seasonal dating of burials from the presence of fly pupae. Am. Antiquity 32, 534–535. Goff, M. L. (1992). Problems in estimation of postmortem interval resulting from wrapping of the corpse: A case study from Hawaii. J. Agri. Entomol. 9, 237–243. Goff, M. L. and Catts, E. P. (1990). Arthropod basics-structure and biology, in Entomology and Death: A Procedural Guide (E. P. Catts and N. H. Haskell, Eds.). Clemson, SC: Joyce’s Print Shop, 41–71. Hall, R. D. (1990). Medicocriminal entomology, in Entomology and Death: A Procedural Guide (E. P. Catts and N. H. Haskell, Eds.). Clemson, SC: Joyce’s Print Shop, 1–8. Hall, R. D. (2001). Introduction: Perceptions and status of forensic entomology, in Forensic Entomology: The Utility of Arthropods in Legal Investigations (J. H. Byrd and J. L. Castner, Eds.). Boca Raton, FL: CRC Press, 1–15. Harvey, M. L., Mansell, M. W., Villet, M. H., and Dadour, I. R. (2003). Molecular identification of some forensically important Calliphoridae (Diptera) of southern Africa and Australia. Med. Vet. Entomol. 17, 363–369. Johnson, M. D. (1975). Seasonal and microseral variations in the insect populations on carrion. Am. Mid. Nat. 93, 79–90. Kaufmann, R. R. U. (1937a). Investigation on beetles associated with carrion in Pannal Ash, near Harrogate: I. Entomol Mon. Mag. 73, 78–81. Kaufmann, R. R. U. (1937b). Investigation on beetles associated with carrion in Pannal Ash, near Harrogate: II. Entomol. Mon. Mag. 73, 227–233. Kaufmann, R. R. U. (1937c). Investigation on beetles associated with carrion in Pannal Ash, near Harrogate: III. Entomol. Mon. Mag. 73, 268–272. Kühnelt, W. (1950). Bodenbiologie. Vienna, Austria: Herold.

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122 Ian R. Dadour and Michelle L. Harvey Kulshrestha, P. and Satpathy, D. K. (2001). Use of beetles in forensic entomology. Forensic Sci. Int. 120, 15–17. Leblanc, H. N. and Strongman, D. B. (2002). Carrion insects associated with small pig carcasses during fall in Nova Scotia. Can. Soc. Forensic Sci. J. 35, 145–152. Lord, W. D. (1990). Case histories of the use of insects in investigations, in Entomology and Death: A Procedural Guide (E. P. H. Catts and N. H. Haskell, Eds.). Clemson, SC: Joyce’s Print Shop Inc., 9–37. Lord, W. D. and Stevenson, J. R. (1986). Directory of Forensic Entomologists, 2d ed. Washington, DC: Defense Pest Management Information Analysis Center, Walter Reed Army Medical Center. Lundt, H. (1964). Ecological observations about the invasion of insects into carcasses buried in soil. Pedobiologia 4, 158–180. Morris, B. and Dadour, I. R. (2005). Forensic entomology: The use of insects in legal cases, in Expert Evidence (I. Freckelton and H. Selby, Eds.). Andover, UK: Thomson Publishing Services. Motter, M. G. (1898). A contribution to the study of the fauna of the grave: A study of one hundred and fifty disinterments, with some additional experimental observations. J. NY Entomol. Soc. 6, 201–231. Payne, J. A. (1965). A summer carrion study of the baby pig Sus scrofa Linnaeus. Ecology 46, 592–602. Payne, J. A. and King, E. W. (1969). Coleoptera associated with pig carrion. Entomol. Mon. Mag. 105, 224–232. Putman, R. J. (1978). The role of carrion-frequenting arthropods in the decay process. Ecol. Entomol. 3, 133–139. Reed, H. B. (1958). A study of dog carcass communities in Tennessee, with special reference to the insects. Am. Mid. Nat. 59, 213–245. Rodriguez, W. C. and Bass, W. M. (1985). Decomposition of buried bodies and methods that may aid in their location. J. Forensic Sci. 30, 836–852. Ruppert, E. E. and Barnes, R. D. (1994). Invertebrate Zoology, 6th ed. Philadelphia: Saunders Publishing. Shubeck, P. P. (1970). Ecological studies of carrion beetles in Hutcheson Memorial Forest. J. NY Entomol. Soc. 77, 138–151. Simpson, G. and Strongman, D. B. (2002). Carrion insects on pig carcasses at a rural and an urban site in Nova Scotia. Can. Soc. Forensic Sci. J. 35, 123–143. Smith, K. G. V. (1986). A Manual of Forensic Entomology. Oxford: British Museum (Natural History) and Cornell University Press. Steele, B. F. (1927). Notes on the feeding habits of carrion beetles. J. N Y Entomol. Soc. 35, 77–81. VanLaerhoven, S. L. and Anderson, G. S. (1999). Insect succession on buried carrion in two biogeoclimatic zones of British Columbia. J. Forensic Sci. 44, 32–43. Wallman, J. F., Leys, R., and Hogendoorn, K. (2005). Molecular systematics of Australian carrion–breeding blowflies (Diptera: Calliphoridae) based on mitochondrial DNA. Invert. Syst. 19, 1–15. Wells, J. D. and Sperling, F. A. H. (2001). DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae). Forensic Sci. Int. 120, 110–115.

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The Decomposition of Hair in the Buried Body Environment

6

Andrew S. Wilson

Contents 6.1  6.2  6.3  6.4  6.5 

Introduction............................................................................................... 123 The Structure, Growth, and Function of Hair...................................... 125 Weathering, Contaminants, and Color Change to Hair Fibers......... 128 Structural Alteration to the Hair Fiber.................................................. 130 Keratinophilic Fungi—Their Geographic Distribution and Ecological Factors Influencing Keratinolytic Activity..........................131 6.6  Th  e Mechanism of Microbial Degradation of Hair Keratin.............. 133 6.7  Histological Alteration to the Hair Shaft............................................... 134 6.8  Hair in Association with a Buried Body................................................ 137 6.9  Summary and Appropriate Measures for Safeguarding Evidence.... 139 References............................................................................................................. 140

6.1  Introduction Although hair can survive for millennia, it may also degrade in a matter of weeks—an important consideration now that it is possible to obtain a considerable amount of valuable information from hair (Wilson 2005; Wilson and Gilbert 2007). In forensic science, hair along with other fibers has long been regarded as important trace evidence (Robertson 1999; Seta, Sato, and Miyake 1988), with the current potential to derive important genetic information (Bonnichsen et al. 2001; Gilbert, Tobin, and Wilson in press), as well as dietary, seasonality, and location information (Bonnichsen et al. 1996; Pain 1998; Tam 2000; Toribara and Muhs 1984; Wilson, Dixon, Dodson, et al. 2001) as evidence for toxicology and drug use (Baez et al. 2000; Counsell, Lunt, and Sutherland 2000) and as a trap for particulate evidence such as gunshot residue (MacCrehan, Layman, and Secl 2003). Routine morphological means of characterizing hair samples (Ogle and Fox 1999) have been supplanted by individualization using DNA (Houck and Budowle 2002; Rowe 2001; Taupin 2004). In addition to the routine 123

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importance of largely fresh shed or plucked hairs as forensic trace evidence where survival of the root bulb is important for the recovery of nuclear DNA (Hedman and Jangblad 2003; Nozawa et al. 1999), the hair shaft (Allen et al. 1998; Butler and Levin 1998; Wilson, DiZinno, et al. 1995; Wilson, Polanskey, et al. 1995) is now considered to be a good source of mitochondrial DNA (mtDNA), with the oldest authentic DNA from hair being on a par with that recovered from bone or teeth (Gilbert et al. 2004; Gilberet et al. 2007). Even in circumstances when soft tissues are gone or decomposed, hair can still survive and may be of vital importance in individualization or determining systemic poisoning and drug abuse (Kelly et al. 2000; Kintz 1996; Moeller, Fey, and Sachs 1993; Nakahara 1999; Sachs and Kintz 1998; Tsatsakis et al. 2001). For instance, morphine was found in hair from two Italian women exhumed seven months after burial (Mari and Bertol 1997), and cocaine and lidocaine were detected in scalp and pubic hair from a man and a woman exhumed after seven months and two months of burial, respectively (Arado et al. 2001). A series of papers based on the discovery of hair in cave deposits suggested that hair survival should be widespread (Bonnichsen and Schneider 1995; Bonnichsen et al. 1996) and that hair should be recovered from soil deposits more frequently if closer attention was paid to retrieving this biomaterial (Morell 1994). A dispersal technique for soil aggregates was proposed (Beatty and Bonnichsen 1994), and this has been successfully used in the retrieval of fibers from large quantities of garden soil in forensic casework. However, the claim that hair survives almost universally is difficult to accept in light of a comprehensive literature that discusses the degradation of hair by specific groups of microorganisms (English 1963, 1965; Kushwaha and Guarro 2000). Despite an extensive literature on the taphonomy of bone and teeth, there has been far less written about hair. Hair degradation must be considered separately from other biomaterials because of the disparate structural biology of hair, bone, and teeth and the contrasting information that these biomaterials can provide. In bone, histological changes are considered to occur relatively rapidly from the outset, before bone attains some degree of equilibrium (Millard 2001), highlighting the need to study degradation over short timescales to understand degradation over the medium to longer term. In general, hair is less resistant to degradation than bone or teeth, largely because the organic fraction of bone and teeth (Pfeiffer 1992) is protected by the mineral phase of these tissues, whereas hair has no mineral phase. Hair is largely composed of keratin proteins—cystine is the most abundant amino acid in hair, composing roughly 18% (Halal 2002)—whereas the great bulk of protein in fresh bone, cement, and dentine is type I collagen, with glycine making up roughly one third of the amino acid content. In teeth, 90% of

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the organic component of enamel is amelogenin, a protein unique to enamel matrix (proline is about a quarter of its amino acid content) (Hillson 1996). Hair is commonly recovered in association with human remains such as victims of accidental death, homicide victims, or exhumed cemetery remains—a combination of surface-exposed or -concealed or burial environments. At the soil surface hair may be dispersed by small mammals or birds and used as nesting material (Rodriguez 1997). Since typically bodies and not skeletons are buried, the byproducts of soft-tissue decomposition are significant to hair survival and degradation. The impact of the buried body environment on organic fiber degradation was first discussed in relation to textile remains and the preservation of textile fragments in metal artefact corrosion products (mineral preserved organics) found most notably in Anglo-Saxon graves in the United Kingdom (Janaway 1985). Depending on the prevailing environmental conditions and the postmortem interval, softtissue remains may have undergone some degree of putrefaction and hair may have started to detach from the cranium as part of skin slippage. The decomposition of hair and soft-tissue remains is summarized in this chapter through archaeological and forensic observations, with particular reference to decomposition studies using both human cadavers and animal models and long-term experiments.

6.2  The Structure, Growth, and Function of Hair The main functions of hair are protection, insulation, sexual display, sensory organs, and a waste sink for toxic substances. Human hair exists in three main forms: (1) as thick, long, terminal hairs growing on the scalp, eyebrows, and eyelashes after birth and on the face, chest, arms, and groin after puberty; (2) as fine downy relatively unpigmented vellus body hair; and (3) as lanugo hair, which develops in utero from about 3 months and is shed at approximately 36 weeks of pregnancy (Gray, Dawber, and Whiting 1997). This chapter is concerned solely with a study of terminal hair. Although the average daily growth of hair will vary to some extent with age (Serri and Cerimele 1990), sex hormones (Schweikert and Wilson 1974a, 1974b; Thornton et al. 1998), race (Loussouarn 2001), time of year (Randall and Ebling 1991), and genetic disorders (Birnbaum and Baden 1987; de Berker and Sinclair 2001), human scalp hair grows on average at a rate of about 1 cm per month. This characteristic is of great importance since it means that time-resolved differences in, for example, diet or drug use can be accurately determined. Hair follows a growth cycle that can be divided into three main phases (Paus 1998) with active shedding adding another phase (Milner et al. 2002). Anagen is the growth phase, lasting on average three years but as long as six to ten years (Forslind 1990), during which time the

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126 Andrew S. Wilson a Cuticle Cortex Co

b P

En Ex

Medulla

δ A

Cu

Pigment Granule

Ep

Cortical cell

β Nuclear Remnant Cortical cell Membrane Complex

Macrofibril c Microfibril Protofibrils

Keratin Alpha-helical Structure

N

CmC

P M

Figure 6.1  Schematic diagram showing the main structural components of the hair shaft in transverse section: high-resolution light micrograph (a) bar equals 10 μm; transmission electron micrograph of the hair cuticle (b: bar equals 120 nm); transmission electron micrograph of the hair cortex (c: bar equals 1.5 μm). Co, cortex; Cu, cuticle; P, pigment granule; En, endocuticle; Ex, exocuticle; A, A layer; Ep, Epicuticle; β, β layers of the cell membrane complex; δ, δ layer of the cell membrane complex; M, macrofibril; N, nuclear remnant; CmC, cell membrance complex. (Line drawing adapted from Ryder 1973 Hair. Studies in Biology no. 41. London: The Institute of Biology.)

hair shaft is actively produced and melanin synthesis occurs. Catagen is the regression phase, lasting roughly three weeks, during which the lower 70% of the anagen hair follicle is resorbed. Telogen is the resting phase, lasting on average three months, during which no significant tissue remodeling occurs. Human hair growth follows a mosaic rather than a synchronous (i.e., moulting) growth pattern from about 1 year of age onward. Humans may have up to 150,000 scalp hair follicles, and up to 100 scalp hairs may be shed each day (Sperling 1991). The hair shaft (Figure 6.1) comprises three main structures: (1) the outer cuticle responsible for the main optical and frictional properties of the fiber; (2) the cortex, responsible for the bulk fiber mechanical properties such as strength and flexibility; and (3) the porous medulla, which is more prominent in gray hair, but otherwise may or may not be present. Cuticle thickness varies markedly between species. Though much of our understanding of hair structural biology is derived from the study of wool, this homologous

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tissue does differ from human hair in several key aspects. Wool frequently has a cuticle layer only one cell thick, whereas human hair may have up to ten layers of cuticle cells. In addition, the type and amount of high-sulfur proteins differ between hair and wool (Gillespie 1983). In human scalp hair the overlapping cuticles point toward the distal tip. Each cuticle cell (up to 0.7 µm thick) has a laminate structure. Underlying the outermost epicuticle (~5–7 nm thick), the sulfur rich A-layer (~ 40 nm thick) forms the uppermost part of the exocuticle (~0.2 µm thick). An irregular boundary separates the exocuticle from the less resistant endocuticle (~0.1 µm thick) comprising remnants of various cell organelles (Jones 2001). Each cuticle cell is separated from the next by lipid-rich membranes. Covalently-bound C21 saturated fatty acids are located on the outer hydrophobic surface layer of the cuticle cell and constitute the F-layer (0.9 nm thick) linked to the protein by thioester linkages as an integral part of the epicuticle (Wertz 1997). Sebum secreted from the sebaceous gland attached to the hair follicle coats the cuticle but is not bonded to it (Gray et al. 1997). The protein of the epicuticle is highly crosslinked by both disulfide and isopeptide bonds (Powell and Rogers 1997), with large quantities of lysine and glutamic acid forming a particularly insoluble protein complex (Jones 2001). The cortex is made up of bundles of spindle-shaped cortical cells (~100 µm in length and 5–10 µm in diameter at their widest point) aligned to the fiber long axis. Each cortical cell comprises bundles of macrofibrils (~300 nm in diameter), osmophilic (i.e., heavily stained by the electron microscopy stain osmium tetroxide) nuclear remnants, and pigment granules (melanosomes) (~0.5–1 µm in diameter). Pigment granules may also be found occasionally within the endocuticle (Puccinelli, Caputo, and Ceccarelli 1967). Each macrofibril is in turn made up of keratin intermediate filaments, also known as microfibrils (~8–10 nm in diameter). These are estimated to contain thirty-two keratin protein chains embedded in a high-sulfur intermicrofibrillar matrix of cysteine-rich and glycine/tyrosine-rich keratin-associated proteins (Powell and Rogers 1997), giving the characteristic fingerprint pattern of the macrofibrils. Hair curl is due to two different cortex types (more clearly defined in wool fibers) found in the same transverse section. These are ortho-cortex in which macrofibrils are clearly separated by nonkeratin intermacrofibrillar matrix and para-cortex in which the macrofibrils are in close apposition. Toward the root end, the fiber margins and cortical cells become more irregularly shaped in transverse section. Both the cuticle and cortical cells are bounded by cell membranes that, together with the intercellular material, are known as the cell membrane complex (Marshall, Orwin, and Gillespie 1991). This consists of the β-layers, protein-lipid complexes (~5 nm thick) on either side of the δ-layer, and an intercellular cement (~15 nm thick) (Baden 1990) rich in amino and carboxyl

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functional groups (Swift 1997) and probably containing glycoproteins (Allen, Ellis, and Rivett 1991). Where present, the central medulla is a loosely packed spongy structure that may be continuous or interrupted along the length of the fiber and may be either air or fluid filled. The medulla comprises medullary trichohyalin granules, filling the cells with amorphous deposits that do not contain any filaments (Rogers, Fietz, and Fratini 1991). Trichohyalin is a keratin-associated protein found only in the hair shaft medulla. It consists of a mass of fused protein granules cross-linked by isopeptide bonds (O’Keefe et al. 1993; Powell and Rogers 1997). Trichohyalin is extensively cross-linked by transglutaminase to itself as well as to the keratin intermediate filaments (Bertolino and O’Guin 1994). The integrity of hair is largely defined by its composition of keratins and keratin-associated proteins. Crystalline alpha-keratin intermediate filaments (microfibrils) are embedded in a sulfur-rich matrix of amorphous keratinassociated proteins (inter-microfibrillar matrix) that comprise about 40% of the protein content of the cell (Harding and Rogers 1999). The sulfur-rich matrix constitutes one of the richest sources of cystine found in nature (Gillespie 1983), with about 80% of the disulfide linkages in human hair accommodated within the sulfur-rich matrix (Marshall et al. 1991). The strong covalent disulfide bonds in cystine are key to the structural stability of keratin. Though the mechanical strength, elastic properties and chemical and biological resistance of keratins stem directly from the three-dimensional helical structure stabilized by disulfide bonds (ibid.), the structural stability of hair also depends on salt bridges between side-group chains (electrovalent unions of acidic and basic side-chain residues); hydrogen bonds between neighboring groups (between the oxygen and hydrogen atoms of the carboxy- and amino-terminal groups in the polypeptide chain); and hydrophobic interactions (Bertolino and O’Guin 1994).

6.3  Weathering, Contaminants, and Color Change to Hair Fibers Hair being superficially distributed on the body may be subject to physical and chemical alteration and exposure to environmental contaminants in life as well as postmortem. Grooming practice (Gamez 1998; Schramm and Kuhnel 1992) and cosmetic treatment, ranging from frequency of washing and choice of shampoo (Andrasko and Stocklassa 1990; DiPietro et al. 1989; LeBlanc, Dumas, and LeFebvre 1999) to dyeing, bleaching, perming, and environmental exposure (Doi et al. 1988; Raghupathy et al. 1988; Robbins 1979) all potentially have an impact on hair condition and may introduce

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contaminants (Skopp, Potsch, and Moeller 1997). Repeated mechanical and chemical damage termed weathering produces progressive changes to the cuticle, with the distal tip most severely affected (Bottoms, Wyatt, and Comaish 1972). Weathering will accelerate damage, lifting, and loss of cuticle scales, further exposing the cortex to damage and resulting in split or brush ends (Robinson 1976). The appearance of buried or shed hair fibers will also be affected by sample condition, since damage to the outer cuticle will affect the reflectance properties of the fiber. Certain environments, such as those producing bog bodies, are known to yield hair of a red-brown color (Brothwell 1986; Brothwell and Gill-Robinson 2002), but it has commonly been assumed that this happens to all long-buried hair. This concept has been perpetuated by popular nicknames such as “Ginger”—affectionately given to a naturally mummified predynastic individual with red hair on display in the mummy rooms at the British Museum (Rae 1993). However, it is virtually impossible to claim that hair has decolored (Lubec et al. 1987) without comparative reference samples. Potential change in hair color can be explained by examining the biochemistry of melanin. All hair contains a mixture of both black-brown eumelanin and red-yellow phaeomelanin pigments, which are susceptible to differential chemical change under certain conditions. Importantly, phaeomelanin is much more stable to environmental conditions than eumelanin; hence, the reactions occurring in the burial environment favor the preservation of phaeomelanin, revealing and enhancing the red-yellow color of hairs containing this pigment (Wilson, Dixon, Dodson, et al. 2001). Long-term degradation of hair in an arid environment will be influenced by the action of oxygen on the chemical integrity of the hair protein both in vivo and in the burial environment, although this may be confined to the fiber surface because of the low diffusivity of oxygen in dry keratin (Smith 1995). Additionally, in waterlogged bog environments, the breakdown of organic matter to humic acids under the Maillard reaction imparts a brown color to recovered remains (Painter 1991). Color change occurs far more slowly under dry oxidizing conditions than wet anoxic conditions. Ultraviolet and visible light are known to significantly modify the physico-chemical properties of hair keratins and melanin. The most noticeable examples of the effects of light in melanin are photo-induced free radical formation and photo-modification of the redox properties of melanin (Sarna and Swartz 1998), resulting in bleaching following reaction with singlet oxygen (Smith 1995). It has been suggested that black hair is largely protected from the damaging effects of sunlight by the quantities of eumelanin present and that, in contrast, blond hair is detectably photochemically damaged due to the lower pigment concentration and presence of phaeomelanin (Hoting and Zimmerman 1997). From a forensic standpoint it is important to note

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a

b

Figure 6.2  Fading of hair color caused by 6 months of exposure to sunlight at the ground surface in a temperate environment: faded upper surface (a); lower surface shows original brown coloration (b); transverse section showing histological alteration to the uppermost margin of the hair fiber (c: bar equals 10μm).

that hair color will fade due to photooxidation if left exposed in direct sunlight at the ground surface (Figure 6.2). Although the physical strength of hair is maintained by the highly crosslinked disulfide bonds in cystine, this bond is also one of the hair fiber’s most reactive sites (Sibley and Jakes 1984). It has been suggested that the cleavage of disulfide bonds increases the reaction of hair with heavy metals (Jones 2001). Researchers analyzing hair from the Karluk archaeological site in Alaska for trace elements make the distinction between the highly organic soil deposits (containing tannic acids from rotting wood), from which the hair was recovered and other frozen and dry environments in Arctic regions from which hair has also been recovered and analyzed. They caution that the Karluk samples may have thus been subject to a variety of environmental conditions that may have compromised the chemical composition of the hair strands and that use of hair from more protected environments may provide more reliable data (Egeland et al. 1999); however, this is a problem common to most buried remains.

6.4  Structural Alteration to the Hair Fiber Oxidative damage to hair, associated with weathering and cosmetic treatment, has been investigated by the cosmetics industry using diamond-cell

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Fourier transform-infrared spectroscopy (FT-IR). Oxidative damage to hair is evidenced by the intensity of the S=O band at 1040 cm-1, correspondent with the formation of cysteic acid (Brenner et al. 1985; Strassburger and Breuer 1985), and the additional presence of cysteine-S-sulfonate at 1022 cm-1. Difference in absorption spectra between ancient and modern hair at wavelengths 3,080 cm-1 and 3,259 cm-1 can be explained by desiccation of these samples (Lubec et al. 1987). FT-Raman spectroscopy has been used to examine changes in hair from various disparate depositional environments ranging from arid desert conditions to metal coffins (Edwards, Farwell, and Wilson 1998; Wilson, Janaway, and Tobin 1999), and over long curation periods (Edwards, Hassan, and Wilson 2004). Degradative change was evidenced by alteration to the amide I and III modes near wavelengths 1651 cm-1 and 1128 cm-1, respectively, and loss of definition to the (C-C) skeletal backbone. The presence of environmental contaminants derived from specific burial contexts, such as lead carbonates from lead-lined coffins and calcium carbonates from a cave environment were noted (Edwards et al. 1998; Wilson, Edwards, Farwell et al. 1999). Bulk amino acid analysis is well documented for wool and hair (Van Sande 1972; Zahn and Gattner 1997). Protein oxidation and amino acid racemization have been used to determine the extent of degradation in long-buried hair samples subject to extreme environmental conditions (Hrdy 1978; Lubec, Weninger, and Anderson 1994; Lubec et al. 1997; Macko et al. 1999). Glutamic acid, as the most abundant hair amino acid, has been used to estimate the decay and denaturation of proteins (Lubec et al. 1994). Scalp hair from a Romano-British burial from Dorchester, United Kingdom, showed extensive loss of glycine (Eastoe 1981). Similarly, slight decreases in the absolute abundances of the less stable amino acids (e.g., serine, threonine) occurred in hair from Egyptian Coptic mummies and the Iceman compared to modern hair (Macko et al. 1999). Minimal alteration of the hydrophobic amino acids (e.g., leucine, alanine, glycine, valine, isoleucine) in the Iceman’s hair may in part account for the hair’s resistance to hydrolysis during burial (Macko et al. 1999).

6.5  Keratinophilic Fungi—Their Geographic Distribution and Ecological Factors Influencing Keratinolytic Activity Despite optimistic claims in the archaeological literature that “no microbial system has been described that degrades native hair” (Lubec et al. 1987, p. 119) and that hair can therefore be recovered almost universally (Bonnichsen and Schneider 1995; Morell 1994), environmental, medical, and veterinary

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microbiology have long known that keratin can be exploited by specialized microorganisms. The literature falls in two main subject areas dealing with the action of dermatophytes (Raubitschek and Evron 1963; Tanaka et al. 1992; Vanbreuseghem 1952) (concerned in particular with microorganisms of pathogenic significance for man and animals) and composting/bioconversion (Rodale 1974) of waste keratins, for example, digestion of poultry waste (Hood and Healy 1994; Kaul and Sumbali 1997; Kornillowicz-Kowalska 1997a; Onifade et al. 1998; Shih 1993) and gelatin factory byproducts (Malviya et al. 1992; Rajak et al. 1991). Many different microorganisms may be implicated in the degradation of keratin-containing biomaterials, involving a variety of different mechanisms. Two terms, keratinolytic and keratinophilic, have been used interchangeably in the literature to describe microorganisms capable of degrading biomaterials composed of keratin. Though microorganisms may be unable to initiate hair decomposition, some are able to utilize the products of degradation brought about by keratinolytic fungi (Ulfig 1996) or chemical alteration to hair. Both types of microoganisms are of relevance to long-buried hair since hair keratin can be denatured by oxidation and hydrolysis in the depositional environment. The geographic distribution of keratinolytic fungi is widespread (Philpot 1978). The range of environments harboring keratinolytic fungi include desert soils (Al-Musallam 1988; Mubahser et al. 1992), surface waters (Ulfig and Ulfig 1990), and areas frequented by people and animals, such as home gardens, parks, and animal yards (Jain, Shukla, and Srivastava 1985; McAleer 1980). The airborne spread of keratinolytic fungi has also been reported (Filipello Marchisio, Fusconi, and Rigo 1994; Hawks and Rowe 1988). This widespread reporting of fungi able to colonize keratin substrates is hardly surprising since it has been suggested that keratinolytic activity is relatively widespread among common fungi (Friedrich et al. 1999). It is recognized that there may be two main groups of keratinolytic fungi (Kaul and Sumbali 1999). This distinction may relate to different adaptations to keratin such as pathogenic dermatophytes (Deshmukh and Agrawal 1985; Rebell and Taplin 1970; Sinski 1974; Suhonen, Dawber, and Ellis 1999) versus more generalized saprophytic keratinophytes (Deshmukh and Agrawal 1982; English 1963, 1965; Mathison 1964; Oyeka and Okoli 2003; Safranek and Goos 1982), although Kunert (1989b) suggested that there is no significant difference in the keratinolytic activity of pathogenic and saprophytic keratinolytic fungi. Keratinolytic fungi may also be further divided as to the source from which they are most commonly isolated, for example, anthropophilic (associated with humans) (Rippon 1985; Weitzman and Summerbell 1995), zoophilic (associated with animals) (Hainer 2003), or geophilic (found in soil) (Cooke and Rayner 1984; Kushwaha and Nigam 1996), although these categories will almost certainly overlap. Keratinolytic fungi have frequently been isolated from soil where they are known to colonize various

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keratin-rich substrates, degrade them and add to the mineral content of soil (Malviya et al. 1992). Though the occurrence of keratinolytic fungi in soil has generally been found to be proportional to the amount of soil organic matter, weak acid to weakly alkaline soils are optimal for keratinolytic fungi, regardless of soil organic content (Kaul and Sumbali 1999).

6.6  The Mechanism of Microbial Degradation of Hair Keratin Keratinolytic fungi have the ability to utilize cystine not only as a source of sulfur but also of carbon and nitrogen (Kunert 1988). This ability is not exclusive to keratinolytic fungi (Kunert 1989a, 1989b). However, in the course of adaptation to the degradation of proteins rich in cystine, keratinolytic microorganisms have gained the ability to regulate the ratio of both byproducts of organic sulfur oxidation: sulfate and sulfite (Kunert 1989c). Keratinolysis in fungi does not just involve proteolysis but also includes denaturation of the substrate through the cleavage of disulfide bridges by excreting sulfite. In neutral or alkaline solution, sulfite reacts with disulfides cleaving them to thiols and S-sulfocompounds (Kunert 1989a). Consider the following (Maclaren and Milligan 1981):

RSSR + SO32- D RS- + RSSO3-

The sulfitolysis of disulfide bonds is a key reaction of keratinolysis in fungi. Once keratin is denatured by sulfitolysis it can then be hydrolyzed by extracellular fungal proteases. Keratinolytic fungi release excess nitrogen from hair by deamination and ammonia excretion. In doing so, they create an alkaline environment around the mycelium accelerating sulfitolysis (Kunert 1987). A decrease in pH later in keratinolysis is caused possibly by neutralization of ammonium ions by the production of highly acidic sulfur compounds such as inorganic sulfate, thiosulfate, and S-sulfocysteine from sulfur oxidation (ibid.). Sulfate excretion is observed for all keratinolytic species with sulfur released as sulfur-containing amino acids and other intermediate products of sulfitolysis, such as sulfite and thiosulfate (Kornillowicz-Kowalska 1997b). Microorganisms have different mechanisms for exploiting keratin. The ability to attack lipids seems to be widespread among wool-colonizing fungi, and it has been suggested that this biochemical activity may be a prerequisite for the successful colonization of keratin fibers by these microorganisms (AlMusallam and Radwan 1990). Though much of the literature is concerned with the keratinolytic ability of fungi, keratinolysis by bacteria (Brady et al.

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1990; Macdiarmid and Burrell 1992) such as those used to process poultry waste (Kornillowicz-Kowalska 1997a) and actinomycetes have also been discussed (Bockle and Muller 1997; Kunert and Stransky 1988), and fibers incubated for thirty days with an actinomycete lost their luster and looked dull at a gross level (Brady et al. 1990). On detailed examination these fibers had suffered enzyme attack and structural damage. Loss of the protective cuticle exposed the underlying cortex to enzymatic attack, resulting in separation of individual cortical cells.

6.7  Histological Alteration to the Hair Shaft Limited research has been targeted at understanding degradative change in hair recovered from forensic casework (Rowe 1991; Widy and Andreas-Ludwicka 1970). Specific morphological changes to hair caused by keratinolytic fungi include surface erosion by hyphal fronds, which extend across the surface of the hair (Filipello Marchisio et al. 1994), and the radial penetration of boring hyphae into the hair shaft resulting in structures that have been variously described as invaginations (Hsu and Volz 1975) and fungal tunnels (DeGaetano, Kempton, and Rowe 1992; Rowe 1997) (Figure 6.3). Infection in which arthrospores are confined within the hair are termed endothrix, whereas clusters on the outer surface of the hair shaft are termed ectothrix (Campbell et al. 1996). Hair fibers recovered from a murder victim, buried for 3 weeks before her body was exhumed, appeared to the naked eye to be clean and free from mold growth. However, microscopic examination of the hair shaft showed that some of the hair fibers had extensive fungal damage whereas others had suffered localized fungal tunneling. Whereas the scalp hair showed evidence of localized fungal damage, pubic hair from the same individual did not, possibly because of the physical protection offered by clothing (DeGaetano et al. 1992). Postmortem changes at the hair root (proximal) end in 22 forensic hair samples derived from decomposed scalps included fibers with dark stained bands located toward the proximal end, defined as “postmortem root banding,” as well as unstained fibers with hard points or brush-like ends (Linch and Prahlow 2001). There remains little evidence as to how these different microscopic features originate, but postmortem root banding probably results from putrefaction and is not noted in older hair from archeological remains. Much of our understanding of hair degradation is derived from evidence of degradation observed in clothing and textile fibers, particularly wool (Cooke and Lomas 1990; Kempton et al. 1994; Zeronian et al. 1986) and from archeological (Janaway 2002; Sibley and Jakes 1984), or experimental studies using soil burial (Kundrat and Rowe 1989; Lasko 1983; Serowik and Rowe 1987; Wilson et al. 2003), immersion in water (Kupferschmid, Van Dyke, and

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135 a

b

Figure 6.3  Fungal damage to hair recovered from soil: scanning electron micrograph of the hair shaft, with detail, showing the eroded margins of a fungal tunnel with hyphal remnant (a: bar equals 4 μm); lactophenol blue-stained fiber whole mount shows fungal tunnels and remnant hyphae (b: bar equals 20 μm); high-resolution light micrograph longitudinal section through a fungal tunnel stained with toluidine blue shows extensive erosion and lateral movement of fungal hyphae (c: bar equals 10 μm).

Rowe 1994), or exposure to airborne fungi (Hawks and Rowe 1988). Gross visual observations of hair condition and examination of fiber whole-mounts using low-power microscopy provide an important overview of sample integrity; however, without the use of staining techniques, adherent structures such as fungal hyphae or adherent soil may not be clearly distinguished. Evidence of degradation provided by light and scanning electron microscopy (SEM) of hair from diverse burial environments include the presence of adherent deposits and brittle fractures (Hayashiba et al. 1983), an irregularly shaped cross-section profile with radiating fissures (Paterson 1981), surface pitting (ibid.), loss of cuticle scales with concomitant porosity to the fiber surface (Conti-Fuhrman and Rabino Massa 1972), and fibrillation (separation of cortical cells) (Ferguson 1992), as well as the presence of fungal mycelia and evidence of partial dissolution of keratin fibers even in some of the best preservation environments such as the grave of Petty Officer John Torrington from the ill-fated Franklin expedition, buried in permafrost (Schweger and Kerr 1987). Insect macrofauna such as Dermestid beetles (Anthrenus spp.) or Clothes moth larvae (Tineola bissiella) can exploit hair, producing

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b

Figure 6.4  Cuspate lesions to the hair shaft characteristic of insect damage to hair (bar equals 20 μm).

characteristic cuspate damage to the hair shaft (Figure 6.4) (Appleyard 1972; Robinson, Ciccotosto, and Sparrow 1993; Widy and Janiszewska 1970). A small number of studies have attempted to image the cut or broken fiber cross-section of long-buried hairs using SEM, but interpretation can be difficult. Images described as showing excellent preservation (Benfer et al. 1978) ignore characteristics that appear similar to the deformation of the internal structure (Hayashiba et al. 1983) and porosity (Chiarelli, ContiFuhrman, and Rabino Massa 1970; Conti-Fuhrman and Rabino Massa 1972; Rabino Massa 1976; Rabino Massa, Masali, and Conti-Furhman 1980) observed by others. Although the use of sectioning techniques has a long history in anthropology to assess hair form to characterize racial affinity (Trotter 1938), internal histological examination of hair fibers has not been widely adopted as a technique to assess hair sample condition. In the earliest study of the condition of long-buried hair, a single fiber from medieval deposits in Hythe, United Kingdom, was sectioned longitudinally and examined by transmission electron microscopy (TEM) (Brothwell and Spearman 1963). In subsequent studies relatively few images have been reproduced (Baez et al. 2000; Birkett, Gummer, and Dawber 1986; Hess et al. 1998). The relative paucity of published histological data reflects difficulty in deriving histological sections of hair. Yet histological assessment is regarded as one of the most informative techniques in studying archeological bone preservation with a six-point scale of histological condition now in general usage (Millard 2001; Hedges, Millard, and Pike 1995). Hair can be almost entirely destroyed by enzymatic digestion after only a month of continuous in vitro exposure to certain dermatophytes such as

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Trichophyton mentagrophytes (Guarro, Figueras, and Cano 1988). However, different fungal species have varied capacity to exploit hair. An attempt was made to classify fungi according to their ability to attack human hair using ten species of soil keratinolytic fungi and sixteen species of nonkeratinolytic fungi (Kunert 1989b), but the categories are loosely defined and need to be evaluated against the fact that keratinolytic microoganisms have been cultured and classified by both environmental and medical microbiologists for different purposes. When hair is subject to the action of consortia of unknown microorganisms, as is usually the case in forensic science, a description of the progress and extent of degradation is more useful. Various researchers have recognized that the progress of hair degradation corresponds to the level of keratinization (cystine content) of different hair structures; in other words the structural components of hair are attacked in sequence, beginning in the low sulfur regions of the cuticle and cortex (Baxter and Mann 1969; Fusconi and Filipello Marchisio 1991; Guarro et al. 1988; Kunert and Krajci 1981). This has led to the development of a scheme for the histological assessment of hair that has been fixed, embedded in resin, sectioned using an ultramicrotome, and examined using either high-resolution light microscopy or TEM (Wilson et al. 2004). These sectioning techniques provide key information because differential preservation can result in severe alteration to the interior of the hair fiber while the fiber exterior remains relatively well preserved (Wilson et al. 1999; Wilson, Dixon, Dodson, et al. 2001; Wilson, Dixon, Edwards, et al. 2001; Wilson, Janaway, et al. 2001). Differential degradation of the hair (Wilson et al. 2007) is particularly evidenced by the survival of melanin pigment granules, which are usually not damaged by keratinolytic microorganisms (Figure 6.5) (Cano, Guarro, and Figueras 1991; Guarro et al. 1988; Hsu and Volz 1975; Kanbe and Tanaka 1982). Despite their action on keratin proteins, only certain pathogenic keratinolytic fungi appear capable of exploiting hair melanin, for example, extracellular enzymes of Trichophyton mentagrophytes (Hsu and Volz 1975) and Aspergillus fumigatus (Luther and Lipke 1980) have the ability to affect hair pigment granules in contrast to the nonpathogenic fungus Trichophyton terrestre (ibid.).

6.8  Hair in Association with a Buried Body Hair is found in association with human remains that may vary in their overall state of preservation. Even under conditions generally assumed to favor tissue survival, significant changes do occur. Environments where microbial activity is restricted, such as permafrost (Beattie 1992; Hart Hansen, Meldgaard, and Nordqvist 1985; Rudenko 1970), aridity (Galloway et al. 1989), hypersalinity, waterlogged peat conditions (Brothwell and Gill-Robinson 2002; Fischer 1998), and use of metal, or metal-shell coffins (Owsley

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a

b

Co

F P

P

m

F

Cu

Co

F

Figure 6.5  Differential damage to hair: transmission electron micrograph showing cortex damage beneath a surviving cuticle (a); involving the breakdown of the intermacrofibrillar matrix and separation of individual macrofibrils (b: bar equals 1.5 μm); destruction of the keratinaceous structures of the cortex resulting in fiber collapse and aggregation of surviving melanin pigment granules. Cu,l cuticle; Co, cortex; m, macrofibrils; P, pigment granules; F, fungal structures.

1992; Owsley and Compton 1997; Rogers et al. 1997; Torre and Cardellini 1981) may ensure excellent morphological preservation. However, they are not always conducive to the preservation of other macromolecules such as lipids or DNA. Preservation is often selective and although one macromolecule may survive it does not necessarily guarantee the persistence of another (Poinar and Stankiewicz 1999). Histological condition has important implications for the preservation of forensic evidence such as DNA survival (Gilbert, Janaway, et al. 2006) and condition (Gilbert, Menez, et al. 2006). It is conceivable that any surviving hair may be partially degraded. Hair survives when either keratinolytic microorganisms are absent from the depositional environment or when the processes of microbial degradation are inhibited. An understanding of hair degradation must be based on a thorough knowledge of the interaction of human remains with the depositional environment. Although it has been suggested that the decomposition rate of a buried corpse is approximately eight times slower than above ground (Grupe 2001), the complexities of the dynamic burial environment cannot realistically permit such assertions to be made. Processes leading to preservation must be considered on a case basis. The bulk of our understanding of taphonomic processes involving human cadavers is based on observations

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Table 6.1  Comparison of Mechanisms of Keratin Degradation by a Dermatophyte Fungus with That of an Actinomycete Microsporum Gypseum (Dermatophyte Fungus) Sulfitolysis occurs simultaneously with proteolysis in lytic holes surrounding the mycelium (tunneling) Sulfur excreted as sulfate Proteolytic enzymes more active in the presence of reducing agents No thiosulfate found

Streptomyces Fradiae (Actinomycete) Keratin denatured through direct reduction of the disulfide bonds No evidence of sulfitolysis Excess nitrogen eliminated by deamination of peptides/amino acids and production of ammonia Inorganic thiosulfate formed as final product of metabolism of sulfur from cystine

Sources: Kunert and Stransky 1988; Kunert 1989a; Kunert 1992.

of tissue survival or breakdown in both archaeology and forensic casework where a number of different variables are likely to affect the decomposition of a buried body and hence influence hair survival. Field experiments are in progress to model hair degradation at contrasting sites in temperate and arid environments, replicating the conditions brought about by a decomposing cadaver using donated bodies at the Anthropological Research Facility, University of Tennessee, Knoxville and elsewhere using pigs (Sus scrofa) as human body analogs (Janaway et al. 2003; Wilson et al. 2003, 2007). During active putrefaction the burial microenvironment changes markedly with localized alteration to the soil pH, temperature, moisture content, and, hence, microbiological activity and redox conditions. Such changes vary with the soil depth profile of the grave but will initially limit the activity of keratinolytic fungi at the base of the grave relative to similar depths in pits without a body analog. Hence, over relatively short time frames hair may be found preserved in the base of a grave, whereas hair may be considerably degraded in the upper levels of the grave.

6.9  Summary and Appropriate Measures for Safeguarding Evidence In forensic taphonomy we are familiar with the concept of differential survival of biomaterials. Hair does not survive universally, suggesting that degradation or contamination will be variable in recovered hair fibers. As with many biomaterials, hair degradation occurs most rapidly immediately postdeposition. The progress of hair degradation is largely defined by variables within the depositional environment. Add to that burial environment the

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presence of a decomposing body and a unique set of parameters defines the extent of degradation in hair. All hair recovered from decomposed or partially decomposed human remains should be assumed to have undergone some alteration. Furthermore, microbial degradation that may have been inhibited during burial can be reactivated by poor packaging and storage conditions on recovery. Hence, appropriate measures for safeguarding evidence from hair following recovery from a crime scene include either immediate processing or storage under frozen conditions. Similarly, histology has an important screening role in the assessment of hair condition.

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Benfer, R. A., Typpo, J. T., Graf, V. B., and Pickett, E. E. (1978). Mineral analysis of ancient Peruvian hair. Amer. J. Phys. Anthropol. 48, 277–282. Bertolino, A. P. and O’Guin, W. M. (1994). Differentiation of the hair shaft, in Disorders of Hair Growth: Diagnosis and Treatment (E. A. Olsen, Ed.). London: McGraw-Hill, 21–37. Birkett, D. A., Gummer, C. L., and Dawber, R. P. R. (1986). Preservation of the subcellular ultra structure of ancient hair, in Science in Egyptology (R. A. David, Ed.). Manchester, UK: Manchester University Press, 367–369. Birnbaum, P. S. and Baden, H. P. (1987). Heritable disorders of hair. Dermatol. Clinics 5, 137–153. Bockle, B. and Muller, R. (1997). Reduction of disulphide bonds by Streptomyces pactum during growth on chicken feathers. Appl. Environ. Microbiol. 63, 790–792. Bonnichsen, R., Beatty, M. T., Turner, M. D., and Stoneking, M. (Eds.) (1996). What Can Be Learned from Hair? A Hair Record from the Mammoth Meadow Locus, Southwestern Montana. Oxford: Oxford Science Publications. Bonnichsen, R., Hodges, L., Ream, W., Field, K. G., Kirner, D. L., Selsor, K., et al. (2001). Methods for the study of ancient hair: Radiocarbon dates and gene sequences from individual hairs. J. Archaeol. Sci. 28, 775–785. Bonnichsen, R. and Schneider, A. L. (1995, May–June). Roots. The Sciences, 26–31. Bottoms, E., Wyatt, E., and Comaish, S. (1972). Progressive changes in cuticular pattern along the shafts of human hair as seen by scanning electron microscopy. Brit. J. Dermatol. 86, 379–384. Brady, D., Duncan, J. R., Cross, R. H. M., and Russell, A. E. (1990). Scanning electron microscopy of wool fibre degradation by Streptomyces bacteria. S. Afr. J. Anim. Sci. 20, 136–140. Brenner, L., Squires, P. L., Garry, M., and Tumosa, C. S. (1985). A measurement of human hair oxidation by Fourier transform infrared spectroscopy. J. Forensic Sci. 30, 420–426. Brothwell, D. (1986). The Bog Man and the Archaeology of People. London: British Museum Press. Brothwell, D. R. and Gill-Robinson, H. (2002). Taphonomic and forensic aspects of bog bodies, in Advances in Forensic Taphonomy: Method, Theory, and Archaeological Perspectives (W. D. Haglund and M. Sorg, Eds.). Boca Raton, FL: CRC Press, 119–132. Brothwell, D. R. and Spearman, R. (1963). The hair of earlier peoples, in Science in Archaeology (D. Brothwell and E. Higgs, Eds.). Bristol: Thames & Hudson, 427–436. Butler, J. M. and Levin, B. C. (1998). Forensic applications of mitochondrial DNA. Trends Biotechnol. 16, 158–162. Campbell, C. K., Johnson, E. M., Philpot, C. M., and Warnock, D. W. (1996). Identification of Pathogenic Fungi. London: Public Health Laboratory Service. Cano, J., Guarro, J., and Figueras, M. J. (1991). Study of the invasion of human hair in vitro by Aphanoascus spp. Mycoses 34, 145–152. Chiarelli, B., Conti Fuhrman, A. C., and Rabino Massa, E. (1970). Nota preliminare sulla ultra struttura dei capelli di mummia egiziana al microscopio elettronico a scansione. Riv. Antropol. 57, 275–278.

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150 Andrew S. Wilson Strassburger, J. and Breuer, M. M. (1985). Quantitative Fourier transform infrared spectroscopy of oxidised hair. J. Soc. Cosmetic Chem. 36, 61–74. Suhonen, R. E., Dawber, R. P. R., and Ellis, D. H. (1999). Fungal Infections of the Skin, Hair and Nails. London: Martin Dunitz. Swift, J. A. (1997). Morphology and histochemistry of human hair, in Formation and Structure of Human Hair (P. Jolles, H. Zahn, and H. Hocker, Eds.). Basel: Birkhauser Verlag, 149–175. Tam, P. (2000). They were what they ate. Wellcome News Q3, 14–15. Tanaka, S., Summerbell, R. C., Tsubop, R., Kaaman, T., Sohnle, P. G., Matsumoto, T., et al. (1992). Advances in dermatophytes and dermatophytosis. J. Med. Vet. Mycol. 30, 29–39. Taupin, J. M. (2004). Forensic hair morphology comparison—A dying art or junk science? Sci. Justice 44, 95–100. Thornton, M. J., Hamada, K., Messenger, A. G., and Randall, V. A. (1998). Androgen-dependent beard dermal papilla cells secrete autocrine growth factor(s) in response to testosterone unlike scalp cells. J. Invest. Dermatol. 111, 727–732. Toribara, T. and Muhs, A. (1984). Hair: keeper of history. Arctic Anthropol. 21, 99–108. Torre, C. and Cardellini, C. (1981). Ultrastructure of human tissues after prolonged interment in metal-lined coffins. J. Forensic Sci. 26, 710–714. Trotter, M. (1938). A review of the classifications of hair. Amer. J. Phys. Anthropol. 24, 105–126. Tsatsakis, A. M., Tzatzarakis, M. N., Psaroulis, D., Levkidis, C., and Michalodimitrakis, M. (2001). Evaluation of the addiction history of a dead woman after exhumation and sectional hair testing. Amer. J. Forensic Med. Path. 22, 73–77. Ulfig, K. (1996). Interactions between selected geophilic fungi and pathogenic dermatophytes. Rocz. Panstw. Zakl. Hig. 47, 137–142. Ulfig, K. and Ulfig, A. (1990). Keratinophilic fungi in bottom sediment of surface waters. J. Med. Vet. Mycol. 28, 419–422. Van Sande, M. (1972). Hair amino acids: Normal values and results in metabolic errors. Monographs Human Genetics 6, 157. Vanbreuseghem, R. (1952). Keratin digestion by dermatophytes: A specific diagnostic method. Mycologia 44, 176–182. Weitzman, I. and Summerbell, R. C. (1995). The dermatophytes. Clin. Microbiol. Rev. 8, 240–259. Wertz, P. W. (1997). Integral lipids of hair and stratum corneum, in Formation and Structure of Human Hair (P. Jolles, H. Zahn, and H. Hocker, Eds.). Basel: Birkhauser Verlag, 227–237. Widy, W. and Andreas-Ludwicka, B. (1970). Przyczynek do zmian obserwowanych we wlosach pobranych ze zwlok [Changes observed in the hair of corpses]. Przeg. Derm. 57, 159–161. Widy, W. and Janiszewska, M. (1970). Zmiany morfologiczne wlosa ludzkiego wywolane przez larwe mola. Przeg. Derm. 57, 297–298. Wilson, A. S. (2005). Hair as a bioresource in archaeological study, in Hair in Toxicology: An Important Biomonitor (D. J. Tobin, Ed.). Cambridge, UK: Royal Society of Chemistry, 321–345.

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Wilson, A. S., Dixon, R. A., Dodson, H. I., Janaway, R. C., Pollard, A. M., Stern, B., et al. (2001). Yesterday’s hair: human hair in archaeology. Biologist (London). 48, 213–217. Wilson, A. S., Dixon, R. A., Edwards, H. G. M., Farwell, D. W., Janaway, R. C., Pollard, A. M., et al. (2001). Toward an understanding of the interaction of hair with the depositional environment. Chungara, Rev. Antropol. Chilena 33, 293–296. Wilson, M. R., DiZinno, J. A., Polanskey, D., Replogle, J., and Budowle, B. (1995a). Validation of mitochondrial DNA sequencing for forensic casework analysis. Int. J. Legal Med. 108, 68–74. Wilson, A. S., Dodson, H. I., Janaway, R. C., Pollard, A. M., and Tobin, D. J. (2003). Survival and alteration: Experiments in hair degradation, in Mummies in a New Millenium (N. Lynnerup, C. Andreasen, and J. Berglund, Eds.). Nuuk, Greenland: Greenland National Museum and Archives, Danish Polar Center, 63–66. Wilson, A. S., Dodson, H. I., Janaway, R. C., Pollard, A. M., and Tobin, D. J. (2004). The development of a histological index for assessing the condition of hair from archaeological or forensic contexts. J. German Soc. Dermatol. 2, 515. Wilson, A. S., Dodson, H. I., Janaway, R. C., Pollard, A. M., and Tobin, D. J. Selective biodegradation in hair shafts derived from archaeological, forensic and experimental contexts. Brit. J. Dermatol. 157(3): 450–457. Wilson, A. S., Edwards, H. G. M., Farwell, D. W., and Janaway, R. C. (1999). Fourier transform Raman spectroscopy: Evaluation as a non-destructive technique for studying the degradation of human hair from archaeological and forensic environments. J. Raman Spectrosc. 30, 367–373. Wilson, M. R., Polanskey, D., Butler, J., DiZinno, J. A., Replogle, J., and Budowle, B. (1995b). Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18, 662–669. Wilson, A. S., Janaway, R. C., Holland, A. D., Dodson, H. I., Baran, E., Pollard, A. M., et al. (2007). Modelling the buried human body environment in upland climes using three contrasting field sites. Forensic Sci. Int. 169, 6–18. Wilson, A. S., Janaway, R. C., Pollard, A. M., Dixon, R. A., and Tobin, D. J. (2001). Survival of human hair: The impact of the burial environment, in Human Remains, Conservation, Retrieval and Analysis, vol. S934 (E. Williams, Ed.). Oxford: British Archaeology Reports, 119–128. Wilson, A. S., Janaway, R. C., and Tobin, D. J. (1999). Effect of the burial environment on hair shaft morphology: Relevance for archaeology and medico-legal investigations. J. Investigat. Dermatol. Symposium Proceedings 4, 353. Zahn, H. and Gattner, H. G. (1997). Hair sulphur amino acid analysis, in Formation and Structure of Human Hair (P. Jolles, H. Zahn, and H. Hocker, Eds.). Basel: Birkhaurser Verlag, 239–258. Zeronian, S. H., Alger, K. W., Ellison, M. S., and Al-Khayatt, S. M. (1986). Studying the cause and type of fibre damage in textile materials by scanning electron microscopy, in Historic Textile and Paper Materials, Conservation and Characterisation, vol. 212 (H. L. Needles and S. H. Zeronian, Eds.). Washington, DC: American Chemical Society, 77–94.

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Robert C. Janaway Contents 7.1  Introduction................................................................................................ 154 7.2  Textile Materials........................................................................................ 158 7.2.1  Natural Protein Fibers: Wool and Silk....................................... 158 7.2.2  Natural Cellulose Fibers: Cotton................................................ 160 7.2.3  Regenerated Cellulose: Viscose and Viscose Rayon................ 160 7.2.4  Synthetic Fibers: Nylon, Polyesters, Acrylics, Elastane............161 7.3  Soil as a Burial Environment................................................................... 163 7.3.1  Agents of Decomposition............................................................. 164 7.3.2  Degradation of the Body and Its Effect on Associated Materials........................................................................................ 166 7.4  Decomposition of Textiles and Leather.................................................. 166 7.4.1  Assessment of Textile Deterioration........................................... 167 7.4.2  Degradation of Textiles and Clothing........................................ 168 7.4.3  Degradation of Natural Fibers.................................................... 169 7.4.4  Degradation of Synthetic Fibers.................................................. 170 7.4.5  Degradation of Leather................................................................ 170 7.4.6  Casework Examples...................................................................... 172 7.4.6.1  Case Study: Duvet Cover in Woods........................... 172 7.4.6.2  Case Study: Woman Buried in Pantyhose.................174 7.4.6.3  Case Study: Differential Decay of Clothing on a Skeletonized Body..........................................................174 7.5  Corrosion of Metals................................................................................... 175 7.5.1  Dry Corrosion.................................................................................176 7.5.2  Aqueous Corrosion........................................................................176 7.5.3  Metal-Preserved Organics........................................................... 178 7.5.4  Casework Examples...................................................................... 179 7.5.4.1  Case Study: The Mummified Body of a Woman...... 179 7.6  Textile Degradation Experiments........................................................... 180 7.6.1  Experiments in Forensic Taphonomy........................................ 180 7.6.2  Blue Denim Textiles and Metal Zippers, Rivets, and Fasteners.........................................................................................181 153

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7.6.3 

The Effect of Cadaveric Decomposition on Differential Degradation of Textile Materials: Bradford Pig Experiments.................................................................................. 184 7.7  Summary and Conclusions...................................................................... 190 References............................................................................................................. 190 Appendices: Review of Clothing Based on Current U.K. Experience......... 195 Appendix A. Men’s Clothes............................................................................... 196 Appendix B. Women’s Clothes.......................................................................... 197 Appendix C. Household Fabrics....................................................................... 199

7.1  Introduction A buried or dumped body may be accompanied by a range of materials, including clothing and other textiles, metals such as tools and weapons, as well as plastics and paper products. This chapter concentrates on clothing and metal fastenings associated with clothing. Bodies that have been subject to clandestine disposal may be clothed, semiclothed, or naked. Reconstructing the nature and position of this clothing is critical to understanding the circumstance of disposal as well as perhaps to assisting in establishing motive and offender behavior. In addition, clothing and personal effects may provide assistance in establishing identity, although this will need confirmation by dental records or DNA. Modern clothing, footwear, and accessories are made from a range of materials: natural and synthetic textiles, leather, plastic, and metal. Along with the body they may be subject to a range of depositional environment, including surface disposal and burial in a range of soil types and microclimates. These materials will respond and degrade at different rates often leading to differential preservation. It has been recognized that the presence of clothing will have an effect on the decomposition of the body, for instance by restricting access by insects (Mant 1987; Micozzi 1991; Miller 2002), but also the decomposition of the body will modify the burial environment and will affect decomposition rates of textiles and associated materials (Janaway 1987, 2002; Wilson et al. 2007). It is first necessary to consider the response of a range of clothing materials to soil burial and then to consider the modification of the burial environment by cadaveric decomposition. In addition to the interaction of textiles, the body, and the depositional environment, they may also be in close association with corroding metal. These metal–organic material interactions can result in a phenomenon that has been termed metal preserved organics (MPOs) (Figure 7.1). Although these have been most frequently documented in the archaeological literature (Arnold, Janaway, and Keepax 1983; Biek 1963;

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(a)

(b) Figure 7.1 Archaeological textile remains preserved in iron corrosion, Macro photograph (a) with scanning electron micrograph (b) (original magnification 2000×). The iron corrosion products have formed a negative cast around the wool fibers prior to their degradation many centuries before examination. (Photo: R. C. Janaway.) (See color insert following p. 178.)

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Figure 7.2 Textiles recovered from a mass grave at Kasr-el-yahud in the Jordan valley. This was the result of an act of intercommunal violence in 614 AD. The bodies were skeletonized with surviving cotton and linen clothing. (Photo: R. C. Janaway.) (See color insert following p. 178.)

Cameron 1991; Janaway 1983; Janaway and Scott 1989; Keepax 1975), they do, however, have potential relevance to forensic investigations. In forensic contexts MPOs may relate to corroded metal dress fastenings such as jean rivets and zippers or might consist of a buried weapon that has been wrapped in cloth for burial—for instance, a steel machete wrapped in a cotton T-shirt. The survival of vulnerable evidential material is dependent on a combination of how aggressive the depositional environment is to that material and how long it has been exposed to those conditions. In some forensic cases very short depositional timescales may be the predominant factor in the recovery of vulnerable evidential material. Under longer and more aggressive conditions all but the most robust material will be lost. However, there are environmental conditions that can lead to the long-term preservation textile materials usually due to freezing, desiccation, or lack of oxygen due to waterlogging. The resultant inhibition of microbial activity can result in the preservation of textiles and other vulnerable organic material over hundreds of years (Brothwell, 1986; Janaway, 1996b) (Figure 7.2 and 7.3). Over both shorter and longer timescales buried material will decay at different rates, leading to differential preservation that reflects a combination of the material the environment and the timescale. For instance, a cotton T-shirt burial in garden topsoil may have significantly decayed in a matter of months, whereas an acrylic pullover buried with it may survive largely intact over the same

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(a)

(b) Figure 7.3 Archaeological woolen textile preserved in anoxic waterlogged conditions for approximately 1,000 years. (a) As found; (b) macro photograph of weave structure after processing. (Photo: R. C. Janaway.)

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timescale. Modern leather shoes may survive in even the most aggressive environments for years.

7.2  Textile Materials Until the mid 20th century only natural textile fibers were generally available, and most garments were made of either wool or cotton. From the 1940s onward natural fibers were more commonly partly replaced by synthetic fibers used either on their own or in mixed natural and synthetic yarn. By the late 20th century natural fiber use had dropped worldwide to approximately 35%. Despite this, cotton remains the most widely used textile fiber (Miller 1992). The second half of the 20th century saw a massive expansion not only in the use of synthetic fibers but also in natural–synthetic fiber mixtures. Natural fibers are directly derived from plant (i.e., cellulose) or animal (i.e., protein) sources. Though at the upper end of the clothing market specialist fabrics such as silk, angora, cashmere, and linen have been used, the natural fibers still used in mass clothing are sheep’s wool and cotton. During the first half of the 20th century wool cloth was still widely used for trousers, dresses, and jackets in temperate countries, which reflected its dominance as a clothing fabric stretching back into the European Middle Ages and earlier. In Europe, linen and cotton were important for undergarments, shirts, and the like, with cotton being used for the majority of clothing fabrics in warmer parts of the world (Barber 1991; Crowfoot, Pritchard, and Staniland 2006; Forster et al. 2005; Harris 1993). The introduction of synthetic yarn mixtures during the twentieth century produced cloth with better use characteristics such a longevity, crease resistance, and ease of washing. By the early 21st century, woven woolen textiles (except for specialized, up-market fabrics) were usually mixtures and had lost dominance in the popular wardrobe. Knitted textiles currently range from 100% natural (e.g., wool) to 100% synthetic (e.g., acrylic) or a mixture (e.g., 80% wool, 20% viscose). 7.2.1  Natural Protein Fibers: Wool and Silk Wool fibers are derived from sheep fleece, with 99% of the fiber composed of keratin with less than 1% being composed of fats, sterols, and lipids (Ryder and Stephenson 1968). Woolen fabrics need careful laundering, as overheating in hot water can cause accidental shrinkage and felting. This coupled with the relatively high expense of production has caused a decline in overall market share. Though wool fibers are vulnerable to strong alkalis, they are resistant to acidic conditions and are decomposed by keratinolytic microorganisms under a range of depositional environments (Mathison 1964; Safranek and Goos 1982). Wool is subject to degradation given sufficient time

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Figure 7.4  Wool fibers colonized by fungal hyphae.

under aerated conditions, with similar patterns of attack to hair (see Chapter 6). Figure 7.4 is a scanning electron microscope (SEM) micrograph of wool colonized by an active fungal colony. Under acidic, anoxic, waterlogged conditions biodegradation will be inhibited. And even under adverse conditions of shallow burial in a biologically active soil, the decay rates of keratin-based wool is slower than cellulose-based cotton (Janaway 1987; Janaway et al. 2003). For typical use of wool and synthetic mix fabrics, see Table 7.1. Silk is not a common fiber in contemporary Western textiles. Due to its labor-intensive production, it has always been used in luxury fabrics (Barber 1991; Greenhalgh 1986; Harris 1993; Warner 1921). The larvae of a number of moths produce silk, although the cultivated Mulberry silkworm (Bombyx mori) is used for commercial silk. Prior to pupation the larvae produce silk fibers from a pair of glands called spinnerets. The fibers are composed of a protein sericin. During processing, silk is sometimes weighted, meaning that it is treated with metallic salts (Anstey and Weston 1997; Stout 1960). In the past these treatments included lead, zinc, and tin, although more toxic metals such as lead are no longer used. Silk is a highly hygroscopic fiber and is generally resistant to rapid degradation. In the burial environment silk Table 7.1  Typical Use of Wool-Synthetic Mix Fabrics    Fabric Wool, acrylic Wool, Viscose Wool, Nylon

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Garment type Knit wear (pullovers etc.) Knit wear (pullovers etc.) Socks

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Table 7.2  Examples of Garments Made from Cotton and Cotton Mix Fabrics    Fabric 100% cotton Cotton Lycra Cotton Polyester

Garment type T shirts, Jeans Stretch Jeans, stretch T shirts Shirts

will be lost from aerobic soils, although it can survive for centuries under anaerobic conditions (Sibley and Jakes 1984). No specific long-term burial experiments have examined the loss of silk under damp, aerated conditions. The survival of silk from 19th-century burial contexts (Janaway 1993, 1998) may be complicated by the use of weighted silk. 7.2.2  Natural Cellulose Fibers: Cotton Today cotton has replaced wool as the worldwide predominant natural textile fiber (Farnie 1979; Kenough 1971; Rivoli 2005; Shepherd 1969). It is used as a pure fiber and in a range of cotton and synthetic mixtures. Cotton consists of 90%–99% cellulose and, as such, is highly vulnerable to decomposition. Under favorable conditions, for instance in a shallow burial in damp, aerated, and biologically active soil, undyed cotton may completely decay in a matter of months. Cotton fibers are very absorbent and swell when exposed to water. Cotton is more vulnerable to acids than alkalis. As cotton is prone to mildew attack, commercial finishes can be applied to inhibit its growth. Long-term preservation of cotton is mainly associated with desiccation or freezing; archaeological cotton textiles have been recovered from desert soils and permafrost (Barber 1991; Betts et al. 1994; Janaway 2002). For examples of garments made from cotton and cotton mix fabrics, see Table 7.2. 7.2.3  Regenerated Cellulose: Viscose and Viscose Rayon Regenerated cellulose is known as rayon in the United States and some other countries. Viscose rayon is made with purified wood pulp, which is initially broken down into alkali. Following further processing, the resultant liquid is extruded through spinnerets into a coagulating medium (Beer 1962; Hunlich 1939; Mwyer-Larson 1972). During the late 19th century chemical technologists began working on the chemical modification of natural products such as vulcanized rubber. During the 1890s and early 1900s, work on modified cellulose led to nitrocellulose fiber woven into cloth at the Paris Exposition in 1889, and the first commercial production of a semisynthetic fiber by Courthauld’s appeared in 1905 (Hardie and Pratt 1966). The fiber is produced by an extrusion process and swells in the presence of water. The

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reaction of viscose is similar to cotton, being vulnerable to most cellulose decomposers and subject to microbial attack of both the fiber and the starch size used in finishing. Being cellulose based, rayon is especially vulnerable to degradation during soil burial, especially in biologically active acid to neutral soils. Acetate is also derived from wood pulp, is treated in acetone, and then is extruded through a spinneret into a stream of warm air. In triacetate the hydroxyl groups of the cellulose molecules are completely replaced by acetyl groups, which further alter the properties of the fiber, giving it fewer cellulose characteristics. It has a higher resistance to microorganisms than viscose and has been used to impart easy-care properties to fabrics (Cowan and Jungerman 1962). 7.2.4  Synthetic Fibers: Nylon, Polyesters, Acrylics, Elastane Nylon (alternatively named polyamide) was developed in the United States by DuPont in the late 1930s, with nylon yarn being used in experimental production of stockings in 1937. During World War II it was used for the manufacture of parachutes, glider tow ropes, nets, tents, and clothing (Hardie and Pratt 1966). It is formed from a long-chain polyamide with recurring amide groups as an integral part of the polymer chain. Nylon is hydrophobic, is not attacked by insects, and is resistant to microbial attack (Cowan and Jungerman 1962). Polyester textile fibers were developed during the 1940s. After World War II Imperial Chemical Industries (ICI) bought world rights to produce this fiber under the name of Terylene in the United Kingdom, whereas DuPont purchased the rights in the United States and produced the fiber under the name Dacron. The fiber consists of long-chain synthetic polymers composed of at least 85% by weight of an ester of dihydric alcohol and terphalic acid (P-HOOC-C6H4-COOH). It is used both on its own or often in mixed yarns with either wool or cotton. Figure 7.5 is an SEM micrograph of a mixed cotton polyester yarn cloth. In recent years its low water absorbency means it is often used as the basis of technical fabrics such a fleece jackets, where it is often marketed under the trade name Polartec. It is not attacked by moth larvae and is resistant to microbial activity, although mildew may form on some sizes and starches used in finishing. Although this will not weaken the fiber, this can cause discoloration (Hardie and Pratt 1966). Acrylic yarn or acrylonitrile (vinyl cyanide, CH2=CH.CN) began being used in fiber making during the early 1950s. These fibers of long-chain synthetic polymers are composed of at least 85% by weight of acrylonitrile units and have been marketed under a number of trade names including acrilan, courtelle, and orlon (Hardie and Pratt 1966). Acrylics have low water absorbency and since the 1960s have been used for easy-to-wash and easy-to-dry

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Spot WD Mag Sig Det HV 5.0 10.3 mm 600x SE LFD 20.0 kV

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Figure 7.5 SEM micrograph of undegraded cotton polyester fabric (original magnification 600×). (Photo: R. C. Janaway.)

fabrics. They are highly resistant to chemical degradation, and the fiber is moth resistant on its own; however, in wool-acrylic mixtures moth larvae will attack through acrylic to get to the wool. Acrylics are naturally resistant to mildew and fungi. Elastane is often known under the trade names of Spandex or Lycra. Elastane is used to make highly elastic yarns (Anstey and Weeston 1997; Cowan and Jungerman 1962). Current use has been in highly flexible, close-fitting sports clothing. Despite the generic term Lycra for these garments, they are often mixed with other fibers to give better wear characteristics similar to elastain-nylon mixtures. Elastane fibers are also combined with cotton to provide garments such as underwear with some stretch. The fiber is formed from a long-chain synthetic polymer composed of at least 85% of segmented polyurethane. In 1958 the first spandex fibers were produced by DuPont. In 1959 the name LycraTM was used in commercial production starting in 1960–61. Figure 7.6 shows an undegraded knitted cotton Lycra fabric.

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100.0 µm

Figure 7.6 SEM micrograph of knitted cotton/Lycra fabric (original magnification 600×) Cotton fibers dominate the image. (Photo: R. C. Janaway.)

7.3  Soil as a Burial Environment Soil provides an aggressive environment that will promote the biodeterioration of organic materials and the corrosion of most metals. In the United Kingdom, clandestine single burials are often, but not exclusively, shallow, generally less than 50–60 cm deep. There are exceptional cases where the perpetrator has buried the body at a depth of more than 1 m within subsoils such as clay. Soils can be defined by their biological, chemical, and physical characteristics (see Chapter 1 and 11). These characteristics can influence the decomposition of clothing and cadaveric materials (see Carter and Tibbett, this volume). Experimental work with pig cadavers has shown that cadaveric decomposition can result in a significant shift of pH and redox. These can occur over short timescales as well as in areas of high, fluctuating watertables (Wilson et al. 2007). This is discussed in greater detail in the following section.

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7.3.1  Agents of Decomposition The breakdown of organic matter in soil is primarily driven by biological agents. Cellulose is the most abundant biomolecule on the planet, with a wide range of decomposer organisms in the natural environment capable of utilizing it as a source of carbon under a range of conditions (Cronyn 1990). Other naturally occurring biomolecules—such as keratin, found in hair, wool, hoof, and nail—may be broken down by a more select group of organisms (Paul and Clark 1996; Tate 2000). The ease with which synthetic polymers are broken down will be, to some extent, dependent on their chemical similarity to abundant naturally occurring biomolecules. In general, cellulose derived materials are the most easily decomposed, followed by keratin, with manmade synthetic polymers such as nylon being the most robust under a range of depositional environments. In the soil ecosystem, biological activity is influenced by the distribution of nutrients, water, and composition of soil atmosphere (including oxygen and carbon dioxide). Typically, there will be a greater level of biological activity at the surface and in the upper soil layers because of the greater availability of oxygen and food (Lawson et al. 2000). In addition, burial at depth may result in the deposited material’s being either constantly or periodically below a fluctuating water table. This can restrict oxygen availability and thus, can retard decomposition. Thus, the depth of burial will influence the decomposition of organic materials and the oxidative corrosion of metals with greater depth impeding decay. Soil biota that are principally involved in biodegradation are microbes and the mesofauna. The mesofauna comprise a large number of species that are visible to the naked eye and consume organic matter, in particular Collembola (springtails). However, their feeding patterns are complex consisting largely of partially broken down organic material, faecal pellets, and fungal mycelia. Thus, they do not necessarily have a primary role in the breakdown of buried textile materials. More significant is the role of microorganisms, specifically the fungi and bacteria. These organisms are highly destructive to buried organic material composed of cellulose, lignin, hemicellulose, keratin, and collagen. Fungi are aerobic and, thus, will be retarded by reduced oxygen in the substrate (Hudson 1980, 1986). Bacteria, however, can be classed into three groups according to their oxygen requirements: aerobes, obligate anaerobes, and facultative anaerobes (Paul and Clark, 1996; Tate 2000). Aerobes are active only in the presence of oxygen and are completely dependent on aerobic respiration as a source of energy. Obligate anaerobes depend on fermentation or anaerobic respiration as an energy source under conditions of little or no oxygen. Facultative anaerobes are active under either aerobic or anaerobic conditions. Because populations of microorganisms in any given

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environment will compete for access to nutrients the most active groups will be those that are most efficient under those conditions. For instance, aerobes will dominate aerated conditions, and specialist anaerobes will have advantage over facultative anaerobes under strongly reducing conditions. Certain biomolecules require specific groups of organisms for complete degradation. Inhibition of these organisms can lead to partial or differential preservation. For instance, the keratin in wool and hair is broken down under aerobic conditions but under anaerobic conditions can be preserved for hundreds of years (Hald 1980; Janaway 2002; Pritchard 1984; Walton 1989). The inhibition of microbial decay of organic material will occur due to a range of environmental conditions. In certain instances this can be sufficient to preserve material over long, even archaeological, timescales. In more marginal circumstances the rate and extent of decay may have been slowed down, and the condition of the material will be dependent on how long it has been at the deposition site. Waterlogged soils result in anaerobic conditions, excluding aerobes, including all fungi (Clark 1967). This will result in impeded breakdown of keratin and lignin. Thus anoxic burials tend to retain ligno-cellulosic material (e.g., wood with a high lignin content) and textiles such as wool. In burial locations with fluctuating water tables, where periods of high rainfall cover the buried materials, periodic anoxic conditions will persist. Although these conditions may not lead to organic preservation over archaeological timescales, they may retard degradation significantly in forensic cases. Most soil microorganisms are inhibited by cold and freezing conditions. Low, but not freezing, temperature will slow bacterial activity. For biodeterioration to be inhibited over longer timescales, soil conditions need to be well below freezing. In the 1980s the frozen bodies of three seamen who had been buried in the Canadian arctic were exhumed. The bodies exhibited extensive soft-tissue preservation, and the textile remains were in excellent condition (Beattie 1992; Beattie and Geiger 1987). Microorganisms require a substrate with significant moisture content. Thus, textile remains may rapidly decay in a damp, aerated soil but will not decay if the same soil is dried out. Thus, decay rates may exhibit seasonal fluctuations in some regions. Generally environmental conditions that will desiccate a substrate are hot dry or cold dry (Dzierzykray-Rogalski 1986; Hansen, Meldgaard, and Nordqvist 1991). Archaeological textile remains have frequently been recovered from high mountains such as the Andes and desert regions (Ceruti 2002). Except for the short-term burial, highly vulnerable textiles made of cotton are generally only found preserved in either frozen or desiccated conditions. Damp, aerated soils are highly vulnerable to degradation.

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7.3.2  Degradation of the Body and Its Effect on Associated Materials Cadaver decomposition is subject to two principle processes: autolysis and putrefaction (see Chapter 8). During autolysis tissue breakdown is abiotic and is due to the action of internal enzymes released through cell lysis. Putrefaction results in widespread destructive changes due to the metabolism of anaerobic bacteria. This results in the conversion of soft tissue into liquids and gases. As the soft tissues of the body putrefy, the immediate burial environment will be modified (Janaway 1987). There will be a marked increase in microbial activity, pH, and redox potential, which will influence the corrosion of metals as well as the biodeterioration of adjacent organic materials. As the tissues liquefy, the depositional environment at the base of the grave is chemically and biologically dominated by the semiliquid remains. Semidecomposed soft tissue is depleted in oxygen and is dominated by anaerobic bacterial activity (Janaway et al. 2003; Wilson 2002; Wilson et al. 2007). The rate of putrefactive change is governed by a number of interrelated factors including the initial condition of the body, time interval between death and burial, and the nature of the burial environment (Mant 1987; Micozzi 1991). Treatment of a cadaver prior to burial can have a key effect (e.g., a complete cadaver or cadaver components that have been sealed in polythene soon after death); thus, providing a closed environment will decompose at a slower rate than human remains in open bags or buried directly in the soil. Likewise, drainage can have a significant effect on decomposition (Wilson et al. 2007).

7.4  Decomposition of Textiles and Leather When buried in a moist, oxygenated soil environment, most textile materials are subject to rapid decomposition by fungi and bacteria. The vulnerability of any particular fabric is dependent on the fiber–yarn composition as well as possible effects of dyes and finishes. Modern textile fabrics such as those used in clothes are generally either made totally of natural fibers (e.g., 100% cotton), totally of synthetic fibers (e.g., 100% acrylic), or a natural–synthetic fiber mix (e.g., 50% cotton, 50% polyester). In general, natural fibers are more vulnerable to rapid decay than synthetic fabrics. Figure 7.7 is an SEM micrograph of a degraded cotton polyester fabric. Microbial activity has focused on the cotton fibers as a more accessible substrate than the polyester. Fabrics made from mixed fibers can be in two forms: (1) a mixture of natural and synthetic fibers used to form the yarn; or (2) fabric woven or knitted from yarns of distinct fibers. Although differential preservation will occur in both types, it will be most apparent in the latter,

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Spot WD Mag Sig Det HV 3.0 11.9 mm 453x SE ETD 20.0 kV

167

100.0 µm

Figure 7.7 SEM degraded cotton polyester. (Photo: R. C. Janaway.)

often accompanied by failure of the fabric into slits where the yarns in one direction remain intact whereas the more vulnerable are broken or lost. It should be noted that that even a garment labeled “100% cotton” may contain sewing thread that is a cotton-polyester mix and labels (i.e., manufacturer and care) that are also synthetic. Thus, the stitching and labels may survive in better condition than the base fabric. The rate of textile biodegradation may also be affected by the use of dyes and surface finishes. A number of synthetic dyes may act to inhibit microbial action, as demonstrated in soil burial experiments comparing the decomposition rates of cotton dyed with synthetic indigo compared with undyed cotton (Janaway2002). The study by McGrath (1999) demonstrated variable decomposition rates in various colors of denim fabrics after short-term soil burial. 7.4.1  Assessment of Textile Deterioration From a forensic point of view, our interest is usually focused on whether textile fiber remains are morphologically preserved sufficient to be identified,

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whether the loss of a garment is due to differential decay or offender actions, or whether the garments on a body will remain sufficiently intact to influence cadaver breakdown. Although tensile strength has been used in a number of industry-led textile deterioration studies (Saville 1999), it is notoriously difficult to get reproducible figures from buried textile test samples (Holland 2000). Of greater interest is whether sufficient textiles of a specific type survive in an identifiable state. Though tensile testing might have utility in assessing whether the knees of a pair of work trousers will fail after limited wear, it does not assist the crime scene investigator in determining whether the badly decomposed body of a victim was deposited wearing underwear— or whether that underwear was white 100% cotton and is likely to be differentially decomposed compared to a dyed, mixed-synthetic-fiber top. The use of a systematic condition score has generally been more useful when assessing the effect of textile decomposition following burial in, or deposition on, soil (Bell, Fowler, and Hilson 1996; Holland 2000). This can be based on staining or percent loss of fabric. The use of scanning electron microscopy has been used to document changes in fiber morphology and fungal colonization. In addition, Fourier transform infrared (FTIR) spectroscopy has been used to document degradation at a molecular level that may not be apparent either macroscopically or microscopically (Hardman 1996). 7.4.2  Degradation of Textiles and Clothing Though a number of generalizations can be made about the degradation of different fabrics under different soil conditions, the vast range of fabrics, fiber mixes, dyes, and finishes means that each specific set of textiles need to be considered in light of the specific depositional environment (see Table 7.3). For instance, a cotton–lycra stretch fabric used in the manufacture of a pair of girls’ trousers will not only stretch differently but will also degrade differently if one yarn set is cotton rich whereas the other is lycra rich compared with a fabric in which the warp and weft are made of the same cotton–lycra yarn. In the case of the former, the lycra-rich yarns will remain largely intact, with total loss of the cotton-rich yarns in places. Figure 7.8 is an SEM micrograph of degraded cotton lycra; although some cotton fibers are present, they are morphologically compromised, unlike the lycra yarns. Burial in biologically active soils has long been recognized as one of the most aggressive environments for textiles (Lloyd 1968). Though vulnerable textiles may be lost in a matter of months, more resistant fabric may differentially survive for much longer. Clearly the essential factor here is the time interval between burial by the perpetrator and excavation by the forensic team.

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Table 7.3  Textile Fabrics Classified by Vulnerability to Decomposition after Burial for 6–36 months in a Biologically Active Soil Vulnerability to Decomposition Most vulnerable Highly vulnerable Vulnerable to decay Resistant to decay

Fabrics Undyed cotton, some light dyed cotton fabrics Rayon Dyed cotton, including denim Wool Silk Cotton–polyester (depending on mix) Nylon Acrylic Polyester Elastain

7.4.3  Degradation of Natural Fibers

Spot WD Mag Sig Det HV 3.0 10.1 mm 500x SE ETD 20.0 kV

100.0 µm

Figure 7.8 SEM micrograph degraded cotton/lycra. There has been differential loss of the microbiologically vulnerable cotton compared with the more robust Lycra. (Photo: R. C. Janaway.)

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Figure 7.9  Wool from Overton Down experimental earthwork recovered after 32 years of burial in a chalk environment; inset SEM micrograph showing fungal attack. (Photo: Experimental Earthworks Committee/R. C. Janaway. With permission.) (See color insert following p. 178.)

The two most common natural textile fibers encountered in modern fabrics have contrasting responses to soil burial. Under most soil burial conditions cellulose will degrade rapidly whereas wool will decay at a slower rate. These phenomena are demonstrated by the degradation of textile fibers from the Experimental Earthworks Project (Janaway 1996a). Figures 7.9 and 7.10 compare wool and linen buried in the chalk environments at Overton Down for 32 years. The linen is denatured to the point that there is little surviving morphology, whereas the wool retained some fiber structure. 7.4.4  Degradation of Synthetic Fibers Synthetic fibers have been characterized by a resistance to degradation over forensically relevant timescales (Table 7.4). Nylon (polyamide), polyester, and acrylic fibers show considerable resistance to soil burial. Regenerated cellulose fibers (rayon viscose), however, share the vulnerability of natural cellulose to decomposition (Rowe 1997). However, they do show a higher degree of resistance to biodegradation compared with natural fibers or regenerated cellulose, with the exception of triacetate. 7.4.5  Degradation of Leather Leather items that are most likely to be recovered from a crime scene include footwear and belts. Modern tanned leather is highly resistant to decay. In the

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Figure 7.10 Linen from Overton Down experimental earthwork recovered after 32 years of burial in a chalk environment. (Photo: Experimental Earthworks Committee/R. C. Janaway. With permission.) (See color insert following p. 178.)

Table 7.4  Deterioration of Synthetic Fibers Buried in Well-Watered Soil, after Rowe (49) Fiber Type Rayon Acetate Triacetate Nylon Polyester Acrylic

No Deterioration

Slight Deterioration

Definite Deterioration

After 2 months After 2 months

After 4 months After 3 months

After 9 months After 9 months After 9 months After 9 months

Source: Rowe, W. F. (1997). Biodeterioration of hairs and fibers, in Forensic Taphonomy: The Postmortem Fate of Human Remains (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press

case of a 3-year-old boy covered with stones from a wall in Yorkshire, U.K., for 26 years, the only surviving clothing was his sandals (Hunter and Dockrill 1996). The robust nature of tanned leather buried for nearly 100 years is amply illustrated by recent excavations of burials from the Western Front of the First World War, where bodies had completely skeletonized and the rest of the nonmetallic uniform items had decayed, but the boots remained recognizable on the feet. Experimental samples of tanned leather from the Overton Down Experimental Earthwork (Bell et al. 1996) buried for 32 years

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Figure 7.11 Chrome tanned leather from Overton Down experimental earthwork after 32 years of burial in a chalk environment. (Photo: Experimental Earthworks Committee/R. C. Janaway. With permission.) (See color insert following p. 178.)

were recovered in excellent condition. Figure 7.11 shows a sample of chrome tanned leather after burial for 32 years in the chalk bank of the Overton Down Experimental Earthwork. Modern footwear is rarely constructed of 100% leather, with nailed and stitched leather soles as was common in the past. A range of synthetic materials are used for the soles, along with synthetic textile liners. In cheaper footwear, plastic leather substitutes are also used. Whether made of leather or synthetics, footwear represents one of the most durable clothing elements liable to be recovered on a body. One factor of an intact shoe that encloses the foot is that it is likely to retain the small bones of the foot and remnants of the sock even in a skeletonized individual. This is the case even when a boot has become detached from the rest of the body. Figure 7.12 shows a detached boot recovered from waterlogged soils in Flanders dating to World War I. The leather is in good condition, and the waterlogged conditions have also preserved the woolen sock. The ferrous hobnails on the sole have extensively corroded, and the bones of the foot remain inside the sock. 7.4.6  Casework Examples 7.4.6.1  Case Study: Duvet Cover in Woods The body of a teenage girl was discovered in coniferous woods in the north of England. It had been wrapped in multiple large plastic bags that had

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Figure 7.12  World War I leather boot and woolen sock from waterlogged trench in Hooge, Flanders. (Photo: R. C. Janaway.) (See color insert following p. 178.)

originally been individually sealed and then contained in a cotton–polyester duvet cover. The body bundle was adjacent to a shallow grave and partially covered by branches. The bags had been ripped open shortly before being reported to the police, allowing body decomposition products to run onto parts of the duvet cover. Subsequently, a man was arrested and during the interview admitted to disposing of the body in the woods. According to the suspect’s statements during the interview, the duvet cover (which was far from new) was placed around the body from the outset, then it was placed in the mud and water in the back garden of his house. It was left there for 2 to 3 weeks, with the body and duvet cover subsequently being buried in the soil and pine needles in the woods. According to the suspect it had remained there for 8 months (although the police have continued to suspect that this time frame is incorrect and another form of storage was used). Examination of the duvet was carried out to question whether the degree of soiling and degradation is consistent with the suspect’s statement. There is only limited damage to the fabric, despite allegedly being exposed in the wood for 8 months. Soiling by body decomposition products was likely to be associated with disturbance of the body, whereas the degree of soiling of the fabric from contact with soil was not consistent with the stated time frames. Degradation of the cotton component of the textile was apparent but not

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conclusive either way. During the subsequent trial, other evidence was put forward that strongly suggested the body had been stored elsewhere for part of the 8 months. 7.4.6.2  Case Study: Woman Buried in Pantyhose The body of a woman who had been buried for 16 years was excavated from a back garden in northern England. The grave site was concealed under a concrete terrace. The grave was 0.8 m deep and cut into the clay subsoil, with the base of the grave was filled with glutinous wet clay. When excavated, the soft tissues were adipose; no other clothing had been present or survived, but her nylon pantyhose were intact and holding the lower part of her body together. 7.4.6.3  Case Study: Differential Decay of Clothing on a Skeletonized Body The excavation of a clandestine grave had revealed the largely skeletonized remains of a young man who had been buried for 5 years in a biologically active soil. The subsoil was clay, with the grave cut being water-filled at the time of the excavation due to a fractured field drain. It was covered by a stack of horse manure used as agricultural fertilizer that had considerably modified the burial conditions. The body had been buried clothed, and items of textile were recovered with the human remains. These included cloth, metal, and leather that had been subject to considerable differential preservation. A leather belt was still in place on the body and was fastened with a metal buckle and keeper. Both of these were cast in a nonferrous alloy and were originally plated with a copper alloy. The leather was in good condition, being intact but lacking flexibility due to burial. The metal had corroded to form a corrosion crust that was consistent with burial duration and conditions. The shoes were intact, consisting of leather uppers, synthetic sole and containing the remains of dyed cotton socks and the small bones of the foot. A mass of degraded cloth was presented for examination. This comprised sewing thread, pocket linings, and garment labels. The cotton fabric—confirmed by analysis and from the manufacturer’s labels—was in a highly degraded and fragmentary condition. The condition of these remains is consistent with largely cellulose-based (i.e., cotton) garments being buried in an acidic, biologically active burial environment. The sewing thread, which is largely synthetic, showed more resistance to degradation. Garment labels giving detail of the maker, size, and laundry instructions were all well preserved compared with the base fabric. Recovered with the textiles was a number of corroded jeans-style reinforcing rivets, metal stud buttons, and plastic buttons. Remains of the jeansstyle trousers and jacket also showed differential preservation, including cotton–polyester pocket linings, stitching, and two types of highly degraded

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blue-dyed cotton fabrics. From analysis of the fabrics, labels, and stitching it was possible to identify the remains of blue-dyed cotton jeans-style trousers, matching jacket, and cotton–synthetic fiber-mix shirt worn underneath. The loose plastic buttons that were recovered matched this garment. There was no evidence of underpants either in terms of fabric remains or maker’s label. Though it is possible that undyed cotton underwear may not have survived burial, all the other garment fabric labels had survived. It is probable that the deceased was not wearing underwear when buried. Better preserved than the other textile items were the remains of a machine knitted pullover made from a cotton–acrylic mix. As would be expected, the cotton component of the yarn was preserved in worse condition than the acrylic component. The appearance and origins of fragmentary garments such as the jacket and jeans were traceable through the manufacturer’s product codes on the clothing labels.

7.5  Corrosion of Metals The rate of metallic corrosion depends on a number of different factors, including the composition and structure of the metal artefact, the chemical nature of the burial environment, and the interval of burial. Metals can be divided into three groups according to their susceptibility to corrosion: (1) corrosion resistant metals (e.g., gold; surgical steel used in body piecing studs); (2) metals that after initial rapid corrosion form a layer of stable corrosion products and, thus, become resistant to further attack (in most burial environment these will have an extensive metallic core even after burial for hundreds of years, such as copper); or (3) metals that corrode rapidly but do not form a layer of protective corrosion products. In aggressive environments over long timescales these may be either totally lost from the burial environment or characterized by a mass of corrosion that may cover a much reduced metallic core (e.g., iron). The stability of buried metals largely depends on a combination of pH and redox (Edwards 1996). Under high redox values (oxidizing conditions) most metals will easily corrode, whereas under low redox values (reducing conditions) they will tend to remain as uncorroded metal. In addition, acidic conditions (low pH) will assist corrosion, whereas alkaline conditions will tend result in the formation of a stable corrosion matrix in most metals. Thus, in a well-drained, acidic sand or gravel site, all metals except the most inert (e.g., gold) will corrode rapidly and extensively. However, under most other burial conditions, most metal will be capable of recovery, albeit in a corroded state even after many centuries.

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176

7.5.1  Dry Corrosion Since nearly all environments that involve burial or deposition on soil–vegetation surfaces involve some water, dry corrosion is usually superseded by aqueous corrosion. However, many metal objects will have undergone dry corrosion prior to deposition. When a freshly polished, bright metal is left exposed to a dry atmosphere, it may become dull and tarnished. For instance, a new copper alloy coin will form a layer largely composed of red–brown copper (I) oxide, cuprite (Cu2O). 7.5.2  Aqueous Corrosion Metals are subject to electrochemical corrosion in the presence of water: Metal atoms lose electrons to become positively charged metal ions that go into solution. These then react with other chemical species in the soil groundwater to form solid corrosion products (e.g., metal oxides, hydroxides, sulfates). It is these solid corrosion products that often form a colored matrix with soil particles around the corroding object (Cronyn 1990). The initial formation of the metal ions takes place at a site on the metal known as the anode, whereas the electrons produced consumed by another reaction with an electron acceptor (the cathode). Due to the electrical conductivity of metals the location of the anode and cathode can be at different locations on the metal surface. In the presence of water and oxygen the cathodic reaction is

O2 + 2H2O + 4e- → 4(OH)-

Where there are depleted oxygen levels, hydrogen ions act as the electron acceptors:

2H+ + 2e- → H2

In the absence of oxygen, unless there is an abundance of hydrogen ions (e.g., in an acidic environment of pH 4 or below), corrosion rates are generally slow. This is because the reaction at the cathode determines corrosion rate. In addition to the metal itself, metallic corrosion is largely influenced by two key environmental parameters: redox potential and pH. These will determine whether the metal ions form and, if they do form, whether they remain in solution and are dissipated away from the metal surface or form stable corrosion films over the surface. Where the ions do not form is termed immunity. Where ions dissipate and the metal continues to corrode is termed corrosion. Where stable films are formed, preventing further corrosion, is termed passivation. Marcel Pourbaix (1966) developed a series of equilibrium potential pH diagrams that predict the likelihood of corrosion based on thermodynamic

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stability. Figure 7.13 is a simplified version of an iron/water Pourbiax diagram (Edwards 1996). This predicts that at low redox potentials metallic iron (Fe) will be the stable form (i.e., immunity). At higher redox potentials that are 2 Fe3+

EH(V)

1 Fe2O3 Fe2+

0

–1

Fe3O4

Fe

7

0

14

pH

(a)

2

EH(V)

1 Passivation

Corrosion 0

Immunity

–1

0

7 pH

14

(b) Figure 7.13 Simplified Pourbaix diagram for iron/water.

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acidic, ferrous and ferric ions will be the stable forms (Fe2+ and Fe3+: corrosion), whereas at higher redox, but more alkaline conditions, this will result in the formation of haematite Fe2O3 or magnetite Fe3O4: passivation). Due to the importance of pH and redox, the influence of cadaveric decomposition is critical. Where decomposition lowers redox and raises pH, for instance in the base of the grave, corrosion of most metals will be inhibited during the active phase of the decomposition. 7.5.3  Metal-Preserved Organics In most depositional and burial environments organic materials will be subject to degradation over comparatively short time frames. For instance, plain cotton textiles may be lost from a well-aerated soil in a matter of months. The interaction of corroding metal in association with vulnerable organic materials has long been recognized by scientists working with archaeological material (Janaway and Scott 1989) whereas the preservation of organic structures within a corrosion matrix was observed by Faraday (1836). The bulk of the research into these mechanisms was conducted during the mid to late twentieth century (Arnold et al. 1983; Biek 1963; Cameron 1991; Cronyn 1990; Janaway 1983; Janaway and Scott 1989; Keepax 1975). In the case of metals that have a biocidal action, such as copper, electrochemical corrosion results in metal ion solubilization and highly concentrated deposition in the organic material in contact with the metal surface (Janaway 1983). Clearly, burial conditions have to be such that sufficient metal ions need to be transported to the organic material (a function of pH and redox), and some organic structures will capture metal ions more readily than others. For instance, cotton yarns will readily take up metal ions in solution. The result is that a small area of organic material will have sufficient metal ions to inhibit microbial activity and that small traces of the organic material can be preserved over long timescales (hundreds of years). Typical archaeological examples consist of copper alloy brooches that have been pinned through clothing. Forensic examples could include copper bracelets worn on the wrist. Analyses of MPOs associated with copper alloy using FTIR (Gillard et al. 1994) indicate that substantially intact organic structures can be preserved over archaeological timescales. Ferrous corrosion products do not have a biocidal effect; however, iron corrosion tends to form a much more extensive and encapsulating matrix. This corrosion matrix, which is principally formed of iron oxides, hydroxides, and soil minerals, will encapsulate any organic materials. This usually takes the form of a negative cast (Keepax 1975), provided that the primary layer of corrosion product has been laid down prior to any extensive degradation of the organic material, then fine surface detail will be preserved in the corrosion cast. Because the iron corrosion will not inhibit degradation

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Figure 7.14  World War I British brass buttons and webbing fittings from plowed soil in Somme, France. (Photo: D. Charlton. With permission.) (See color insert following p. 178.)

of the organic material, all that is usually left over longer timescales is the hollow casts. Generally, the most detailed corrosion preserved organic structures associated with ferrous artefacts are derived from materials that are more resistant to decay in aerated, neutral-to-acid burial conditions. Clearly, if the organic material has lost morphological definition before sufficient corrosion matrix has been deposited no latent structure will be recognizable. Figure 7.14 shows a corroded brass button and webbing belt fittings excavated from plowed soil over chalk in the Somme region of France. These have been buried since the First World War. The brass, while retaining a substantial metallic core, has developed a corrosion matrix that partly encapsulates textile information. The cotton webbing belt has entirely degraded, except within the strap end. 7.5.4  Casework Examples 7.5.4.1  Case Study: The Mummified Body of a Woman The mummified body of woman was found dumped in the grounds of a house in a town in northern England. The presence of loft insulation fibers, along with the mummification, suggested that it had been stored for a number of years in the attic of a house. It had probably been found shortly after being dumped outside. On the woman’s wrist was a digital watch; the steel back had

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corroded, and ferrous corrosion products were adhering to the outer casing. Within the corrosion were fragments of a cotton–polyester shirt. Over the remains of the fragmentary shirt was an acrylic sweater; this was much more intact. Some acrylic fibers were also encapsulated within the metal corrosion.

7.6  Textile Degradation Experiments 7.6.1  Experiments in Forensic Taphonomy Because of the importance of the gut microflora in putrefactive change (Garland and Janaway 1989; Hopkins, Wiltshire, and Turner 2000), most experiments in forensic taphonomy have used complete cadavers. Although very useful preliminary work has been done using isolated tissue, incubated under laboratory conditions (Aturaliya and Lukasewycz 1999), the bulk of experimental work has been conducted using pigs as human body analogs. Buried pigs have been used as geophysics targets (Bray 1996; France et al. 1992), to investigate the effect of maggot masses in the movement of clothing (Komar and Beattie 1998) and have been extensively used in other entomological experiments (Goff and Odom 1987; Goff, Omori, and Gunatilake 1988; Lopes de Carvalho and Linhares 2001; Payne 1965). Haskell (2000) experimentally compared the insect activity on animal and human carcasses. Pigs have been used as human cadaver analogues for soft-tissue studies, as they have comparable body mass and skin structure and a similar fat-to-muscle ratio (Schoenly, Griest, and Rhine 1991). Their fat composition is similar, but not identical, to humans, having four times as much stearic acid (Gunstone 1967). Though other animals have been used, such as dogs and rats, these suffer from having large surface-area-to-volume ratios and small body masses, which provide poor analogs for adult human bodies, and having very different skin structures. Currently pigs remain the most popular human cadaver analogs used in forensic experiments. Worldwide, the use of human subjects for taphonomic work has been confined to review of case files (Galloway et al. 1989) and work at the Anthropological Research Facility of the University of Tennessee–Knoxville (Mann, Bass, and Meadows 1990). This facility remains unique in its program of long-term research using donated human cadavers. It was established in 1972 and has underpinned key research into cadaveric decomposition (Bass and Jefferson 2003; Mann et al. 1990; Rodriguez and Bass 1983, 1985; Vass et al. 1992). However, though the use of human cadavers overcomes problems of interspecies comparisons, the work is not without difficulty—not least because donated bodies do not arrive at the facility to order, making subjects capable of replication at a premium. In addition, the donated cadavers are individuals who are considerably older than many victims of violent

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crime. A further difficulty rests in the unique nature of the facility at a single location within the United States. This difficulty should be somewhat alleviated with the advent of the Donated Body Program at the University of California–Davis. However, to assess the effect of different climates, insect populations, and burial conditions, the use of animal analogs are necessary throughout the United States and the rest of the world. 7.6.2  Blue Denim Textiles and Metal Zippers, Rivets, and Fasteners Denim-based clothing, jeans trousers and jackets, are characterized by some common features. Though the denim fabric itself can vary, they tend to have riveted stud buttons, reinforcing rivets, and leather or synthetic leather manufacture labels. As was demonstrated in the case study in Section 7.4.6.3, though the cotton fabric can decay quite extensively in aggressive depositional environments zippers, buttons, rivets, and so forth can be much more durable. This durability of the zippers and fastenings compared with synthetic, indigo-blue-dyed, cotton denim fabric is illustrated by a simple set of experiments (Tigg 2005). 30 cm × 30 cm square commercial blue denim fabric had sections of brass and aluminum zippers sewn on using polyester thread. In addition, nickel-coated brass and plain brass rivets and stud buttons were also attached. These samples were exposed to three contrasting depositional environments for 15 weeks. Location 1 consisted of an agricultural small holding with topsoil pH 6 and yellow clay subsoil at a depth of 40 cm. Location 2 consisted of well-tilled garden topsoil, pH 5–6. Location 3 was the surface underneath a conifer hedge. This was very dry, as the leaf canopy prevented direct precipitation and was covered by shed needles. The results are summarized in Table 7.5 and are illustrated in Figures 7.15–7.18. Although there was limited corrosion of the brass metal, there was not sufficient mobilization of copper ions to protect the cotton denim from significant biodegradation. The aluminum did not exhibit significant corrosion. There was considerable variation in degradation of the denim between locations. At Location 1 the denim fabric showed degradation at both 30 cm and 60 cm depth, whereas burial within the clay layer at 60 cm (Figure 7.15) retarded degradation relative to 30 cm at the same location (Figure 7.16). The turnover of cotton textile was more advanced from Location 2 (the garden soil). In this instance no denim remained, with only the polyester webbing of the zippers surviving (Figure 7.17). The surface under the conifer hedge provided a very specific set of conditions that severely retarded cotton decomposition. The key factor here is desiccation (Figure 7.18).

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Table 7.5  Results Summary Table Blue Denim, Zippers and Studs Depth Location 1

30 cm

Horizon/ Soil Type

Denim

A Humic topsoil Humic topsoil

Degraded

60 cm

C Orange clay

Severely degraded

Location 2

30 cm

Almost total loss

Location 3

0 cm

A Humic topsoil Shed conifer needles

Degraded

Negligible decay

Brass Zipper Extensive surface oxidation Extensive surface oxidation Extensive surface oxidation Extensive surface oxidation Negligible corrosion

Nickel Plating Some loss of nickel plating Some loss of nickel plating Nickel plating almost entirely lost Some loss of Nickel plating Negligible corrosion

Aluminum Zipper No significant corrosion No significant corrosion No significant corrosion No significant corrosion Negligible corrosion

Figure 7.15  Denim test fabric buried at 60 cm in clay at location 1. (Photo: D. Charlton. With permission.) (See color insert following p. 178.)

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Figure 7.16  Denim test fabric buried at 30 cm at location 1. (Photo: D. Charlton. With permission.) (See color insert following p. 178.)

Figure 7.17  Denim test fabric buried at 30 cm in garden soil location 2. (Photo: D. Charlton. With permission.) (See color insert following p. 178.)

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Figure 7.18  Denim test fabric laid on soil surface under conifer hedge. (Photo: D. Charlton. With permission.) (See color insert following p. 178.)

7.6.3  The Effect of Cadaveric Decomposition on Differential Degradation of Textile Materials: Bradford Pig Experiments A series of experiments were set up in West Yorkshire, U.K., to test the relationships among the decomposition of buried hair, textiles, metal, and cadavers (pig) under a range of conditions relevant to regional depositional environments (Holland 2000; Wilson et al. 2007). Replicated cadaver and control graves (i.e., graves with experimental materials but without cadavers) were dug and exhumed after 6, 12, and 24 months. Three experimental sites were used: pasture, moorland, and woodland. These were chosen to complement each other in terms of altitude, soils, and drainage (Wilson 2002). The moorland field site is at 430 m above sea level with peat soils overlying boulder clay/weathered Millstone grit. It is characterized by a high and fluctuating water table. The soils at this location are reducing and wet, with the upper layers becoming more aerated during drier periods. Measurements of the water table on the site indicated that the bases of the graves were covered for most of the year, resulting in slow decomposition rates with an average soil pH of 4.2.

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The woodland field site is in deciduous woodland at an elevation of 140 m above sea level, with brown earth soil over Millstone grit. The site is much freer draining than the moorland site; the soils are much more biologically active, with an average soil pH of 4.6. Preliminary experiments using cotton test strips in plain soil burial demonstrated a much more rapid turnover at the woodland site compared to the moorland site (Wilson 2002). The pasture field site is under pasture at 220 m above sea level, with brown earth soil and ironwork waste resulting in an average soil pH of 7.3. The site is free draining. At the pasture site, an early study utilized two pig cadavers (A and B) with a mass of ~50 kg and a length of 150 cm. Cadavers were buried in two graves (175 cm × 75 cm × 60cm). Between these graves was a control grave (no cadaver) of similar dimensions. Cadaver A had died 3 days before cadaver B and had been left lying on its side in the concrete yard. Cadaver A had a distended abdomen and had begun to purge fluid through its mouth prior to burial. From case experience it was acknowledged that cadaver A would decompose at a faster rate than cadaver B because it was exposed to a longer interval between death and burial. In addition, cadaver A was subject to limited fox scavenging just after burial. This caused damage to the forelimbs and opened up the grave. Although the grave was reinstated before further damage could occur, it was predicted that this would further increase the decomposition rate of this cadaver A. When the graves were excavated (24 months later), cadaver A was much more skeletonized than B, which exhibited extensive adipocere (Janaway et al. 2003; Wilson 2002). Grave-soil carbon dioxide respiration was measured after exhumation. This confirmed greater microbial activity in the cadaver graves relative to control graves (Wilson et al. 2007). However, this also confirmed that cadaver B was much more actively putrefying than cadaver A, which nonetheless showed enhanced activity over the control. The base of each pig grave showed higher microbial activity levels than the top of each grave. Further experiments were conducted with paired cadaver and control graves at moorland and woodland sites: To date, these have been excavated at 6- and 12-month intervals, with an as yet undefined long-term interval that is still ongoing. The methodology had been refined by these experiments, not least in the successful exclusion of foxes and other scavengers. At the moorland site, which was known to be a wet site, piezometer pipes were installed to measure fluctuations in the water table across the site, and both manual and automated temperature recording was installed (Wilson et al. 2007). The pasture site cadavers, which were in a much more free-draining soil, had decayed to a greater extent than the Oxenhope cadavers. Piezometer readings at the moorland site had confirmed variable values across the site; however, during the wet periods at the start of the experiment the water table was above the base of the graves but had dropped below for the summer months

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and by October had risen again to above the level of the cadavers (Wilson et al. 2007). The active decomposition of the cadavers modified the immediate burial environment causing shifts in pH and redox. The pH shifts were easily documented with a mean soil pH of 4.2 at moorland site rising to 6.3 after 12 months’ burial and a corresponding shift of 4.6 to 7.2 at the pasture site. These data are similar to rises of soil pH associated with decomposing pig cadavers buried in a clay soil during an experimental project in Hertfordshire. In the Anthropological Research facility in Knoxville, Tennessee, soil pH has been observed to increase in the early and intermediate stages of cadaveric decomposition, with a corresponding drop in pH as the fermentation of soft-tissue ceases (Rodriguez and Bass 1985; Vass et al. 1992). Though it has been possible to measure redox directly in gravesoil, redox probes have proved of variable reliability and were not used in these early experiments. Thus, grave-soil redox shift due to cadaveric decomposition was more difficult to quantify. Although simplistic, polished-copper billets placed under cadavers that have remained in a reduced condition (exhibiting immunity in terms of electrochemical corrosion) were in accordance with the Pourbaix diagram for the activity of copper in water (Figure 7.19) at the measured pH ranges indicated (Holland 2000; Janaway 1987). In an initial study at the pasture site, replicate samples of dyed polyester, undyed wool, undyed cotton, and synthetic indigo-dyed denim were placed above (30 cm depth) and below (60 cm depth) cadavers and at 30 cm and 60 cm depth in control graves. When exhumed at 24 months, cadaver B was still actively decomposing with extensive adipocere formation at the base of the grave. This was confirmed by determination of carbon dioxide respiration rates for soil samples taken at the time of recovery (Wilson 2002). It was clear that decomposition of the cadaver had influenced the decomposition rates of the textiles depending on location in the grave after 24 months of burial (Table 7.6). The results from these experiments are consistent with a number of principles. During active decomposition the pig cadaver had modified the burial environment sufficiently to affect the preservation of vulnerable textile materials. During this time period the synthetic polyester was resistant to decay, resulting in no major loss of the fabric. The natural fibers were highly vulnerable to decay in this well-drained, nonacidic, biologically active soil, with total loss of all textiles from the control grave. The undyed cotton was the most vulnerable to turnover by soil microorganisms. However, a small amount of material did survive under the reducing conditions formed under the pig at the base of the grave. The blue denim fabric was heavier, and the dye afforded some protection to the fabric resulting in good preservation under the pig and some preservation over the pig. A similar pattern was observed with the wool.

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2

CuO

1

CuO22

–

EH(V)

Cu2+ Cu2O 0

Cu

–1

0

7 pH

14

Passivation

2

1

Corrosion

(a)

EH(V)

Corrosion 0

Immunity

–1

0

7 pH

14



(b) Figure 7.19 Simplified Pourbaix diagram for copper/water.

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Table 7.6  Condition of Textile Samples after 24 Months’ Burial at the Pasture Site Sample Position

Dyed Polyester

Undyed Wool

Pig B

Above 30 cm

No discernible loss

Total loss

Pig B

Below 60 cm

No discernible loss

Control

Above 30 cm

60–90% loss of fabric area Total loss

Up to 60% loss of fabric area No discernible loss Total loss

Control

Below 60 cm

No discernible loss No discernible loss

60–90% loss of fabric area No discernible loss Total loss Total loss

Total loss

Total loss

Undyed Cotton

Indigo dyed Denim

When similar experiments were repeated at the moorland site using undyed cotton, blue-dyed denim, and undyed wool fabric, similar results were obtained. At the moorland site the underlying rock is Millstone grit, overlaid with boulder clay. This is overlaid by peat 0.4–1.2 m depth with surface vegetation of moorland grass and rush. There was a high, fluctuating water table with soil pH ranging from 3.3 to 3.7. Graves (1.8 m × 1 m × 0.6 m) were dug, allowing roughly 0.30 m soil depth above each cadaver. A control grave (no cadaver) was dug for each cadaver grave. Two cadaver and two control graves were examined after 12 months. Water levels across the site were measured using eight piezometers. Analysis of the piezometer records indicated variable values across the site. However, some general trends were indicated. During wet periods at the start of the experiment, the water levels were above the bases of the graves but dropped below the bases during the dry summer period. By October, levels had risen above the level of the pig. Soil temperatures within the grave decreased slightly with depth. Five textile fabrics (i.e., undyed wool, commercial blue-dyed denim, heavy undyed cotton, medium undyed cotton, and light undyed cotton) were included in each grave. The textile samples were cut into 3 cm × 15 cm strips and sewn into nylon net bags to assist recovery. The mesh size (2 mm) was sufficient to allow access to all but the largest fauna. Nylon was used because it would not significantly deteriorate over the timescale of the experiment. Sets of textiles were buried in replicates of three above and below the pig and at the same levels in the control grave (30 cm and 60 cm), allowing differentiation between decay rates due to the presence of the pig cadavers from those that are purely a function of depth. After 12 months of burial, two pigs and two controls were excavated. The bones of feet and forelimbs were starting to separate, and the skin was weak, although the rest of the cadaver was intact. When the body was exhumed, it was capable of being turned onto a

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Decomposition of Materials Associated with Buried Cadavers Best Preserved Wool

Denim

189

Least Preserved Heavy Cotton

Medium Cotton Light Cotton

Figure 7.20 General trends for textile preservation from cadaver and control graves.

body sheet in the grave. In a more advanced stage of decomposition, such handling would have caused the body to break apart. The fat had degraded into a soft, wet material. At the moorland site the acidic, semiwaterlogged soils resulted in very slow turnover of textile samples compared with better-drained, more biologically active soils such as those at the woodland site. Nonetheless, the area below the cadavers exhumed after 12 months retarded decomposition, although this was exacerbated by the base of the control graves being below the water table for part of the experiment. Wool is the most resistant to the acidic burial conditions, being best preserved over a range of conditions. The denim was again better preserved than the undyed cotton, which gave the most sensitive response to the other variables tested. In the control graves comparable textiles were better preserved at 60 cm than at 30 cm. The general trends for the condition of a range of recovered textiles are presented in Figure 7.20. Wool is resistant to wet, acidic conditions. However, body fluids raise the pH sufficiently to effect its preservation such that even though the appearance is unchanged, tensile strength is decreased. The better preservation of denim over other cotton textiles is consistent with work on the inhibition of cotton decay by synthetic indigo. The undyed cotton gives the most sensitive response to the other variables tested in this experiment. In the control graves comparable textiles are better preserved at 60 cm than 30 cm. Although depth of burial generally follows this trend, at this site, the relatively longer periods during which the base of the graves was below the water table is thought to be significant. The decay of the cadaver raised the pH, lowered redox values (especially underneath), and affected the degradation rate of vulnerable textiles underneath the body, at least while active decomposition is taking place. Equivalent control textiles are more degraded than those with the pig, whereas those below are better preserved than those from on top. This reflects a greater turnover of vulnerable textile in the soil, as opposed to buried with a body under comparable circumstances. Though variation in decomposition rates with depth is expected in the case of the moorland site, this is exacerbated by the water table covering the base of the grave for significant parts of the year. Within 12 months, soft-tissue decomposition had a marked effect on the degradation of associated textiles, as cadaver decomposition products

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dominate and modify the immediate burial environment. Although textiles from the base of the control graves were in good condition, the textiles from underneath the cadaver were in the best condition. The decomposition of cadavers and textiles at the moorland site was much less advanced that at the other experimental sites. These data generally support trends seen from archaeological finds and forensic cases in comparable upland peat environments. Importantly, in a forensic context, the inhibition of biodeterioration of both cadaver and associated clothing needs to be considered when ascertaining postmortem interval.

7.7  Summary and Conclusions The investigations of modern clandestine graves have the potential to recover a wide range of textiles and other materials. These will have a range of responses to depositional environments, resulting in differential preservation and loss. The experiment with denim test cloth (Section 7.6.2) demonstrates that even a single cloth type exposed over a short time frame (fifteen weeks) can have significantly different decomposition rates. However, because modern garments, even those of environmentally vulnerable undyed cotton, are often made with polyester thread (case study in Section 7.4.6.3), evidence from missing garments can be recovered. Corroding metal artifacts in contact with textiles have the potential to preserve evidence of degraded textiles. Though these MPOs are more often documented in the archaeological literature, it can have relevance to the investigation of criminal cases (case study in Section 7.5.4.1). During the active decay of buried bodies, decomposition products will modify the immediate grave environment in terms of pH and redox (Section 7.6.3), and this will affect the response of corroding metals and the decay of most vulnerable textile materials (Table 7.6).

References Anstey, H. and Weston, T. (1997). The Anstey Weston Guide to Textile Terms. Leeds, UK: Weston Publishing. Arnold, C. J., Janaway, R. C., and Keepax, C. (1983). A detailed study of the metalwork from the Christchurch Anglo-Saxon cemetery, including analysis of the textile and wood, in Excavations in Christchurch, 1969–1980 (K. S. Jarvis, Ed.). Dorchester, UK: Dorset Natural & Archaeological Society Monograph Series, no.5, 111–129. Aturaliya, S. and Lukasewycz, A. (1999). Experimental forensic and bioanthropological aspects of soft tissue taphonomy: 1: Factors influencing postmortem tissue desiccation rate. J. Forensic Sci. 44, 893–896.

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Barber, E. J. W. (1991). Prehistoric Textiles: The Development of Cloth in the Neolithic and Bronze Ages with Special Reference to the Aegean. Princeton, NJ: Princeton University Press. Bass, W. M. and Jefferson, J. (2003). Death’s Acre. London: Time Warner. Beattie, O. B. (1992). The results of multidisciplinary research into preserved human tissues from the Franklin Arctic Expedition of 1845, in Proceedings of the 1st World Congress of Mummy Studies. Cabildo de Tenerife: Organismo Autonomo de Museos y Centros, 579–586. Beattie, O. B. and Geiger, J. (1987). Frozen in Time: The Fate of the Franklin Expedition. London: Grafton. Beer, E. J. (1962). The Beginning of Rayon. London: Phoebe Beer. Bell, M., Fowler, P. W., and Hilson, S. W. (Eds.) (1996). The Experimental Earthwork Project, 1960–1992. York, UK: Council for British Archaeology Research Report 100, Council for British Archaeology. Betts, A., van der Borg, K., de Jong, A., McClintock, C., and van Strydonck, M. (1994). Early cotton in northern Arabia. J. Archaeol. Sci. 21, 489–499. Biek, L. (1963). Archaeology and the Microscope: The Scientific Examination of Archaeological Evidence. London: Lutterworth Press. Bray, E. (1996). The use of geophysics for the detection of clandestine burials: some research and experimentation. Unpublished M.A. dissertation, University of Bradford, Bradford, UK. Brothwell, D. (1986). The Bog Man and the Archaeology of People. London: British Museum Press. Cameron, E. (1991). Identification of skin and leather preserved by iron corrosion products. J. Archaeol. Sci. 18, 25–33. Ceruti, M. C. (2002). Archaeological finds of three frozen mummies and offerings at the Inca ceremonial complex on mount Llullaillaco (Northwest Argentina), in Mummies in a New Millennium: Proceedings of the 4th World Congress of Mummy Studies, Nuuk, Greenland (N. Lynnerup, C. Andreasen, and J. Berglund, Eds.). Copenhagen: Greenland National Museum and Archives and the Danish Polar Centre, 178–182. Clark, F. E. (1967). Bacteria in soil, in Soil Biology (A. Burges and F. Raw, Eds.). London: Academic Press, 25–49. Cowan, M. L. and Jungerman, M. E. (1962). Introduction to Textiles. New York: Appleton-Century-Crofts. Cronyn, J. (1990). Elements of Archaeological Conservation. London: Routledge, 18. Crowfoot, E., Pritchard, F., and Staniland, K. (2006). Textiles and Clothing, c. 1150– c. 1450: Medieval Finds from Excavations in London. London: Boydell Press. Dzierzykray-Rogalski, T. (1986). Natural mummification, in Science in Egyptology (A. R. David, Ed.). Manchester, UK: Manchester University Press, 101–112. Edwards, R. (1996). The effect of changes in groundwater geochemistry on the survival of buried metal artefacts, in Preserving Archaeological Remains in situ (M. Corfield, P. Hinton, T. Nixon, and M. Pollard, Eds.). London: Museum of London Archaeological Service, 87–92. Faraday, M. (1836). Letter on recent discovery of Roman Sepulchral relics in one of the greater barrows at Bartlow, in the parish of Ashdon. Essex Archaeologia 26, 306–310.

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Hudson, H. (1986). Fungal Biology. London: Edward Arnold. Hunlich, R. (1939). Textile Fibres and Materials: Their Properties and Identification with Special Reference to Rayon and Staple Fibre. London: Skinner. Hunter, J. R. and Dockrill, S. (1996). Recovering human remains, in Studies in Crime: An introduction to Forensic Archaeology (J. R. Hunter, C. A. Roberts, and A. Martin, Eds.). London: Batsford, 55–56. Janaway, R. C. (1983). Textile fibre characteristics preserved by metal corrosion: The potential of SEM studies. Conservator 7, 48–52. Janaway, R. C. (1987). The preservation of organic materials in association with metal artefacts in inhumation graves, in Death, Decay and Reconstruction: Approaches to Archaeology and Forensic Science (A. Boddington, A. N. Garland, and R. C. Janaway, Eds.). Manchester, UK: Manchester University Press, 127–148. Janaway, R. C. (1993). The textiles, in The Spitalfields Project: Volume 1—The Archaeology (J. Reeve and M. Adams, Eds.). York, UK: Council for British Archaeology Research Report 85, 93–119. Janaway, R. C. (1996a). Textiles, in The Experimental Earthwork Project, 1960–1992 (M. Bell, P. W. Fowler, and S. W. Hillson, Eds.). York, UK: Council for British Archaeology Research Report 100, Council for British Archaeology, 160–168. Janaway, R. C. (1996b). The decay of buried human remains and their associated materials, in Studies in Crime: An Introduction to Forensic Archaeology (J. Hunter, C. A. Roberts, and A. Martin, Eds.). London: B. T. Batsford, 58–85. Janaway, R. C. (1998). An introductory guide to textiles from 18th and 19th century burials, in Grave Concerns: Death and Burial in England, 1700–1850 (M. Cox, Ed.). York, UK: Council for British Archaeology Research Report 113, 17–32. Janaway, R. C. (2002). Degradation of clothing and other dress materials associated with buried bodies of both archaeological and forensic interest, in Advances in Forensic Taphonomy: Method, Theory, and Archaeological Perspectives (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 279–402. Janaway, R. C. and Scott, B. (Eds.) (1989). Evidence preserved in corrosion products: New fields in artifact studies, United Kingdom Institute for Conservation Occasional Paper Number 8, London. Janaway, R. C., Wilson, A. S., Holland, A. D., and Baran, E. (2003). Taphonomic changes to the buried body and associated materials in an upland peat environment: Experiments using pig carcasses as human body analogues, in Mummies in a New Millennium: Proceedings of the 4th World Congress of Mummy Studies, Nuuk, Greenland (N. Lynnerup, C. Andreasen, and J. Berglund, Eds.). Copenhagen: Greenland National Museum and Archives and the Danish Polar Centre, 56–59. Keepax, C. (1975). Scanning electron microscopy of wood replaced by iron corrosion products. J. Archaeol. Sci. 2, 145–150. Kenough, L. (1971). Export of Cotton-Type Textiles from Developing Countries to the United States. Washington, DC: International Bank for Reconstruction and Development. Komar, D. and Beattie, O. (1998). Post-mortem insect activity may mimic peri-mortem sexual assault clothing patterns. J. Forensic Sci. 43, 792–796.

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Lawson, T., Hopkins, D. W., Chudek, J. A., Janaway, R. C., and Bell, M.G. (2000). Experimental earthwork at Wareham, Dorset after 33 years: 3: Interaction of soil organisms with buried materials. J. Archaeol. Sci. 27, 273–285. Lloyd, A. O. (1968). The evaluation of rot resistance of cellulosic textiles, in Biodeterioration of Materials: Microbial and Allied Aspects (A. H. Walters and J. J. Elphick, Eds.). Amsterdam: Elsevier, 170–177. Lopes de Carvalho, L. M. and Linhares, A. X. (2001). Seasonality of insect succession and pig carcass decomposition in a natural forest area in southeastern Brazil. J. Forensic Sci. 46, 604–608. Mann, R. W., Bass, W. M, and Meadows, L. (1990). Time since death and decomposition of the human body: Variables and observations in case and experimental field studies. J. Forensic Sci. 35, 103–111. Mant, A. K. (1987). Knowledge acquired from post-War exhumations, in Death, Decay and Reconstruction: Approaches to Archaeology and Forensic Science (A. Boddington, A. N. Garland, and R. C. Janaway, Eds.). Manchester, UK: Manchester University Press, 65–80. Mathison, G. E. (1964). The microbiological decomposition of keratin. Ann. Soc. Belg. Med. Trop. 44, 767–791. McGrath, C. (1999). Biodeterioration of cotton denim in soil burial environments. Unpublished M.Sc. Dissertation, University of Bradford, Bradford, UK. Micozzi, M. S. (1991). Postmortem Changes in Human and Animal Remains: a Systematic Approach. Springfield, IL: Charles Thomas. Miller, E. (1992). Textiles: Properties and Behaviour in Clothing Use. London: B. T. Batsford. Miller, R. A. (2002). The effects of clothing on human decomposition implications for estimating time since death. Unpublished M.A. thesis, University of Tennessee–Knoxville. Mwyer-Larson, W. (1972). Man Made Fibres. Hamburg: International Rayon and Synthetic Fibres Committee, Rowohlt. Paul, E. A. and Clark, F. E. (1996). Soil Microbiology and Biochemistry. San Diego: Academic Press. Payne, J. A. (1965). A summer carrion study of the baby pig Sus Scrofa Linnaeus. Ecology 46, 592–602. Pourbaix, M. (1966). Atlas of Electrochemical Equilibria in Aqueous Solutions. London: Pergamon Press. Pritchard, F. A. (1984). Late Saxon Textiles from the City of London. Mediev. Archaeol. 28, 46–76. Rivoli, P. (2005). The Travels of a T-Shirt in the Global Economy: An Economist Examines the Markets, Power, and Politics of World Trade. Hoboken, NJ: John Wiley. Rodriguez, W. C. and Bass, W. M. (1983). Insect activity and its relationship to decay rates of human cadavers in east Tennessee. J. Forensic Sci. 28, 423–432. Rodriguez, W.C. and Bass, W. M. (1985). Decomposition of buried bodies and methods that may aid in their location. J. Forensic Sci. 30, 836–852. Rowe, W. F. (1997). Biodeterioration of hairs and fibers, in Forensic Taphonomy: The Postmortem Fate of Human Remains (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 337–346. Ryder, M. L. and Stephenson, S. K. (1968). Wool Growth. London: Academic Press.

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Safranek, W. W. and Goos, R. D. (1982). Degradation of wool by saprotrophic fungi. Can. J. Microbiol. 28, 137–140. Saville, B. P. (1999). Physical Testing of Textiles. Boca Raton, FL: CRC Press. Schoenly, K., Griest, K., and Rhine, S. (1991). An experimental protocol for investigating the post-mortem interval using multidisciplinary indicators. J. Forensic Sci. 36, 1395–1415. Shepherd, G. (1969). Export of Cotton Textiles from Developing Countries to the European Economic Community and the United Kingdom, 1958–1967. Washington, DC: International Bank for Reconstruction and Development. Sibley, P. and Jakes, K. A. (1984). Survival of protein fibres in the archaeological context. Sci. Archaeol. 26, 17–27. Stout, E. E. (1960). Introduction to Textiles. London: John Wiley. Tate, R. L. (2000). Soil Microbiology. New York: Wiley. Tigg, A. E. (2005). The corrosion of metals over forensic timescales. Unpublished M.Sc. Dissertation, University of Bradford, Bradford, UK. Vass, A. A., Bass W. M., Wolt, J. D. Foss, J. E., and Ammons, T. (1992). Time since death: determination of human cadavers using soil solution. J. Forensic Sci. 37, 1236–1253. Walton, P. (1989). Textiles, Cordage and Raw Fibre from 16–22 Coppergate (The Archaeology of York). York, UK: Council for British Archaeology. Warner, F. (1921). The Silk Industry of the United Kingdom: Its Origin and Development. London: Drane. Wilson, A. S. (2002). Biodegradation of hair and its effect on the quality of biogenic information in hair from inhumation burials. Unpublished Ph.D. thesis, University of Bradford, Bradford, UK. Wilson, A. S., Janaway, R. C., Holland, A. D., Dodson, H. I., Barran, E., Pollard, A. M., et al. (2007). Modelling the buried human body environment in upland climes using three contrasting field sites. Forensic Sci. Int. 169, 6–18.

Appendices: Review of Clothing Based on Current U.K. Experience Currently, potentially hundreds of different combinations of natural and synthetic yarns are available to manufacturers to weave or knit into cloth. Early 21st-century clothing comprises a broad mix of purely natural, purely synthetic, and mixed fabrics. Fabric content will influence response of the garment to different depositional environments and will likely lead to differential preservation of the garment sets. The latter half of the 20th century saw the development of unisex clothing items in which the general style and 

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There is considerable diversity of fabric combinations and composition used in modern garments. The examples used in this section are based on garments currently available as both mail order and in large retail outlets, including supermarkets in the UK. This is not a comprehensive study reflecting, for instance, the construction of every bra available in the UK.

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fabric are similar between men and women’s items. The obvious garments in this category are denim jeans. Traditional denim, whether dyed blue, black, or red, is a 100% cotton twill fabric. A variation that was introduced was stretch denim formed by a mixture of cotton and Elastane, which is available to both men and women. However, examination of the stock of popular retail outlets and mail-order catalogs reveals that there is generally a greater range of stretch denim and trouser fabrics in the women’s ranges.

Appendix A. Men’s Clothes A1. T-Shirts/Short-Sleeved Casual Shirts Most are either 100% cotton or a high percentage cotton–polyester blend (e.g., 85%/15%). A2. Formal Shirts These can be 100% cotton, although these fabrics have a tendency to crease and need careful ironing; therefore most of this style of shirt for sale is a polyester–cotton mixture (e.g., 65% polyester, 35% cotton). A3. Pullovers Pullovers have one of the largest ranges of fabrics within a single men’s garment type (Table 7.7). A4. Microfleece These have become a popular alternative to traditional knitted pullovers. The vast majority are 100% polyester, although there some specialized textures made of acrylic–polyester mixtures (e.g., 46%/54%).

Table 7.7  Typical Yarn Composition in Currently Available Pullovers (U.K.)   Item Pullover 1 Pullover 2 Pullover 3 Pullover 4 Pullover 5

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Fabric Composition 100% Cotton 100% Acrylic 50% Cotton–50% Acrylic 100% Wool 80% Wool–20% Viscose

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A5. Trousers Wool polyester mixtures are common for suits and better-quality trousers. Common mixtures are 54% polyester, 44% wool, although the better-quality fabrics have a higher wool content. One hundred percent wool is rare and subject to wear. Cheaper, crease-free trousers are 100% polyester, whereas cotton/polyester mixtures are common for hard wearing. Noncreasing fabric such as uniform or overalls are high polyester/cottons (e.g., 65%/35%). Jeans and casual trousers are often 100% cotton. Stretch fabrics usually consist of mixtures including polyester or Elastane. (e.g., 64% polyester, 34% Viscose, 2% Elastane; 95% cotton, 5% Elastane). A6. Underwear The bulk of men’s underwear currently for sale is either 100% cotton or polyester–cotton (typically 65%/35%), although there are some stretch fabrics (e.g., 95% cotton, 5% Elastane). Stretch components may be confined to the waistband, or in some garments the base fabric also has stretch characteristics. A7. Socks The majority of socks are cotton mixed with synthetic fibers (Table 7.8) to give more durable wear characteristic and stretch. Wool and wool mixture, though common in the past, are currently less common in popular outlets.

Appendix B. Women’s Clothes Though many items of women’s clothing are identical to men’s in terms of fabric use (e.g., microfleece), there are a number of garments that can be substantially different and often contain complex fiber and yarn combinations.

Table 7.8  Some Current Sock Yarn Mixtures (U.K.)   Item Sock 1 Sock 2 Sock 3 Sock 4

Yarn Composition 78% Cotton–20% Nylon–2% Elastain 71% Cotton–15% Nylon–13% Acrylic–1% Elastain 36% Cotton–36% Modal*–26% Polyamide–2% Elastain 70% Cotton–29% Nylon–1% Elastain

* Modal–manmade natural polymer of regenerated cellulose fiber.

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Table 7.9  Range of Fabrics Used in Some Bras Currently Available in the U.K. Bra 1

55% Polyamide

26% Polyester

17% Viscose

Bra 2 Bra 3 Bra 4 Bra 5 Bra 6 Bra 7

38% Polyamide 64% Polyamide 90% Polyamide 77% Polyester 60% Silk 45% Viscose

24% Cotton 11% Cotton 6% Elastain 21% Polyamide 30% Nylon 43% Polyamide

19% Polyurethane 14% Polyester 12% Elastain 8% Polyester 4% Polyester 2% Elastain 10% Elastain 8% Cotton 4% Elastain

2% Elastain

B1. Brassieres There are a huge range of styles and constructions used in bras (Table 7.9). Construction is complex, with as many as 30 different sewing operations. Fabrics are selected for their elastic properties (one-way and two-way stretch), for example, as well as support and comfort. They may also contain underwires, hooks, eyes, and other fastenings. B2. Briefs and Thongs These may be purchased as part of a set, with a bra, or as separates. This can influence the manufacturer’s choice of materials and whether they are intended as a luxury or utility item. Table 7.10 shows the materials in the briefs and thongs that match with the bras presented in Table 7.9. Briefs that are not manufactured as part of set with matching bra can have a much higher natural fiber content (e.g., 95% cotton, 5% Lycra). However, in general, women’s undergarments are less likely to have a high natural fiber content and are more likely to contain significant polyamide content and thus be highly decay resistant. B3. Women’s Hosiery Whereas women’s socks, such as sport socks, are broadly similar to men’s with higher cotton content, women’s pantyhose and tights have a high synthetic Table 7.10  Matching Briefs/Thongs for Bras in Table 7.5 Bra 1 Bra 4 Bra 5 Bra 6 Bra 7

90% Polyamide 85% Polyamide 76% Polyester 89% Silk 48% Polyamide

10% Elastain 15% Elastain 16% Polyamide 6% Elastain 34% Viscose

7% Cotton 4% Nylon 11% Elastain

1% Elastain 1% Cotton 7% Cotton

Note: Briefs that are not manufactured as part of set with matching bra can have a much higher natural fiber content.

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Table 7.11  Yarns Used in Women’s Hose (U.K.)     Item

Fabric Composition

Stretch leggings Tights 1 Tights 2 Tights 3

100% polyester 91% polyamide–9% Elastain 89% polyamide–11% Elastain 100% polyamide

Note: Again, these will have a high resistance to degradation in most depositional

content (Table 7.11). Sheer tights have either a high or exclusively polyamide (Nylon) content sometimes mixed with Elastane. Stretch leggings, which are thicker, are usually 100% polyester. Again, these will have a high resistance to degradation in most depositional environments. As can been seen from the case study (Section 7.1.2) nylon (polyamide)-dominated yarns can be highly resistant to breakdown in burials. However, there are no specific long-term studies of forensic relevance that indicate in what specific circumstances and timescales these will be subject to significant breakdown.

Appendix C. Household Fabrics Household textiles such as bed linens or towels can occur at a crime scene— for instance, a body bundle wrapped in a cotton–polyester duvet cover and deposited in a wood (see case study in Section 7.4.6.1). Biodegradation of the duvet cover will depend on a number of factors including the surface the bundle is placed on, the composition of the fabric, the degree of deterioration of fabric prior to deposition, and whether body decomposition products have access to the textile. Duvet covers, sheets, and pillow covers can have a range of fabric compositions. Most are either cotton or, more commonly, cotton–polyester mixtures of different percentages. Table 7.12 illustrates some currently available in the U.K. through major outlets. Though they can be of fancy weaves (e.g., damask), most are plain weave. Fitted sheets will contain elastic at the corners, whereas duvet covers usually have plastic button or press stud fasteners. Table 7.12  Some Typical Fabric Compositions of Household Textiles    Item Duvet cover 1 Duvet cover 2 Duvet cover 3 Towel

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Towels may have forensic significance due to their highly absorbent nature. The majority are 100% cotton, although cotton–synthetic mixes can occur. C1. Carpet and Carpet Fibers Carpets, or the remains of carpets, have been recovered at a number of deposition sites. These include bodies wrapped in carpets and carpet being partially burned. Carpet remains can be vital to linking the body and the deposition site to a property. Though the identification of trace fibers, including carpet fibers, is a well-established forensic discipline, there has been much less work on the decomposition of carpets of different type in exterior locations. This is further confounded by the wide range of materials and the number of construction techniques used in commonly available carpeting (Table 7.13). Woven pile carpeting consists of a warp yarn that is looped and then cut to form the pile, which is held in place by a woven backing. In some cases a second woven back or an extra synthetic felt may be added to the back. Since the mid 20th century, synthetic foam backing has been popular for a number of applications. The backing is often stiffened with adhesive or size. The nature of this interlace is the difference among Wilton, Axminster, and other such types. In some carpets the warp is not cut, which results in a ridged or looped front surface. It is the failure of the backing that will cause a rotten carpet to fall apart. In the United Kingdom, synthetic fibers dominate the middle and lower end of carpet quality, with wool and wool-synthetic mixtures used in better-quality items. In recent years, however, a specialist market has developed in the use of natural or plant fibers such as jute, sisal, hemp, and seagrass. These sometime incorporate stain-resistant coatings. Nonwoven carpets consist of yarn or fiber adhering to a synthetic rubber back; this backing is often more durable in many depositional environments than woven backing. Carpets can also have synthetic rubber backing to a more traditional woven structure. Typical synthetic carpet fibers used on the face of carpets (on the trodden surface) are nylon, polypropylene, and polyamine due to their wear and stain resistance in normal use. These fibers are resistant to degradation in a range of depositional environments. Whereas traditional carpets were woven using cotton backing yarns, common Table 7.13  Some Typical Carpet Pile Fiber Compositions Carpet 1 Carpet 2 Carpet 3 Carpet 4 Carpet 5

Wool 80% Wool 80% Nylon 100% Polypropylene 100% Polyamine 100% *

Nylon 20% Nylon 10%

Polypropylene 10%

* e.g. Antron™ a Polyamide carpet fiber (du Pont).

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commercial yarns include a range of synthetic and natural and synthetic yarns. Under burial conditions, most modern carpets will fail at the backing, with the carpet becoming fragmentary, but usually the face yarns are more resistant to breakdown and may be recovered from the soil. Due to the carpet being rolled or dumped in layers, the inner layers will be protected by the exterior, leading to variable degradation. Due to the robust nature of many of the materials used in carpets, especially fibers such as nylon or polypropylene, carpet fragments can survive well under unfavorable circumstances. The author has excavated carpet remains that have been partially burned and then left in the ash of a garden bonfire for several years.

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Decomposition Chemistry in a Burial Environment

8

Shari L. Forbes

Contents 8.1  Introduction............................................................................................... 203 8.2  The Chemical Process of Decomposition.............................................. 205 8.2.1  Decomposition Products of Protein........................................... 205 8.2.2  Decomposition Products of Carbohydrates............................. 208 8.2.3  Decomposition Products of Lipids............................................. 208 8.2.3.1  Adipocere........................................................................210 8.3  Liquefaction and Skeletonization............................................................ 213 8.4  Rate of Decomposition of Buried Bodies............................................... 215 8.5  Conclusion...................................................................................................216 References..............................................................................................................217

8.1  Introduction Decomposition is a process that commences immediately following death and, depending on environmental conditions, will proceed until skeletonization has occurred (Evans 1963; Love and Marks 2002). The chemistry associated with decomposition and the destruction of soft tissue is complex (Clark, Worrell, and Pless 1997). Generally, the postmortem process traverses numerous stages before the body is reduced to a skeleton. The process may require days, months, or years to be complete, depending on the surrounding environment (Dix and Graham 2000) and following skeletonization may take hundreds of years for the final bone tissue and mineral phases to disappear (Micozzi 1991). Initially the body will undergo postmortem changes, including livor mortis, rigor mortis, and algor mortis, which occur in the first few hours following death. When the heart ceases to function, blood stops circulating through the body and settles in the dependent capillaries due to gravitational pooling (Geberth 1996). A loss of the usual skin color is observed as the upper surfaces of the body become translucent. Loss of oxygen from the blood causes a change in color from bright red to deep purple as it collects in 203

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the lower parts of the body. This process signals the onset of livor mortis, also known as lividity (Di Maio and Di Maio 2001). Lividity becomes noticeable approximately one to two hours after death, but fixation of lividity may not occur until eight to twelve hours following death (Di Maio and Di Maio 2001; Love and Marks 2002). Fixation of lividity occurs as the body temperature cools, causing solidification of the fat in the dermis surrounding the capillaries and subsequent fixing of the blood within the distended capillaries (Clark et al. 1997). After fixation, the blood will remain in the area where it has settled even if the body is moved (Dix and Graham 2000). This process is used by pathologists for estimating time since death as well as for establishing postmortem repositioning of a body. Rigor mortis is a chemical process that causes the muscles and joints to become increasingly rigid and immobile. It is first noticeable in the facial muscles around 2 to 3 hours postmortem, although all muscles in the body begin to stiffen at the same time after death (Dix and Graham 2000). Within 12 hours rigor mortis will be evident in the entire body, and by 24 to 48 hours rigor will have passed (Spitz 1993). The process of rigor mortis can be used to estimate postmortem interval; however, its onset and duration are significantly affected by environmental conditions. In particular, elevated environmental and internal body temperatures will accelerate the appearance and disappearance of rigor mortis (Di Maio and Di Maio 2001). Conversely, rigor mortis will be retarded in cold temperatures, and the process may be prevented if a body is placed in refrigeration (Varetto and Curto 2005). Rigor mortis will only proceed when the body starts to warm again. However, in most cases following death, the body will progressively lose temperature until it equilibrates with the surrounding environment. The process of normal body cooling is referred to as algor mortis and is most useful for estimating time since death within the early postmortem period (McDowall et al. 1998). Normally the body will maintain a core temperature of approximately 98.6oF during life, and, upon death, the body temperature will decrease until it reaches ambient temperature (Henβge and Madea 2004). As with rigor mortis, the process is strongly dependent on surrounding environmental conditions (Mall et al. 2000). However, in most circumstances, an average size body is estimated to cool at approximately 1.5°F per hour (Clark et al. 1997). The processes of livor, rigor, and algor mortis can be useful indicators of a recently deceased person, but the processes may have expired by the time a body is placed in a shallow or deep grave site. Once these changes have passed, softtissue decomposition proceeds through the processes of autolysis and putrefaction (Fiedler and Graw 2003). In a burial environment, it is these processes that are likely to result in the disintegration of soft tissue and skeletonization.

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8.2  The Chemical Process of Decomposition Autolysis refers to the postmortem disintegration of the cells and tissues in the body (Janssen 1984). The process is not visible with the naked eye but can be observed microscopically as the cellular structure fades and the tissues dissolve by the action of digestive enzymes (Mello de Oliveira and SantosMartin 1995). The destructive hydrolytic enzymes denature molecules and cell membranes, causing cells to detach from each other, and result in the digestion of the major constituents of the body, namely proteins, carbohydrates, and lipids (Gill-King 1997). Macroscopic changes produced by autolysis will become evident within the first few days after death and include skin slippage of the extremities and marbling on the body’s surface (Love and Marks 2002). This phenomenon results from the reaction of degenerated blood with hydrogen sulphide produced by bacteria, causing a black staining of the blood vessels close to the surface that is observed as a marbling effect under the skin (Clark et al. 1997). Putrefaction is the result of microbiological activity and is usually initiated by autolytic processes. It is generally observed later in the postmortem period, but the time frame may vary considerably depending on the surrounding environmental conditions (Prieto, Magna, and Ubelaker 2004). The cessation of autolysis yields a predominantly anaerobic internal environment (Hopkins, Wiltshire, and Turner 2000; Micozzi 1986) that favors the proliferation of bacteria from the intestinal tract. Anaerobic organisms, including Bacteriodes, Clostridia, and Streptococci species, rapidly invade contiguous host cells converting proteins, carbohydrates, and lipids to various acids and gases (Corry 1978; Evans 1963; Gill-King 1997; Janaway 1996). Putrefactive changes are evidenced by bloating of the body, color changes, and distinctive odors that are produced by bacteria (Love and Marks 2002). The process of putrefaction occurs optimally between temperatures of 21°C and 38°C and is considerably retarded at temperatures below 10°C or above 40°C (Mant 1987; Polson, Gee, and Knight 1985). 8.2.1  Decomposition Products of Protein During decomposition, the body’s proteins are degraded by the actions of enzymes through a process known as proteolysis (Evans 1963; Janssen 1984; Polson et al. 1985; Sabucedo and Furton 2003). Like all stages of the postmortem process, proteolysis is affected by environmental variables including moisture, temperature, and the degree of bacterial activity (Sorimachi, Horada, and Yoshida 1996; Vass et al. 2002). The process does not occur at a uniform rate, and, consequently, proteolysis may take place much earlier in some tissues than others (Xiao and Chen 2005). Neuronal and epithelial cells

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are usually the first to be destroyed by putrefaction; these include the lining membrane of the gastrointestinal tract and pancreatic epithelium (Dent, Forbes, and Stuart 2004). The brain, liver, and kidneys will also disintegrate at an early postmortem interval (Gill-King 1997). Conversely, the connective tissues and cartilage are much more resistant to proteolysis and will survive for a longer period of time, although they too will eventually succumb to the effects of putrefaction. Reticulin, epidermis, and muscle protein will resist breakdown for some time, whereas collagen and keratin may survive for longer periods (Linch and Prahlow 2001). Keratin is an insoluble fibrous protein found in the skin, hair, and nails, and its resistance to attack by most proteolytic enzymes (Gupta and Ramnani 2006) is the reason it is often found intact amongst skeletal remains, particularly in burial environments (Macko et al. 1999). In general terms, proteolysis results in the breakdown of proteins to proteoses, peptones, polypeptides, and, ultimately, amino acids. Byproducts that can form during the process include biogenic amines such as histamine, tryptamine, and phenylethylamine (Oliver, Smith, and Williams 1977; Saccani et al. 2005), which are accompanied by the evolution of gases including carbon dioxide and methane (Gill-King 1997). Amino acids containing sulfur atoms can be reduced by desulfhydralation and decomposition to yield ammonia, thiols, pyruvic acid, sulfides, and the toxic hydrogen sulfide gas (Knight and Presnell 2005). The anaerobic conditions of a grave will favor the production of considerable quantities of sulfides (Waksman and Starkey 1931). As mentioned previously, the reaction of hydrogen sulfide with iron present in the body will yield a black precipitate (Shelef and Tan 1998), which is commonly associated with decomposing bodies. Similarly, if hydrogen sulfide reacts with hemoglobin, a green pigment, sulfhemoglobin, will form (Clark et al. 1997; Tatsuno et al. 1986). Furthermore, the reaction of phenyl pyruvic acid with ferric iron in soil will cause a green precipitate to form in the decomposing tissues (Gill-King 1997). These color changes are readily observable at different stages of the decomposition process. Other biogenic amines that are commonly detected as a result of the decomposition of proteins are the distinctive, and aptly named, putrescine and cadaverine (Lange, Thomas, and Wittmann 2002; Pessione et al. 2005). Both decarboxylation products are members of a well-known family of diamines that are highly toxic and that demonstrate a distinctive foul odor. These proteolysis products are usually present in burial environments and represent the characteristic odors of a decomposing body that are often detectable by cadaver dogs (Rebmann et al. 2000). Their accumulation in the abdominal cavity, together with other decompositional gases, cause bloating of the body and subsequent displacement of the earth in a shallow burial (Vass et al. 2002). As decomposition proceeds, the escape of gases and compaction of loosely backfilled soil can cause depression of the soil that is indicative of

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a shallow grave site (Hunter and Martin 1996). This artefact may be used in conjunction with cadaver dogs to detect the presence of a clandestine burial (France et al. 1997; Krogman and Iscan 1986). In a burial environment where the conditions are relatively anaerobic, sulfide products are not transformed further. However, in aerobic environments, sulfides are rapidly oxidized to sulfate and through the assistance of specific bacteria (mainly belonging to the Thiobacillus group) will result in the production of sulfurous acids in the soil (Hartikainen, Ruuskanen, and Martikainen 2001). At the same time, the nitrogen component of proteins is present as a constituent of amino acids and is readily liberated as ammonia to the surrounding environment during the decomposition process (Chapelle 2000). Ammonia (NH3) may be converted to ammonium (NH4+) in the presence of low soil pH and can be subsequently utilized by surrounding plants (Shelef and Tan 1998). A frequent consequence of shallow burials is the degree of vegetation growth that occurs above a buried corpse (France et al. 1997). As decomposition and proteolysis proceeds, the accumulation of ammonium and other decompositional products in the soil provides an enriched environment for vegetative growth. The disturbance of the soil from digging and backfilling the grave results in a looser, more water-retentive medium that can also enhance the vegetative growth above a buried corpse (Hunter and Martin 1996; Krogman and Iscan 1986). As a consequence, the vegetation growing on the burial surface may be more prominent than the surrounding vegetation or may demonstrate differences in fruiting and flowering patterns. Increased vegetative growth is a useful tool for locating buried remains (Figure 8.1). Alternatively, ammonium ions not utilized by plants can undergo nitrification and denitrification (Bolt and Bruggenwert 1978). Various species

Figure 8.1  Increased vegetative growth above a grave site. (See color insert following p. 178.)

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of soil organisms are active in the process of nitrification (i.e., converting ammonia to nitrate). These organisms are divided into two groups, the first of which oxidizes ammonia to nitrite (e.g., Nitrosomonas spp.) and the second of which converts nitrite to nitrate (e.g., Nitrobacter spp.) (Waksman and Starkey 1931). The nitrifying organisms are sensitive to environmental pH with Nitrobacter spp. preferring a pH between 5 and 8 and Nitrosomonas spp. proliferating optimally between pH 7 and 9 (Calli et al. 2003). Denitrification refers to the reduction of nitrate to nitrite, gaseous nitrogen, or nitrous oxide. Although nitrification occurs in an aerobic environment, denitrification requires an anaerobic environment such as a grave site (Chapelle 2000). The bacteria known to be denitrifiers include species from Achromobacter, Bacillus, Micrococcus, and Pseudomonas genera (Bolt and Bruggenwert 1978). Ammonium ions that do not undergo any of the aforementioned processes will be displaced through the soil, subject to adsorption and fixation processes. Anaerobic bacteria present in both the soil and decomposing body are able to cleave the ammonia from amino acids, thus producing large quantities of ammonia in oxygen deficient areas (Chen and Huang 2006). As nitrification is inhibited in anaerobic environments, accumulation of ammonia in burial environments can occur (Carter and Tibbett 2003). 8.2.2  Decomposition Products of Carbohydrates Carbohydrates in the soft tissue also degrade as a result of decomposition. Initially, glycogen, a complex polysaccharide, will convert to glucose monomers by the action of microorganisms (Corry 1978). These sugars may be completely oxidized to carbon dioxide and water or may incompletely decompose to form various organic acids and alcohols (Ellis and Wilson 2003). Sugars decomposed by fungi in an aerobic environment will produce glucuronic, citric, and oxalic acids. Those that are decomposed by bacteria in an anaerobic environment will yield lactic, butyric, and acetic acids (Ueno et al. 2001). Collectively, these organic acid products are responsible for the acidic environment created around a decomposing body. Additional fermentative products include alcohols (e.g., ethanol and butanol), acetone, and distinctive gases including methane, hydrogen, and hydrogen sulfide (Davis et al. 1985). The postmortem bacterial production of ethanol in decomposed remains is a serious problem for pathologists and toxicologists tasked with determining antemortem blood alcohol concentrations (Corry 1978; Collison 2005; De Martinis et al. 2006; Ziavrou, Boumba, and Vougiouklakis 2005). 8.2.3  Decomposition Products of Lipids Lipids represent approximately 60%–85% of the body’s adipose tissue with the other major constituent being water (Reynold and Cahill 1965). Lipids

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comprise approximately 90%–99% triglycerides, which contain numerous fatty acids attached to the glycerol molecule. In the human body, the monounsaturated C18:1 oleic acid is the most widespread in adipose tissue. The polyunsaturated C18:2 linoleic acid and monounsaturated C16:1 palmitoleic acid are also prevalent (McMurry 1994). The corresponding saturated C16:0 palmitic acid is also extensively distributed in fat depots. Following death, the body’s neutral fat is hydrolyzed by intrinsic lipases to yield a mixture of fatty acids that may undergo further hydrolysis or oxidation, depending on the surrounding environment (Gray and Williams 1971; Rothschild, Schmidt, and Schneider 1996). In an aerobic environment, conditions will favor oxidation, and the unsaturated fatty acids released by postmortem hydrolysis will convert to aldehydes and ketones through the oxidative actions of fungi, bacteria, and air (Polson et al. 1985). During the normal course of decomposition, the color of adipose tissue will change from its original yellow hue to a darker yellow as the unsaturated fatty acids accumulate in the tissue. Frequently, the color may be altered by absorption of pigments that are locally available as a result of tissue degradation. Pigments derived from the breakdown products of hemoglobin will cause a pink, red, or blue shade to appear in the fat, whereas the presence of sulfhemoglobin will cause the fatty tissue to take on a greenish hue (Tatsuno et al. 1986). Oxidative changes are less likely to occur than hydrolytic changes in a burial environment because the decomposing body is continually exposed to reducing conditions (Hopkins et al. 2000). In an oxygen-deficient environment, the mixture of unsaturated and saturated fatty acids released during postmortem hydrolysis will undergo further hydrolysis and hydrogenation. The concentration of free fatty acids will increase with a concomitant reduction in neutral fat as decomposition proceeds. Extensive hydrolysis is enhanced by the presence of bacterial enzymes and moisture (Pfeiffer, Milne, and Stevenson 1998). Various Clostridia species produce powerful lipolytic enzymes that significantly aid the anaerobic hydrolysis and hydrogenation of neutral fat under warm conditions. Particularly, Clostridium perfringens (welchii) has been identified as a major agent for decomposition of a cadaver because it resides in the human intestinal tract and has strong saccharolytic, proteolytic, and lipolytic capabilities (Corry 1978; Cotton, Aufderheide, and Goldschmidt 1987). Additionally, C. perfringens is a common inhabitant of soils, and its participation in the decomposition process may also derive from the surrounding burial environment. Moisture is essential for the survival of bacteria and subsequent hydrolysis of neutral fat, and sufficient moisture is usually present in the fatty tissues for the reaction to commence (Sledzik and Micozzi 1997). Providing there is sufficient moisture and enzyme activity, the process will continue until the original fatty tissue is reduced to a mass of fatty acids. If this process occurs under suitable conditions, the formation of adipocere will occur.

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8.2.3.1  Adipocere In recent years there has been considerable discussion regarding the exact chemical process of adipocere formation; however, the essential processes are largely agreed on to be hydrolysis and hydrogenation (Fiedler and Graw 2003; Yan et al. 2001). Following the hydrolysis of triglycerides to free fatty acids, the neutral fats will liquefy and diffuse into the surrounding tissue. Bacterial enzymes convert the unsaturated fatty acids to saturated fatty acids. Hydration and β-oxidation of the oleic acid double bond leads to an increase in palmitic acid (Bereuter et al. 1996), hydrogenation of the double bond produces 10-hydroxy stearic acid (Takatori and Yamaoka 1977a), and subsequent dehydrogenization yields oxo-stearic acid (Takatori and Yamaoka 1977b; Takatori et al. 1986, 1987). The major constituents of adipocere are considered to be a mixture of saturated fatty acids including myristic, palmitic, and stearic acids (Forbes, Stuart, and Dent 2002, 2004). Salts of fatty acids and 10-hydroxystearic acid are regularly identified in adipocere; however, their presence appears to be dependent on the decomposition environment (Corry 1978; Fiedler and Graw 2003; Makristathis et al. 2002). At neutral or slightly alkaline intercellular pH the free fatty acids may attach to sodium or potassium ions present in the interstitial fluid and cell water (Gill-King 1997). When a body is placed in a burial site, the sodium and potassium ions may be displaced by calcium or magnesium ions that are present in the soil. For this reason, calcium salts of fatty acids are often identified as a constituent of adipocere that has been collected from a grave site (Takatori 1996). The previously held theory that adipocere formation was restricted to subcutaneous fat of the cheeks, breasts, and buttocks has since been proven inaccurate (Mant 1957). Adipocere formation may occur in any site whereby fatty tissue or lipids are present prior to death, including internal organs such as the heart, kidney, and liver. Furthermore, adipocere formation can occur in tissues with minimal fat content due to the translocation of liquefied fat and subsequent diffusion into the tissue (Fiedler and Graw 2003; Forbes et al. 2004). Adipocere, when freshly formed in a burial site, has a soft, wet, pastelike appearance (Mant 1957) and can demonstrate a strong ammoniacal odor in a waterlogged environment. It has recently been shown that the odor of adipocere is detectable by cadaver dogs searching for clandestine burials (Rebmann et al. 2000). Older adipocere material becomes dry and brittle (Mant 1957) with a white or gray soapy appearance (Cotton et al. 1987). It is generally more prevalent in individuals with a high fat content, particularly in women and children (Mant 1987). Adipocere formation has been observed in a variety of burial environments, including lead-lined coffins (Mant 1987), peat bogs (Evershed 1992), ice glaciers (Mayer, Reiter, and Bereuter 1997), and submerged locations (Kahana et al. 1999), and consequently numerous principles governing its

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formation have been proposed. The main factor common to all of these environments is their oxygen-depleted nature. Anaerobic environments are necessary to promote the proliferation of C. perfringens and production of lecithinase, which facilitates adipocere formation and preservation of the tissue (Takatori et al. 1986). Studies by the author have demonstrated that the formation of adipocere is possible under a range of burial conditions, providing that the environment is relatively anaerobic and that the fatty tissue contains sufficient moisture and bacteria (Forbes, Dent, and Stuart 2005; Forbes, Stuart, and Dent 2005a, 2005b). Adipocere can form in a range of moist soil textures including sand, silty sand, loam, clay, and sterilized soil (Forbes, Dent, and Stuart 2005a). In a saturated or waterlogged soil environment adipocere will form rapidly; however, adipocere can also form in dry soils, which confirms that sufficient moisture is present in the tissue for the relevant chemical process to occur (Forbes et al. 2005a). The primary exception is an extremely hot, well-drained soil environment that is more conducive to desiccation and mummification (Sledzik and Micozzi 1997). Generally, adipocere formation is promoted in warm environments when the temperature is in the range 22°C–38°C (Forbes et al. 2005a) because the powerful enzymes that aid hydrolysis and hydrogenation of lipids are most effective at these temperatures. Putrefactive activities are retarded when the temperature falls below 10°C or when it exceeds 40°C, and consequently these environments are not as conducive to adipocere formation (Cotton et al. 1987; Mellen, Lowry, and Micozzi 1993). The pH of the soil can also affect adipocere formation, with a mildly alkaline environment being most favorable to its formation (Forbes et al. 2005a). Adipocere can also form in mildly acidic environments, although highly acidic environments will inhibit its formation. Highly alkaline environments that often occur due to the presence of lime in the burial environment (Jarvis 1997; Krogman and Iscan 1986) will prevent decomposition and prohibit adipocere formation (Forbes et al. 2005). The method of burial will also impact the process of decomposition and, subsequently, adipocere formation. Observational studies (Forbes et al. 2005b; Mant 1987) suggest that adipocere forms more readily on a body that is buried directly in the soil as opposed to a body buried in a coffin. Adipocere formation is retarded in a coffin due to the presence of an initial aerobic environment. However, the percentage of available airspace in the coffin is nominal, and calculations suggest that only approximately 150 g–200 g of oxygen gas is available in the grave site for use in the chemical decomposition processes (Dent 2002; Dent et al. 2004). Consequently, decomposition processes in the coffin environment become increasingly anaerobic after a very short period of time, and adipocere formation, although retarded, is still regularly observed in coffin burials.

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Coverings on buried bodies will also considerably retard decomposition and impact the preservation of soft tissue to varying degrees (Mann, Bass, and Meadows 1990). Clothing is a common covering found in burial environments, and parts of the body that are covered by clothing will frequently demonstrate remarkable preservation and adipocere formation (Forbes et al. 2005b). The impact of clothing on decomposition may vary depending on the preservation of the clothing itself (Dix and Graham 2000). Studies have shown the considerable variation that can occur with regard to the deterioration of textiles including cotton, rayon, polyester, nylon, silk, and wool (Janaway 1987, 1989; Morse and Dailey 1985. See also Chapter 7 in this book.). Plastic coverings are often used to wrap the body before burial, both within a coffin or when placed directly in the soil. The use of plastic to cover bodies also greatly retards decomposition; however, its affect on adipocere formation is variable. Some cases report extensive adipocere formation in bodies wrapped only in plastic (Manhein 1997), whereas experimental studies show that tissue wrapped in plastic and buried in soil will result in a semifluid mass of putrefied tissue but no adipocere formation unless clothing is also present (Forbes et al. 2005b). The formation of adipocere can occur in a vast range of burial environments and will proceed by the extraction of moisture from the internal tissues until they become desiccated and mummified (Ubelaker 1995). The dehydration of the internal organs as a result of adipocere formation accounts for the cessation of putrefaction and subsequently decomposition (Fiedler, Schnekenberger, and Graw 2004). In this way, adipocere formation can preserve human remains to an extent that can permit postmortem identification of the body (Polson et al. 1985). Frequently, adipocere formation and mummification will occur simultaneously, making the corpse extremely resistant to decomposition (Bereuter et al. 1996; Makristathis et al. 2002) (Figure 8.2). However, adipocere formation is not an end product, and eventually adipocere will also decompose. The factors necessary for decomposition of adipocere are not well documented. Similar to its formation, the decomposition of adipocere appears to be dependent on the surrounding burial environment (Fiedler et al. 2004). Early studies reported that an aerobic environment was necessary to decay adipocere, suggesting that adipocere would only decompose when a grave site was exposed to air or when an adipocerous body was exhumed to the soil surface (Evans 1963; Mant 1957, 1987). More recent studies suggest that the persistence of adipocere in a burial environment is related to the exclusion of gram positive bacteria, including species of Bacillus, Cellulomonas, and Nocardia, which are able to decompose adipocere (Pfeiffer et al. 1998).

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Figure 8.2  Decomposed porcine remains with partial mummification and adipocere formation. (See color insert following p. 178.)

8.3  Liquefaction and Skeletonization As decomposition proceeds, the body’s tissues and organs liquefy due to the action of digestive enzymes, resulting in a mass of unrecognizable putrefied tissue. Discolored natural fluids are made frothy by the gases emanating from within the decomposing remains and are forced from the natural orifices by the increasing pressure (Clark et al. 1997). A mucus sheath may form around a body buried in soil consisting of the liquefied remains and the fine soil fraction (Janaway 1996). A body buried in a coffin may demonstrate a semifluid mass, although mechanical stress and chemical reactions within will eventually cause the disintegration of the coffin (Forbes et al. 2005b; Ubelaker 1995). Consequently, the liquefaction products will be incorporated into the percolating water in the grave site and will enter the surrounding soil and groundwater systems (Dent et al. 2004). Skeletonization occurs as a result of the disintegration of soft tissue from the body. The rate at which it occurs depends largely on the surrounding environment (Nafte 2000). In a highly aerobic environment, decomposition and the process of skeletonization are rapid. However, in an anaerobic environment, such as a grave site, the process will take considerably longer (Micozzi 1991). As remains decompose to predominantly hard tissue, the process becomes increasingly dependent on bacterial activity (Piepenbrink 1986). The organic collagen phase of bone is eliminated by the action of bacterial collagenases, which cause hydrolysis of the protein–mineral bond to peptides and subsequent degradation to their constituent amino acids (Nawrocki 1995). The inorganic phase is also altered through the loss of mineral hydroxyapatite by inorganic chemical weathering. Removal of the

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organic matrix and mineralized components undermines the protein–mineral bond and significantly weakens the bone structure (Hare 1976). As a result, bone strength and hardness will continue to decrease until the bone disintegrates. Common factors present in a burial environment that cause decomposition and destruction of bone include both abiotic (i.e., water, temperature, soil type) and biotic (i.e., fauna and flora) agents (Nawrocki 1995). The main action of water on bone is by leaching of the organic material into the surrounding soil and groundwater. Bones buried in a waterlogged environment, as a result of a high water table level and poor drainage from the grave site, will generally demonstrate poor preservation. Additionally, the soil type will also affect water content as clay soils retain moisture better than sandy or silty soils (Krogman and Iscan 1986). Soils containing a highly acidic pH will decompose bone rapidly due to the dissolution of the inorganic matrix of hydroxyapatite (Nafte 2000). Consequently, preservation of bone is generally better in soils with neutral or slightly alkaline pH (Nawrocki 1995; Rodriguez 1997). Microorganisms, namely bacteria and fungi, play a role in bone degradation. Microscopic focal destruction of bone is thought to be caused by invading soil microorganisms and results from the fungal invasion of bone tissue (Hackett 1981; Piepenbrink 1986). The effect of their activities mimic pathological changes in bone as does the damage caused by carnivores and other burrowers (Tsokos and Schulz 1999; Tsokos et al. 1999). Small and large mammals can exhume skeletal remains through scavenging and incidental means, resulting in the disturbance, possible removal, or destruction of bone by gnawing (Haglund, Reay, and Swindler 1989; Henderson 1987). Gnawing of bone is not confined to carnivores, and studies have shown that herbivore chewing can also produce spiral fractures that are frequently interpreted as antemortem bone trauma (Ubelaker 1997). Plant roots, where there is surface vegetation above the grave site, can be extremely destructive of bone. Fine roots can travel through the medullary cavity and split long bones lengthwise (Nawrocki 1995). Larger roots can disintegrate the spongy articulations and produce openings in the bone that may be mistaken for fractures (Saul and Saul 2002). Over time, bone is broken down by physical breaking, decalcification, and dissolution due to the factors mentioned previously. Upon exhumation of a body, bones may appear to retain their general shape and form; however, in many instances the bones will readily crumble when touched, and the skeletal remains will disintegrate. Conversely, under suitable conditions, the bones may become fossilized and be preserved for millions of years (Micozzi 1991).

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8.4  Rate of Decomposition of Buried Bodies A body buried in soil will decompose at a significantly slower rate than a body exposed to air (Rodriguez and Bass 1985). The retarded rate of decomposition can be mainly attributed to a lack of insect and animal activity due to the limited access and suboptimal conditions provided by the burial environment. The degree of access to the body is dependent on the depth of burial of the corpse (Krogman and Iscan 1986). Deep burials of more than 1 m will restrict insect and other invertebrate activity and are unlikely to attract the attention of carnivorous animals (Janaway 1996). Shallow burials may allow exposure of the body through cracks that form on the soil surface as a result of bloating of the body and subsequent depression of the grave (Hunter and Martin 1996). An exposed corpse will attract carrion insects that oviposit on the body and accelerate decomposition (Wells and Lamotte 2001). Furthermore, the distinct odors produced by a decomposing corpse will readily permeate through the soil if the body is buried close to the surface. Such odors will attract scavenging animals that will expose and feed on the decomposing remains, thus accelerating the process to skeletonization. The other major factor that considerably retards decomposition of buried bodies is the surrounding soil environment. A body buried in soil is protected from the temperature fluctuations usually experienced in an ambient environment (Galloway, Walsh-Haney, and Byrd 2001). The extent of protection is also dependent on the depth of burial, as temperature will decrease with soil depth. In a deep burial environment, the temperature will be relatively cool, thus slowing the rate of decomposition (Rodriguez and Bass 1985). Shallow burials of less than 1 ft. will experience temperature fluctuations similar to the ambient temperature. Hence, decomposition in a shallow grave will proceed more rapidly than in a deep burial but still at a slower rate than a body decomposing on the soil surface (Rodriguez 1997; Weitzel 2005). Soil particle size is an instrumental factor in determining decomposition rates as it affects water permeability and air exchange within the soil. Clay soils that retain moisture as a result of their small particle size will generally retard decomposition rates to a greater extent than more permeable sandy soils (Bethell and Carver 1987). Once again, the depth of burial plays a role in the extent of moisture present in a grave site. Deep burials generally exhibit more moisture due to a lesser degree of evaporation and their closer proximity to the water table. The presence of excess moisture in a grave site, and subsequent retardation of the decomposition process, can usually be linked to the formation of adipocere (Fiedler et al. 2004). Soil pH is less influential on decomposition processes, with the exception of highly acidic soils. In very acidic sandy and gravelly soils, soft tissue and bone can be reduced to a dark silhouette that is recognized as an outline of the former body at the

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location of interment (Bethell and Carver 1987; Hunter and Dockrill 1996). The phenomenon is often referred to as pseudomorphs or burial silhouettes and is thought to result from the complexing of phosphorus with manganese from the body decomposition products and soil environment, respectively (Bethell and Smith 1989). Other factors that may affect the rate of decomposition include the body type and method of burial. If a body is physically thin or emaciated, as may be observed in a mass grave burial, decomposition and skeletonization will proceed rapidly (Mant 1987). Alternatively, a well-nourished person with excessive body fat at the time of death will more readily form adipocere and thus delay the decomposition process (Nawrocki 1995). The method in which the body is buried will vary depending on the circumstances surrounding death (Mann et al. 1990). The presence of a coffin in the burial environment will noticeably alter the rate of decomposition when compared to a body buried directly in the soil (Forbes et al. 2005b). Liquefaction of the soft tissue will proceed in a coffin until only a semifluid mass remains. This rapid decomposition is thought to be a result of the retention of water in the coffin, a slightly aerobic environment, and the longer length of time elapsed between death and burial (Mant 1987). In a forensic situation, a body is unlikely to be buried in a coffin but may be wrapped in plastic before being buried directly in the soil. The effect of plastic on the rate of decomposition has been discussed previously. The presence of clothing on the body may delay decomposition; where the body is partly clothed, decomposition will be retarded in the covered areas (Galloway et al. 1989; Komar 1998; Weitzel 2005). Clothing acts as a partial barrier between the body and the soil environment, thus negating the effects the surrounding environment has on decomposition. Access to the body by scavengers is also impeded by clothing, and adipocere formation is often encouraged (Galloway et al. 1989). Generally, a clothed body buried in soil will demonstrate better preservation than an unclothed body.

8.5  Conclusion The process of decay of the human body following death passes through many stages including early postmortem changes, autolysis, putrefaction, liquefaction, and, finally, skeletonization. Each of these changes are a result of the chemical processes that occur within the body as decomposition proceeds. The chemical breakdown of the body’s main constituents—namely proteins, carbohydrates, and lipids—is evidenced by distinct color changes and evolution of gases. Important degradation products, such as biogenic amines and adipocere, may also form during the decomposition process if the environmental conditions are favorable. Ultimately, the decomposition products will be released into the surrounding environment, which, in the case of a burial site, is the soil

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matrix. The depth of burial has a major influence on the rate of decomposition by affecting access to scavengers, soil temperatures, moisture levels, and oxygen content. Body type, physical characteristics of the burial environment, and the method of burial will also alter the rate of decomposition.

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McMurry, J. (1994). Fundamentals of Organic Chemistry, 3d ed. Davis, CA: Wadsworth Inc. Mellen, P. F. M., Lowry, M. A., and Micozzi, M. S. (1993). Experimental observations on adipocere formation. J. Forensic Sci. 38, 91–93. Mello de Oliveira, J. A. and Santos-Martin, C. C. (1995). Enzyme histochemistry of the liver in autopsy material at different post-mortem times. Med. Sci. Law. 35, 201–206. Micozzi, M. S. (1986). Experimental study of postmortem change under field conditions: Effects of freezing, thawing, and mechanical injury. J. Forensic Sci. 31, 953–961. Micozzi, M. S. (1991). Postmortem Change in Human and Animal Remains: A Systematic Approach. Springfield, IL: Charles C. Thomas. Morse, D. and Dailey, R .C. (1985). The degree of deterioration of associated death scene materials. J. Forensic Sci. 30, 119–127. Nafte, M. (2000). Flesh and Bone: An Introduction to Forensic Anthropology. Durham, NC: Carolina Academic Press. Nawrocki, S. P. (1995). Taphonomic processes in historic cemeteries, in Bodies of Evidence: Reconstructing History through Skeletal Analysis (A. L. Grauer, Ed.). New York: Wiley-Liss Inc., 49–66. Oliver, J. S., Smith, H., and Williams, D. J. (1977). The detection, identification and measurement of indole, tryptamine, and 2-phenylethylamine, in putrefying human tissue. Forensic Sci. 9, 195–203. Pessione, E., Mazzoli, R., Giuffrida, M.G., Lamberti, C., Garcia-Moruno, E., Barello, C., et al. (2005). A proteomic approach to studying biogenic amine producing lactic acid bacteria. Proteomics 5, 687–698. Pfeiffer, S., Milne, S., and Stevenson, R. M. (1998). The natural decomposition of adipocere. J. Forensic Sci. 43, 368–370. Piepenbrink, H. (1986). Two examples of biogenous dead bone decomposition and their consequences for taphonomic interpretation. J. Archaeol. Sci. 13, 417–430. Polson, C. J., Gee, D. J., and Knight, B. (1985). The Essentials of Forensic Medicine, 4th ed. Oxford: Pergamon Press. Prieto, J. L., Magana, C., and Ubelaker, D. H. (2004). Interpretation of postmortem changes in cadavers in Spain. J. Forensic Sci. 49, 918–923. Rebmann, A. J., Koenig, M., David, E., and Sorg, M. H. (2000). Cadaver Dog Handbook: Forensic Training and Tactics for the Recovery of Human Remains. Boca Raton, FL: CRC Press. Reynold, A. E. and Cahill, G. F. (1965). Handbook of Physiology: Adipose Tissue. Washington, DC: American Physiological Society. Rodriguez, W. C. (1997). Decomposition of buried and submerged bodies, in Forensic Taphonomy: The Postmortem Fate of Human Remains (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 459–467. Rodriguez, W. C. and Bass, W. M. (1985). Decomposition of buried bodies and methods that may aid in their location. J. Forensic Sci. 30, 836–852. Rothschild, M. A., Schmidt, V., and Schneider, V. (1996). Adipocere—Problems in estimating the length of time since death. Med. Law 15, 329–335. Sabucedo, A. J. and Furton, K. G. (2003). Estimation of postmortem interval using the protein marker cardiac Troponin I. Forensic Sci. Int. 134, 11–16.

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222 Shari L. Forbes Saccani, G., Tanzi, E., Pastore, P., Cavalli, S., and Rey, M. (2005). Determination of biogenic amines in fresh and processed meat by suppressed ion chromatography-mass spectrometry using a cation-exchange column. J. Chromatogr. A. 1082, 43–50. Saul, J. M. and Saul, F. P. (2002). Forensics, archaeology, and taphonomy: the symbiotic relationship, in Advances in Forensic Taphonomy: Method, Theory, and Archaeological Perspectives (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 71–97. Shelef, L. A. and Tan, W. (1998). Automated detection of hydrogen sulfide release from thiosulfate by Salmonella spp. J. Food Prot. 61, 620–622. Sledzik, P. S. and Micozzi, M. S. (1997). Autopsied, embalmed, and preserved human remains: Distinguishing features in forensic and historic contexts, in Forensic Taphonomy: The Postmortem Fate of Human Remains (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 483–496. Sorimachi, Y., Harada, K., and Yoshida, K. (1996). Involvement of calpain in postmortem proteolysis in the rat brain. Forensic Sci. Int. 81, 165–174. Spitz, W. U. (1993). Spitz and Fisher’s Medicolegal Investigations of Death—Guidelines for the Application of Pathology to Crime Investigation, 3d ed. Springfield, IL: Charles C. Thomas. Takatori, T. (1996). Investigations on the mechanism of adipocere formation and its relation to other biochemical reactions. Forensic Sci. Int. 80, 49–61. Takatori, T. and Yamaoka, A. (1977a). The mechanism of adipocere formation: I: Identification and chemical properties of hydroxy fatty acids in adipocere. J. Forensic Sci. 9, 63–73. Takatori, T. and Yamaoka, A. (1977b). The mechanism of adipocere formation: II: Separation and identification of oxo fatty acids in adipocere. J. Forensic Sci. 10, 117–125. Takatori, T., Gotouda, H., Terazawa, K., Mizukami, K., and Nagao, M. (1987). The mechanism of experimental adipocere formation: substrate specificity of microbial production of hydroxy and oxo fatty acids. Forensic Sci. Int. 35, 277–281. Takatori, T., Ishiguro, N., Tarao, H., and Matsumiya, H. (1986). Microbial production of hydroxy and oxo fatty acids by several microorganisms as a model of adipocere formation. Forensic Sci. Int. 32, 5–11. Tatsuno, Y., Adachi, J., Mizoi, Y., Fujiwara, S., Nakanishi, K., Taniguchi, T., et al. (1986). Four cases of fatal poisoning by hydrogen sulphide: A study of greenish discoloration of the skin and formation of sulfhemoglobin. Nippon Hoigaku. Zasshi. 40, 308–315. Tsokos, M. and Schulz, F. (1999). Indoor postmortem animal interference by carnivores and rodents: Report of two cases and review of the literature. Int. J. Legal Med. 112, 115–119. Tsokos. M., Matschke, J., Gehl, A., Koops, E., and Puschel, K. (1999). Skin and soft tissue artifacts due to postmortem damage caused by rodents. Forensic Sci. Int. 104, 47–57. Ubelaker, D. H. (1995). Historic cemetery analysis: Practical considerations, in Bodies of Evidence: Reconstructing History Through Skeletal Analysis (A. L. Grauer, Ed.). New York: Wiley-Liss Inc., 37–48.

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Ubelaker, D. H. (1997). Taphonomic applications in forensic anthropology, in Forensic Taphonomy: The Postmortem Fate of Human Remains (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 77–90. Ueno, Y., Haruta, S., Ishii, M., and Igarashi, Y. (2001). Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor. Appl. Microbiol. Biotechnol. 57, 65–73. Varetto, L. and Curto, O. (2005). Long persistence of rigor mortis at constant low temperature. Forensic Sci. Int. 147, 31–34. Vass, A. A., Barshick, S. A., Sega, G., Caton, J., Skeen, J. T., Love, J. C., et al. (2002). Decomposition chemistry of human remains: a new methodology for determining the postmortem interval. J. Forensic Sci. 47, 542–553. Waksman, S. A. and Starkey, R. L. (1931). The Soil and the Microbe. New York: Wiley. Weitzel, M. A. (2005). A report of decomposition rates of a special burial type in Edmonton, Alberta from an experimental field study. J. Forensic Sci. 50, 641–647. Wells, J. D. and Lamotte, L. R. (2001). Estimating the postmortem interval, in Forensic Entomology: The Utility of Arthropods in Legal Investigations (J. H. Byrd and J. L. Castner, Eds.). Boca Raton, FL: CRC Press, 263–285. Xiao, J. H. and Chen, Y. C. (2005). A study on the relationship between the degradation of protein and postmortem interval. Fa. Yi Xue. Za. Zhi.[Chinese] 21, 110–112. Yan, F., McNally, R., Kontanis, E. J., and Sadik, O. A. (2001). Preliminary quantitative investigation of postmortem adipocere formation. J. Forensic Sci. 46, 609–614. Ziavrou, K., Boumba, V. A., and Vougiouklakis, T. G. (2005). Insights into the origin of postmortem ethanol. Int. J. Toxicol. 24, 69–77.

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Shari L. Forbes Contents 9.1  Introduction............................................................................................... 225 9.2  The Body as a PMI Determinant............................................................. 227 9.2.1  Forensic Entomology.................................................................... 228 9.2.1.1  Forensic Entomology and Buried Remains............... 229 9.2.2  Forensic Anthropology................................................................ 231 9.2.2.1  Morphological, Chemical, and Immunological Studies............................................................................ 232 9.2.2.2  Radioisotope Studies.................................................... 234 9.2.3  Forensic Odontology.................................................................... 236 9.2.3.1  Postmortem Tooth Loss as an Indicator of PMI...... 236 9.3  The Burial Environment as a PBI Determinant.................................... 237 9.3.1  Forensic Botany............................................................................. 238 9.3.2  Forensic Palynology...................................................................... 240 9.3.3  Forensic Taphonomy.................................................................... 240 9.4  Conclusion.................................................................................................. 242 References............................................................................................................. 243

9.1  Introduction The term taphonomy refers to the biological, physical, and chemical processes that contribute to fossil preservation (Allison and Briggs 1991). The definition presumed a multidisciplinary approach to the study of death assemblages with regard to ancient buried remains. Early taphonomy represented a subdiscipline of palaeontology (Efremov 1940), which was purely devoted to studying the processes that occur as a result of the decomposition of organic remains (Micozzi 1991). Modern taphonomy relies even more heavily on a multidisciplinary approach and now encompasses the fields of archaeology, palaeoanthropology, and microbiology (Haglund and Sorg 1997a). With the forensic 225

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application of taphonomy, the field has broadened further to incorporate additional disciplines including anthropology, mycology, botany, and entomology. Forensic taphonomy involves the use of experimental and analytical approaches to estimate postmortem interval (PMI) and to determine the cause and manner of death. Applying taphonomic approaches to forensic investigations may also allow for the discrimination of products of human behavior from those created by nature and may provide information regarding pre- and postdeposition activity as well as decomposition and preservation effects in the burial environment (Haglund and Sorg 1997b). Forensic investigations focus on the immediate processes surrounding death and are concerned with the events that occurred in the ante-, peri-, or postmortem period. The postmortem interval can span a period of days, weeks, months, or years. This period of time differs from that conventionally used in taphonomy that spans hundreds to thousands of years and has a much larger margin of error (Haglund and Sorg 1997b). In a forensic investigation, the estimate of postmortem interval needs to be much more precise and may require the use of a specific discipline, or several disciplines, to accurately estimate the time since death. For example, forensic entomology utilizes the development of insects and their succession on the corpse to estimate the postmortem interval within hours, days, and weeks after death (Goff 2000). Alternatively, when the remains are skeletal and insect evidence is absent, the application of forensic anthropology and archaeology may provide a more useful estimate of time since death (Pollard 1996). Postburial interval (PBI) refers to the period that has elapsed between the time of deposition in a burial site and the time of recovery. This differs from postmortem interval in that it represents the time in which a cadaver has been buried in a grave site as opposed to the entire period since death. However, these two time frames may be comparable, particularly in clandestine burials. Taphonomic approaches may also be employed in a forensic investigation to estimate PBI. For example, the application of forensic botany can be used to estimate PBI through the investigation of growth patterns of plants found within the crime scene or burial environment (Haglund and Sorg 1997b). Additionally, the use of postputrefaction fungi has been suggested as a potential tool for identifying clandestine graves and estimating PBI (Carter and Tibbett 2003; Hitosugi et al. 2006). Although taphonomic time is said to be a form of the archaeological concept of relative time (Sorg and Haglund 2002), a forensic investigation is more concerned with chronological time, such as days, weeks, and months. Due to the shorter time frame, forensic taphonomy is concerned with not only bone but also soft-tissue change, particularly with regard to the rate of decomposition and factors affecting their modification. The process of decomposition can yield useful information regarding the fate of soft tissue and skeletonized remains, thus helping to estimate the postmortem interval (Micozzi 1991).

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Furthermore, the burial environment can yield additional information relevant to the estimation of postburial interval. Presently, time since death subsists as one of the most elusive determinants present at a crime scene, and the lack of a simple technique for determining this time frame continues to hinder forensic investigations. Although an estimation of time since death can occur in the early postmortem period based on the progression of rigor, livor, and algor mortis (Di Maio and Di Maio 2001), these conditions can rapidly lose forensic value. Thus, alternative techniques are required for estimation of PMI, particularly in the extended postmortem period. Consequently, considerable research has focused on this issue with the advent of new techniques and the application of old techniques to a forensic context. The aim of such research is to assist forensic investigators in accurately estimating PMI or PBI in later stages of decomposition and skeletonization. This chapter highlights several forensic disciplines that have been applied to the estimation of time since death or deposition and their relevance to forensic taphonomy.

9.2  The Body as a PMI Determinant Decomposition is a complex process whereby the soft tissues disintegrate until skeletonization is achieved. The chemical process has been discussed extensively in previous chapters and will not be referred to here. The relationship of taphonomy to decomposition and the determination of postmortem interval is best summarized using the model proposed by Micozzi (1991) (Table 9.1). In the immediate postmortem interval, which represents minutes to hours following death, enzymatic changes are best observed using the methodology of biochemistry and cell biology. A postmortem interval of hours to one day will demonstrate the triad of pathological phenomena, namely algor mortis, livor mortis, and rigor mortis, and the methods of forensic pathology will be employed to estimate PMI. Approximately one day to one week after death, gross postmortem decomposition will be observed, and the methodology of forensic pathology, ecology (entomology), and taphonomy becomes useful for determining time since death. Disarticulation and skeletonization of the body are generally observed days to months following death, and, at this point, forensic anthropology and archaeology play a role in the postmortem investigation. The presence of soft-tissue or skeletal remains in a burial environment allows for an estimation of postmortem interval using a number of techniques employed in various forensic disciplines. By the time of deposition, the methodologies of forensic pathology are generally redundant; however,

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228 Shari L. Forbes Table 9.1  Relation of Taphonomy to Traditional Determinations of Postmortem Interval Time Interval

Process

I. Minutes to Hours

Enzymatic changes Cellular respiration

II. Hours to 1 Day III. 1 Day–1 Week

“Classic Triad” Gross decomposition (autolysis, putrefaction) Skeletonization, disarticulation

IV. Weeks–Months V. Months–Years VI. Years–Eons

Weathering, burial, pedoturbation Fossilization, diagenesis, trace elements

Methodology Biochemistry Cell Biology (e.g., chemistry of death) Forensic Pathology Forensic Pathology, Taphonomy, Ecology Taphonomy, Anthropology, Archaeology Taphonomy, Anthropology, Archaeology Taphonomy, Archaeology, Palaeontology, Mineralogy

Note: All depend on environmental conditions, which generally (except at extremes) alter rates, but not types, of process. Source: Adapted from Micozzi, M. S., Postmortem Change in Human and Animal Remains: A Systematic Approach, Springfield, IL: Charles C Thomas, 1991.

the methodologies of forensic entomology, anthropology, and odontology can be used to estimate time since death. 9.2.1  Forensic Entomology A cadaver exposed to the environment is subject to degradation by various types of animals, of which insects are often the most predominant. Insects can affect the breakdown of the corpse by augmenting the internal decomposition process (Campobasso, Di Vella, and Introna 2001). The succession and development of some insects that visit a corpse can be used to estimate PMI. Succession data are useful in providing a minimum and maximum estimate of time since death. However, biotic and abiotic factors are known to influence carrion insect growth and activity and need to be considered when estimating PMI (Wells and Lamotte 2001). In a buried environment, the process of decomposition is considerably slowed when compared with a body decomposing on the surface (Rodriguez and Bass 1985; Vanezis, Sims, and Grant 1978). The slowed rate of decomposition results from a number of factors including the exclusion of some microorganisms and vertebrate scavengers, as well as the cooler temperatures that can occur at lower soil depths (Galloway, Walsh-Haney, and Byrd 2001). However, the most influential factor to slow the process of decomposition in buried remains is the partial or complete exclusion of insect activity from the body. The degree of exclusion is generally proportional to the depth

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of burial and soil compactness. Studies and field observations have shown that less than a foot of soil or debris covering a body will exclude the majority of blowfly species (Vanezis, Sims, and Grant 1978). Buried remains, though excluding the insect fauna generally seen on surface remains, may still contain their own unique set of insect fauna within the burial environment. The theory of Mégnin (1887, 1894; see also Motter 1898) that various insects appear in distinct squads at definite and specified periods of cadaveric decomposition, and that they succeed each other in regular order represents one of the earliest references to the arthropods found on corpses buried in tombs and graves. The theory is considered to have advanced the field of forensic entomology by identifying the particular fauna present at certain decomposition stages and thus suggesting a possible correlation with the period since death. Since then, many studies have focused on the estimation of PMI using insect material collected from exposed cadavers, but only a few studies have focused on arthropods associated with buried remains. 9.2.1.1  Forensic Entomology and Buried Remains Initial reports on arthropods associated with buried remains arose from the opportunity provided by cemetery exhumations to identify insect fauna in the grave. One of the first published entomological studies of buried human remains reported on the various insect fauna encountered in 150 disinterments (Motter 1898). The study was useful in providing information on both the fauna of the graves and the stages of decomposition encountered upon exhumation (Bornemissza 1957). The majority of species identified were from orders Diptera and Coleoptera; however, a comprehensive list of other identified species was also included for each disinterment. Although less frequent nowadays, the practice of cemetery exhumations still occurs, and occasionally entomological evidence is recovered. A recent exhumation of twenty-two cadavers in the Lille area of northern France identified three fly species that were regularly found in burials due to their preference for underground or closed environments: Conicera tibialis, Leptocera caenosa, and Ophyra capensis (Bourel et al. 2004). C. tibialis is often referred to as the coffin fly because of its ability to burrow into the soil to a depth of 2 m and oviposit directly on a cadaver enclosed in a coffin. Adults are generally active from April to November and can provide information on the possible season of burial (Bourel et al. 2004). L. caenosa is often associated with organic materials found in underground environments, and O. capensis can colonize confined bodies by laying its eggs on the surface of coffins. The study identified a total of eight Diptera and two Coleoptera species associated with the cadavers in coffins that could be used as indicators of burial or interment. However, due to the length of time associated with cemetery burials, the entomofauna identified in these types of studies can rarely be used to estimate PMI accurately.

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Since forensic investigations generally involve shallow burials, several experimental studies have been conducted in an attempt to identify insect fauna associated with bodies buried in a shallow grave site (Lundt 1964; VanLaerhoven and Anderson 1999). One of the first published studies to investigate the process of decomposition beneath the soil utilized pig carcasses placed in makeshift coffins (Payne, King, and Beinhart 1968). The process was monitored using a transparent coffin lid and photography and gave special reference to the ecology associated with the buried carrion. The study identified five stages of decomposition: (1) fresh; (2) inflation; (3) deflation and decomposition; (4) disintegration; and (5) skeletonization. It reported on the amount of time required to reach each stage. The objective of the research was to determine the rate of underground carrion removal when compared with that above ground. The study was one of the first to demonstrate the slowed rate of decomposition in a buried cadaver based on mass loss due to reduced entomological activity. Additional studies involving pig carcasses has since confirmed that buried carrion demonstrate a distinct difference in the pattern of insect succession compared with what occurs on surface carrion (Turner and Wiltshire 1999). One such study established a database of insect succession and demonstrated the potential of certain species to act as determinants of postmortem interval for buried carcasses (VanLaerhoven and Anderson 1999). Dipteran species, with the exception of the family Calliphoridae, were identified as the most useful indicator species allowing for an estimation of the minimum PMI in a shallow burial environment. This assumption is based on the hypotheses that once insects are able to locate buried remains, they will colonize, feed, and develop in a normal, predictable sequence. The majority of experimental studies in this field have used pig carcasses as models for human decomposition. However, one study has been reported that used human cadavers in an experimental capacity (Rodriguez and Bass 1985). The study conducted in Knoxville, Tennessee, involved the burial of six unembalmed human cadavers at varying depths and subsequent exhumation at varying intervals. Carrion insect activity was only observed on the bodies buried at a depth of approximately 30 cm (1 ft.). The insects were identified as larvae, pupae, and adults of the family Calliphoridae and Sarcophagidae. It was speculated that the adult flies laid their eggs in the small crevices in the soils above the remains and that the larvae then burrowed to the cadaver where further development ensued. The study was able to demonstrate that the depth at which the cadaver was buried directly affected access by carrion insects and subsequently the rate of decomposition. Although the number of studies to investigate arthropods in burial environments is not extensive, the knowledge obtained from such studies has been applied to various investigations. One of the first applications of burial entomofauna occurred in the field of archaeology and was used to identify

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Calliphoridae (blowflies) and Sarcophagidae (flesh flies) associated with 130- to 160-year-old burials (Gilbert and Bass 1967). The presence of pupae in the burial environments seasonally dated the burials from late March to mid October based on the arrival and disappearance of these flies in South Dakota. Although the approximate dates of the burials were previously known, the entomological evidence was able to further confirm the dates by determining the season of burial. Conversely, the effect of taphonomic factors on insect succession in a burial environment can prove problematic to a forensic investigation as demonstrated by a case study in the United Kingdom (Turner and Wiltshire 1999). A male corpse was discovered in a shallow grave that had been partly exposed by large scavengers. Entomological evidence was collected and used to estimate the PMI of the buried remains; however, the estimate provided to the police was in contradiction to the estimations determined from other evidence. The investigation provided an opportunity to conduct an experimental validation on the accuracy of estimating PMI of buried remains using entomological evidence. Replication of the homicide and decomposition process was produced through the use of pig carcasses buried in close proximity to the original shallow grave site, and regular observations were made until the corpses were exhumed. The study showed that insects played no role in the decomposition process until the remains were exposed by scavengers, at which point blowflies oviposited on the exposed tissues. More importantly, the study was able to demonstrate that using only the blowfly larvae to estimate PMI would have resulted in an erroneous estimate of the time since death (ibid.). Although the field of forensic entomology is regularly employed to estimate time since death of exposed remains in forensic investigations, the majority of studies that have investigated arthropod succession on buried corpses suggest that it cannot be used as accurately for estimating PMI of buried remains. Several insect species have been identified as possible indicators of time since death; however, in many instances, insect material will only determine the season of burial and not necessarily the year. Hence, although entomology is useful as an application to forensic taphonomy, additional techniques are required to substantiate any estimate of PMI that may be given to forensic investigators based on entomological evidence. 9.2.2  Forensic Anthropology Forensic anthropology refers to the analysis of human skeletal remains within the context of a legal investigation (Nafte 2000). Anthropology and the use of its techniques in forensic investigations have become increasingly advanced over the last 50 years (Iscan 2001). The assistance of anthropologists is frequently requested in forensic investigations to examine remains that have

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been unearthed during excavations. Ultimately, the goal of the anthropologist is to determine the identity of the individual and to reconstruct the events surrounding their death. In doing so, it is necessary to establish the period of interment (PBI) as well as the interval since death (PMI) of the human remains (Haglund and Sorg 1997a). In many forensic cases, the period of interment will be the same as the time since death (Knight and Lauder 1969), and, as a result, the anthropologist will give an estimate that simply represents the PMI. The interval is important in establishing whether the remains are from a modern or ancient era. The distinction between these two groups can vary but is generally assumed to be in the region of approximately 50 years postmortem (Iscan 2001). Skeletal remains older than 50 years are generally not subjected to a forensic investigation, even if they appear to be a victim of homicide (Knight and Lauder 1969). However, even a simple distinction between modern and ancient bones is difficult, and, as a result, many anthropological studies have focused on identifying reliable methods for dating bone samples. 9.2.2.1  Morphological, Chemical, and Immunological Studies One of the earliest studies to attempt dating of anthropological remains was conducted by Bernard Knight and Ian Lauder, and their results were published in an extensive range of papers (Knight 1968; Knight and Lauder 1967, 1969). Their initial study attempted differentiation on purely morphological and physical characteristics. Observations were made as to the likely appearance of soft-tissue and bone texture over different postmortem periods. However, soft tissues are prone to environmental modifications and do not generally survive the decomposition process, rendering the use of morphological characteristics in dating human remains problematic. A range of chemical studies was then attempted and provided varying success in differentiating between modern (less than 70–100 years) and ancient (more than 70–100 years) bones (Knight 1968; Knight and Lauder 1967, 1969). Analytical criteria including nitrogen content, amino acid identification, and benzidine testing were identified as being useful in distinguishing the two groups. The study noted that environmental conditions were a large source of uncertainty in estimating time since death, and, consequently, the proposed techniques for distinguishing modern from ancient bones could be unreliable indicators if the surrounding conditions were not considered (Knight 1968; Pollard 1996). Determination of nitrogen content showed a progressive decrease with age, and almost all samples less than 50 years old demonstrated nitrogen content of more than 3.5 gm%. The technique was considered advantageous as it was not affected by the age of the individual at death (Knight and Lauder 1967, 1969). Although the degradation of protein is affected by environmental conditions including moisture, temperature, pH, and oxygen content, the

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study reflected a relative independence of environment on nitrogen loss from bone. A later study further confirmed these results by investigating the nitrogen levels in long bones interred for a period of 26 to 90 years (Jarvis 1997). This study improved on the previous method by minimizing errors associated with handling and demonstrating that the intensity of the reaction will decrease with time and that a negative result indicates an older bone sample. The number of amino acids in bone was also observed to decline with postmortem age as bone proteins hydrolyzed and released the amino acids into the environment (Knight 1968). A bone sample that yields seven or more amino acids suggests an age of less than 100 years old, and if it contains both proline and hydroxyproline, it is most likely less than 50 years old (Knight 1968). Benzidine testing of bone surface has been shown to yield a strong positive reaction in recent bone samples (Facchini and Pettener 1977; Knight and Lauder 1969). The intensity of the reaction will decrease with time, and a negative result indicates an older bone sample. As the test relies on the presence of blood, the results are susceptible to the rapid loss of blood remnants due to environmental conditions. However, if these factors are considered, the benzidine reaction can supply a useful indicator for identifying skeletal remains of potential forensic interest. Another test dependent on the presence of blood is luminol testing. As with the benzidine test, the intensity will decrease as the age of the bone increases due to the loss of hemoglobin proteins (Introna, Di Vella, and Campobasso 1999). A negative result is indicative of older bone samples. Luminol testing is perhaps the most recent development in chemical techniques used to date skeletal remains. The method provides a good correlation between the levels of intensity of the chemiluminescence with the difference in time since death of the bones (ibid.). Immunological techniques have focused on the detection of residual serological activity of bone protein as an indicator of postmortem bone age (Berg 1963; Camps and Purchase 1956; Castellano, Villanueva, and von Frenckel 1984; Knight 1968; Knight and Lauder 1967, 1969). Immunological reactions between bone powder and antihuman rabbit serum were evaluated using a gel diffusion test, but inconsistent results were achieved, most likely as a result of nonspecific reactions of serum against bone, soil contamination, and the presence of false-positives arising from unknown contaminating antigens (Brandt, Wiechmann, and Grupe 2002; Lendaro et al. 1991). Additional immunological studies have investigated estimations of fat content in bone (Berg 1963; Castellano et al. 1984; Knight and Lauder 1969), histological bone sections (Berg 1963; Knight and Lauder 1969), and colorimetry techniques (Knight and Lauder 1969); however, the results produced were unreliable, and none of these techniques are considered sufficiently accurate for routine use.

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9.2.2.2  Radioisotope Studies The use of radiocarbon dating is widely accepted as being a suitable method for dating bone material from an archaeological era. However, recently this method has been applied to dating skeletal remains of forensic interest from the post-1950 contemporary era (Ubelaker 2001; Wild et al. 1998, 2000). The theory is based on nuclear weapon testing that was performed during the 1950s and early 1960s and resulted in an increase in the 14C content in the atmosphere compared with prebomb levels. In 1963, a Nuclear Test Ban Treaty was established, and the 14C content in the atmosphere began to decrease. The 14C/12C ratio of an organic material should theoretically be comparable to the atmospheric 14C/12C ratio at the time of death when the uptake ceases. Hence, the chronological change in 14C content—referred to as the bomb pulse—can be used as a calibration curve to date anthropological samples of forensic interest (Forbes 2004). The first forensic application of the method occurred in 1989 and utilized 14C activity as an isotopic tracer to assign human bone samples to one of three periods: a nonmodern period (prior to A.D. 1650), a premodern period (A.D. 1650–1950), and a modern period (A.D. 1950 to present) (Taylor et al. 1989). A bone dated in the nonmodern period was considered to be of no forensic interest; if dated in the premodern period it was considered to be potentially of forensic interest, and a sample dated in the modern period was considered to be of definite forensic interest. The technique was successful in identifying two of the five samples analyzed as being of forensic interest. The advantages of the 14C method were its relative accuracy for the period in question and, more importantly, the fact that it was not affected by environmental variables. However, disadvantages of the method included the cost and amount of time taken to analyze the samples as well as the size of the sample required and the destructive nature of the technique. The following decade saw some of these disadvantages addressed with the introduction of the accelerator mass spectrometry (AMS) method of measuring 14C. The time taken to measure 14C using AMS analysis is now considerably reduced. Furthermore, the required bone sample size has also been reduced, thus allowing analysis of much smaller samples, which is highly beneficial to forensic investigations (Tuniz, Zopi, and Hotchkis 2004). The bomb pulse method is now considered to be one of the most useful techniques for distinguishing between modern and ancient remains (Geyh 2001; Ubelaker, Buccholz, and Stewart 2006; Wild et al. 1998). Attempts to improve the accuracy of the technique have demonstrated the importance of analyzing a bone fraction that contains a rapid turnover rate (Wild et al. 1998). Analysis of the collagen fraction of recent bone has been shown to be less accurate as a result of its relatively long carbon turnover rate compared with other bone fractions (Ubelaker 2001). Lipids from bone and bone

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marrow provide a more reliable estimate of time since death for recent bone samples (Wild et al. 1998, 2000). However, in cases where the lipid fraction has degraded or is not recoverable, hair and dental tissue may also provide a reliable estimate (Geyh 2001; Ubelaker et al. 2006; Wild et al. 1998). Nuclear testing in the 1950s also increased the levels of other radionuclides in the atmosphere, including tritium (3H), strontium-90 (90Sr), and caesium-137 (137Cs) (Papworth and Vennart 1984; Pollard 1996). In particular 90Sr has been intensively studied because it is a bone-seeking element and is not easily lost once stored in bone tissue. Enhanced levels of 90Sr have been detected in fresh postmortem samples compared with archaeological bone samples. However, postburial contamination via uptake from groundwater proved to be a major problem throughout the analyses (MacLaughlin-Black et al. 1992). Analysis of skull bone samples using a 90Sr method demonstrated some success in determining whether a sample dated from a pre- or post1950s period (Neis et al. 1999). However, the study made recommendations for further investigations to establish a correlation between the 90Sr burden and the exact year of death. Lead-210 and polonium-210 are two alternative radionuclides that have recently been suggested as potential indicators of PMI (Swift 1998; Swift et al. 2001). Radiation exposure of 210Pb occurs through inhalation and ingestion of food and water. As this element is not related to nuclear explosions, uptake throughout life remains relatively constant. Once 210Pb enters the bloodstream it is preferentially incorporated into bone by replacing calcium within the matrix. With a half-life of 22 years, 210Pb decays via bismuth-210 (210Bi) to yield the polonium-210 (210Po) isotope. 210Po differs from 210Pb by having a much shorter half-life, and after a certain period of time an equilibrium forms between the two isotopes due to this distinct difference in halflives. This equilibrium remains stable throughout life, but following death the ratio distorts as the isotopes decay exponentially (Forbes 2004). Hence, measurement of 210Pb levels in bone provides possibilities for dating bones from a modern postmortem period. Preliminary studies in this field have evaluated the potential of using 210Po and 210Pb nuclides in conjunction with trace elements to provide a meaningful estimate of time since death (Swift 1998; Swift et al. 2001). The results demonstrated a correlation between certain radionuclide content and time since death as well as an intercorrelation between trace elements and time since death of recent human skeletal remains. However, certain limitations were identified within the study. The study was conducted on skeletal remains of Portuguese male adults collected from a cemetery in Lisbon. Background data for each subject were unavailable, which meant that factors known to increase 210Po levels within a body (e.g., seafood consumption, smoking) during life could not be taken into account. Recommendations were made for future work to include U.K. populations (based on the author’s

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nationality), and similar studies have been conducted in Australia (Howard, Meyer, and Forbes 2006). A large-scale application of this work in a range of countries could provide a potentially international method for measuring the PMI of buried skeletal remains. Extensive anthropological studies have been conducted over the last 50 years in an attempt to accurately identify a technique for dating skeletal remains of forensic interest. The need to estimate this interval is important to both the fields of forensic science and anthropology. In the earlier stages of decomposition when considerable soft tissue is still present, entomological evidence provides one of the most accurate methods of estimating time since death. However, as the soft tissue starts to disintegrate and the body becomes skeletal, the methods of forensic anthropology are more useful in estimating PMI of buried remains. Of the numerous methods discussed, radiocarbon dating is potentially the most beneficial to forensic investigations to date. However, many of the other techniques discussed also have the potential to be used in forensic investigations with refinement and verification. 9.2.3  Forensic Odontology The most common application of forensic odontology is the identification of deceased individuals. Dental identification of human remains may result from a number of situations whereby the body is disfigured to such an extent that visual identification is not possible. Such situations include the victims of fire, violent crime, motor vehicle accidents, mass disaster, bodies found in water, and decomposed remains. Occasionally a body’s dentition may be used for purposes other than identification, and several studies have investigated postmortem tooth loss as a potential indicator of time since death (Hall 1997; McKeown and Bennett 1995). 9.2.3.1  Postmortem Tooth Loss as an Indicator of PMI Tooth enamel represents one of the most resistant skeletal tissues to postdepositional decay in a burial environment (Duric, Rakocevic, and Tuller 2004), and, as a result, teeth are often the only identifying feature of a skeleton to remain. As decomposition proceeds, the loss of soft tissue around the mandible allows the exposed teeth to become dislodged from their original anatomical position. Postmortem tooth loss has been described as a possible indicator of PMI and appears to be dependent on age, periodontal health, seasonality, and location of the body placement (McKeown and Bennett 1995). Cadavers that are deposited in the summer months will undergo a more rapid process of soft-tissue decomposition and thus lose teeth more rapidly than bodies that decompose in the autumn or winter months. Similarly, a cadaver exposed to direct sunlight, or even deposited in a shaded area, will decompose and lose teeth more rapidly than a cadaver that has been buried. The

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rate of decomposition of the soft tissue that binds the tooth into the alveolar bone is directly correlated to postmortem tooth loss when taking into consideration the season and surrounding environment. Hence, tooth loss patterns, in conjunction with other indicators, may provide useful information for estimating PMI (McKeown and Bennett 1995), especially in incidences of mass disaster whereby considerable decomposition has occurred. More recently, the effect of postmortem interval, excavation methods, and root morphology on the rate of postmortem tooth loss has been investigated from both an archaeological and forensic context (Duric et al. 2004). In both contexts, postmortem tooth loss was shown to be a result of soft-tissue decomposition and therefore directly influenced by the postmortem interval. Additionally, the differences in root morphology were determined to be a significant factor related to postmortem tooth loss. However, an expected correlation between postmortem tooth loss and the excavation methods of the burial environment was not identified. Both of these studies represent preliminary research, and further controlled research is required to establish the value of postmortem tooth loss as a PMI indicator. As with all investigations, an accurate estimation of PMI of buried remains is more likely to be achieved using a combination of techniques and forensic disciplines rather than a single method. Although the human body itself provides the best estimate of PMI using pathology, entomology, and anthropology (Micozzi 1991), in the absence of these techniques the burial environment can provide useful information about the burial interval, which is usually comparable to the time since death. The remainder of this chapter focuses on some of the forensic techniques used to estimate PBI.

9.3  The Burial Environment as a PBI Determinant To investigate the role of the burial environment we can return to the model proposed by Micozzi (1991) (Table 9.1) demonstrating the relation of taphonomy to traditional determinations of postmortem interval. The postmortem interval of minutes through to months was discussed in Section 9.2. From Table 9.1 it is noted that the interval of months through to years will be characterized by weathering (of the bone), burial (phenomena), and pedoturbation (of the site). At this stage the methodologies of taphonomy, archaeology, and palaeoecology will be employed to estimate the time interval since deposition. In the final stages, observational phenomena of the period years through to eons will include fossilization, diagenesis, and trace elements and are best analyzed using the methodologies of archaeology, palaeontology, and mineralogy. An extended postburial interval will generally not yield valuable information from the body, particularly if the skeleton has completely disintegrated within the grave site. Consequently, the burial environment itself

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will provide the most useful information when attempting to estimate the time since death or time of deposition. Though several of the disciplines suggested by Micozzi (1991), namely palaeoecology and palaeontology, are not relevant to forensic investigations, the applications of forensic archaeology and taphonomy are regularly utilized. More recently, the field of botany has also been applied to forensic investigations, prompting the application of forensic botany in estimating PBI. 9.3.1  Forensic Botany Forensic botany refers to the analysis of plants in a forensic context. Although the science of botany is well recognized, plant evidence is often overlooked at a crime scene due to the lack of knowledge and training of crime scene investigators with regard to botanical evidence (Hall 1997). Botanical evidence can take many forms and may include roots, stems, branches, leaves, flowers, pollen, and fungi. The evidence has the potential to place a person at a crime scene, to determine the cause of death, and to determine the time of death or deposition. Common uses of botanical evidence include the analysis of stomach contents to determine the last meal of the victim and the rate of digestion to estimate PMI; pollen, fungal, and algae analyses for determining geographic location; and DNA for linking plant remains to a suspect or scene of crime (Courtin and Fairgrieve 2004). In the case of a shallow burial, any plant material that is under or on the human remains as well as any material buried with the remains can be valuable to a forensic investigation (Hall 1997). The action of digging a grave site causes disturbance and damage to the plants in that area, and the refilling of the grave will cause additional damage to surrounding plants. Analysis of the plant material may provide information regarding the season or time the plant was damaged, which can be linked to the time of deposition. Additional seasonal information can be supplied by the presence of flowers, fruits, and pollination (Courtin and Fairgrieve 2004). Determining the time of damage to branches or roots is accomplished by analyzing the annual growth rates in tree rings. The discipline is generally referred to as dendrochronology and provides the basis for most of the published cases in which forensic botany has been used to estimate PBI. The production of the woody structure of a tree will normally show systematic variations across the growth rings unless affected by a change in the growth pattern, such as when the tree is damaged or broken. The abnormal change in the wood structure can then be used to estimate when an event (e.g., digging of a grave) occurred based on its effect on the growth of the tree. Although botany is not used extensively in forensic investigations, three case studies in which forensic botany has been employed are reported here.

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An early case study demonstrates the use of the structure of root wood to estimate the date of a grave (Denne 1977). A skeleton discovered in a shallow grave in a wooded area formed the basis of a forensic investigation. Broken and intact roots were found in the grave and were used to estimate when the body had been buried. Analysis of the roots identified changes in the root growth due to a disturbance that had occurred sometime in the period between late summer 1966 and spring 1967. The skeleton was later identified as that of a young girl who had disappeared in August 1966. It is likely that her body was buried soon after she went missing. In a similar case, the discovery of a fully clothed human skeleton in a shallow burial approximately 1 ft. deep led to a forensic investigation whereby the identification of the deceased and time of death needed to be determined (Vanezis et al. 1978). To estimate time of death, vegetation within the grave site was examined to determine PBI, taking into account root injury, root growth, and branch growth. A root was identified that had sustained injuries consistent with those inflicted by a spade. The damage was estimated to have occurred in the 1969–1971 dormant season. Root growth through the body of the deceased suggested penetration during the 1974 or 1975 growing season. An undamaged branch that had grown across the width of the grave provided evidence that the grave had been dug prior to the 1972 growing season. When combined, these findings were useful in providing a time frame in which the body had been buried and were consistent with burial having occurred soon after her disappearance, approximately 5 years earlier. A unique case that also involved the analysis of annual growth in woody tissue was recently reported (Courtin and Fairgrieve 2004). Although the decomposition environment was above ground rather than buried, the peculiar nature of the case and its application warrants mention here. Human remains in an advanced stage of decomposition were discovered lying across a black spruce (Picae mariana) branch in October 2000. The black spruce had grown up and around the remains, providing an opportunity to estimate PMI using the growth-ring pattern. An anomaly in the pattern of the ring widths suggested displacement of the branch, and hence positioning of the body, occurred between July 1993 and May 1994. The forensic investigation found that the cause of death was suicide rather than homicide, and the deceased was reported missing on August 24, 1993. The aforementioned cases demonstrate the potential of forensic botany and dendrochronology in estimating the postburial interval of human remains. However, as with all forensic disciplines, possible disadvantages of the technique need to be considered. In instances whereby trees grow close together or in shady conditions, production of woody tissue may not occur, thus causing an underestimate of the true age of the annual ring. False rings can be produced by adverse environmental conditions such as drought, fire, or defoliation by insects and chemical sprays and, if present, will cause an

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overestimate in the age of the ring (Denne 1977). Variations in wood structure between trees, and even in parts of the same tree, also need to be considered, particularly if analyzing the wood structure of both roots and stems. When collecting evidence from a burial environment, caution must be exercised to ensure that the botanist knows exactly where the plant material was found. The presence of roots and stems in a burial may have preceded the remains or may have been replaced into the grave during back-filling of the soil. Both types of evidence could potentially lead to an inaccurate estimate of PBI if the surrounding conditions are not considered (Willey and Heilman 1987). It must also be stressed that the number of annual rings represents the minimum time since deposition as it is possible that the roots and stems did not invade the grave at the exact time the body was buried. 9.3.2  Forensic Palynology Forensic palynology refers to the science of deriving legal evidence from pollen and spores of higher plants and cryptograms (Boyd and Hall 1998; Horrocks and Walsh 1998). The main forensic application of palynology lies in its ability to provide associative evidence (i.e., to link a suspect to a crime scene). However, forensic palynology has been previously utilized in an investigation to determine the time of year a burial occurred (Szibor et al. 1998). The case dates back to the mid 1900s and involved a mass grave containing 32 male skeletons discovered in Magdeburg, Germany. The identity of the victims and perpetrators was unclear, and the hypotheses proposed that the victims were either killed by the Gestapo at the end of the Second World War (spring 1945) or were Soviet soldiers killed by the Soviet Secret Police following the revolt in the German Democratic Republic (summer 1953). Pollen analysis was employed to establish the time of death and thus the likely postmortem burial interval. Analysis of the nasal cavities of the corpses was able to indicate the seasonally occurring pollen species at the time of death. The study was able to conclude that some of the victims had inhaled a large quantity of summer pollen shortly before they died. Thus, the postburial interval supported the theory that the mass grave contained the remains of Soviet soldiers who were killed in the 1953 revolt in the German Democratic Republic. 9.3.3  Forensic Taphonomy Decomposing remains are directly affected by the surrounding environment, and, in the case of buried remains, the soil represents the matrix. A burial environment can be defined as the “chemical, biological and geological conditions that prevail in that particular location” (Janaway 1996, p. 58). The chemical conditions can refer to factors such as soil pH, redox potential, ion exchange capacity, and oxygenation. Biological conditions are mostly

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represented by the action of soil microorganisms, particularly bacteria, fungi, algae, and protozoa. Geological conditions refer to the soil mineral particles that are derived from weathered rock (i.e., parent material). Soil types are regularly classified by the soil particle size distribution, which determines whether they are clay, silt, sand, or granules (Craze and Hamilton 1993). Due to their effect on the buried remains, the chemical, biological, and geological conditions of a burial environment may be useful for determining the time of deposition. Chemical investigations have successfully detected microbially produced volatile fatty acids (VFAs) as well as anions and cations in soil solutions beneath decomposing corpses (Vass et al. 1992). A particular set of VFAs— propionic, iso-butyric, n-butyric, iso-valeric, and n-valeric acids—can form and deposit in soil solutions in specific ratios during decomposition. The VFAs are released from the decomposed remains in a set pattern that is dependent on temperature. Hence, information regarding the VFA ratio in the soil solution, combined with temperature data and a gross description of the corpse, can provide the necessary data to determine the stage of decomposition and thus to estimate time since death. Similarly, a range of ions including Na+, Cl–, NH4+, K+, Ca2+, Mg2+, and SO42– will form in the same ratio for a particular stage of decomposition and can also be used to estimate time since death. The advantages to this technique are that environmental conditions, particularly rainfall and soil type, do not appear to appreciably affect the results. The closer the remains are to being skeletal, the more accurate the time since death estimation. Although the study utilized surface decomposition instead of burials, theoretically the technique could also be used to aid in the determination of time since death or deposition of buried remains. The application of field mycology to forensic taphonomic investigations offers an alternative method for estimating PBI (Carter and Tibbett 2003; Hitosugi et al. 2006). Mycological studies have demonstrated the presence of certain fungi, known as postputrefaction fungi, in association with decomposed remains, both human and animal (Carter and Tibbett 2003). The postputrefaction fungi undergo successional periods of fruiting, which can be divided into early and late stages. Early-stage fungi will generally fruit from 1 to 10 months after the release of ammonia during decomposition. Late-stage fungi will fruit from 1 to 4 years following ammonia release into the soil. Although the research is preliminary in nature, postputrefaction fungi have the potential to estimate PBI either up to 1 year or from 1 to 4 years based on early and late-stage fruiting successions. Additionally, postputrefaction fungi can aid as visible grave markers of clandestine burials. In the absence of soil solutions and postputrefaction fungi, textiles and other associated death scene materials may provide information that can be used to estimate the time elapsed between deposition and time of recovery

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of buried remains (Morse and Dailey 1985). An extensive study involving the placement of more than 2,800 perishable items under a range of environmental conditions demonstrated significant differences in the degree of textile deterioration. Recovery of the items occurred at various deposition intervals. Numerous environmental conditions were considered and included temperature, soil type, sunlight, soil pH, depth, moisture, and the presence of a decaying body. Of these factors, temperature was determined to be the most critical factor in causing deterioration. The deterioration sequence for the materials used in the study was rayon, paper, untreated cotton, treated cotton, silk and wool, human hair, cotton–polyester, triacetate, nylon, leather, plastics, and acrylic. The results suggested that an estimation of the degree of deterioration of associated death scene materials could be useful in establishing the time of deposition and thus PBI. To reduce the inaccuracy of the PBI estimation, the deterioration rate of a number of materials should be considered simultaneously. It should be noted that the study was preliminary in nature and that an extensive series of studies has since been conducted to verify the initial work (Janaway 1987, 1996, 2002). Recent studies have found that the decay rate of textiles buried in the ground or in inhumation graves is subject to a range of burial factors that will result in differential degradation. Although at this stage it is not possible to determine a predictive model for estimating PBI based on textile degradation rates, controlled and replicated studies are beginning to show some general patterns in textile degradation in burial environments (Janaway 2002).

9.4  Conclusion The aim of this chapter was to review the techniques and methods currently available to forensic investigators that can potentially estimate postmortem interval or postburial interval. The estimation of time of death or deposition is one of the most important factors that forensic experts are regularly asked to determine. Although numerous methods are available in the early postmortem period (i.e., forensic pathology), once the remains become decomposed the determination of PMI becomes much more difficult to estimate. Furthermore, the methods used to estimate the PMI of exposed remains cannot always be applied to buried remains. As a result, substantial research has been conducted in recent years in an attempt to identify an accurate method for estimating PMI or PBI of remains discovered in burial environments. This chapter has highlighted the potential of forensic entomology, anthropology, and odontology in estimating time since death. It has also reviewed the potential of forensic botany, palynology, and taphonomy to estimate the time of deposition in a grave site. Although the majority of techniques

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proposed were preliminary in nature, refinement and verification of these methods are important. Although they may not be able to conclusively estimate PMI of buried remains on their own, when used in combination these disciplines have the potential to significantly aid forensic investigations requiring postburial interval determination.

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Janaway, R. C. (2002). Degradation of clothing and other dress materials associated with buried bodies of archaeological and forensic interest, in Advances in Forensic Taphonomy: Method, Theory, and Archaeological Perspectives (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 379–402. Jarvis, D. R. (1997). Nitrogen levels in long bones from coffin burials interred for periods of 26–90 years. Forensic Sci. Int. 85, 199–208. Knight, B. (1968). Estimation of the time since death: A survey of practical methods. J. Forensic Sci. Soc. 8, 91–96. Knight, B. and Lauder, I. (1967). Practical methods of dating skeletal remains: A preliminary study. Med. Sci. Law 7, 205–208. Knight, B. and Lauder, I. (1969). Methods of dating skeletal remains. Hum. Biol. 41, 322–341. Lendaro, E., Ippoliti, R., Bellelli, A., Brunori, M., Zito, R., Citro, G., et al. (1991). Brief communication: On the problem of immunological detection of antigens in skeletal remains. Am. J. Phys. Anthropol. 86, 429–432. Lundt, V. H. (1964). Okologische untersuchungen uber die tierische Besiedlung von Aas im Boden. Pedobiologia 4, 158–180. MacLaughlin-Black, S. M., Herd, R. J. M., Wilson, K., and West, I. E. (1992). Strontium-90 as an indicator of time since death: A pilot investigation. Forensic Sci. Int. 57, 51–56. McKeown, A. H. and Bennett, J. L. (1995). A preliminary investigation of postmortem tooth loss. J. Forensic Sci. 40, 755–757. Mégnin, P. (1887). La aune des tombeaux. Compte Rendu Hebdomadaire des Séances de l’Académie des Sciences 105, 948–951. Mégnin, P. (1894). La faune des cadavres. Application de l’Entomologie à la Médecine Légale 96. Paris: Gauthier-Villars. Micozzi, M. S. (1991). Postmortem Change in Human and Animal Remains: A Systematic Approach. Springfield, IL: Charles C Thomas. Morse, D. and Dailey, R. C. (1985). The degree of deterioration of associated death scene material. J. Forensic Sci. 30, 119–127. Motter, M. G. (1898). A contribution to the study of the fauna of the grave: A study of one hundred and fifty disinterments, with some additional experimental observations. J. NY Ento. Soc. 6, 201–231. Nafte, M. (2000). Flesh and Bone: An Introduction to Forensic Anthropology. Durham, NC: Academic Press. Neis, P., Hille, R., Paschke, M., Pilwat, G., Schnabel, A., Niess, C., et al. (1999). Strontium 90 for determination of time since death. Forensic Sci. Int. 99, 47–51. Papworth, D. G. and Vennart, J. (1984). The uptake and turnover of 90Sr in the human skeleton. Phys. Med. Biol. 29, 1045–1061. Payne, J. A., King, E. W., and Beinhart, G. (1968). Arthropod succession and decomposition of buried pigs. Nature 219, 1180–1181. Pollard, A. M. (1996). Dating the time of death, in Studies in Crime: An Introduction to Forensic Archaeology (J. Hunter, C. Roberts, and A. Martin, Eds.). London: B.T. Batsford, 139–155. Rodriguez, W. C. and Bass, W. M. (1985). Decomposition of buried bodies and methods that may aid in their location. J. Forensic Sci. 30, 836–852.

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246 Shari L. Forbes Sorg, M. H. and Haglund, W. D. (2002). Advancing forensic taphonomy: purpose, theory, and process, in Advances in Forensic Taphonomy: Method, Theory, and Archaeological Perspectives (W. D. Haglund and M. H. Sorg, Eds.). Boca Raton, FL: CRC Press, 3–30. Swift, B. (1998). Dating skeletal remains: Investigating the viability of measuring the equilibrium between 210-Po and 210-Pb as a means of estimating the postmortem interval. Forensic Sci. Int. 98, 119–126. Swift, B., Lauder, I., Black, S., and Norris, J. (2001). An estimation of the post-mortem interval in human skeletal remains: A radionuclide and trace element approach. Forensic Sci. Int. 117, 73–87. Szibor, R., Schubert, C., Schoning, R., Krause, D., and Wendt, U. (1998). Pollen analysis reveals murder season. Nature 395, 450–451. Taylor, R. E., Suchey, J. M., Payen, L. A., and Slota, P. J. (1989). The use of radiocarbon (14C) to identify human skeletal materials of forensic science interest. J. Forensic Sci. 34, 1196–1205. Tuniz, C., Zoppi, U., and Hotchkis, M. A. C. (2004). Sherlock Holmes counts the atoms. Nucl. Instrum. Meth. B 213, 469–475. Turner, B. and Wiltshire, P. (1999). Experimental validation of forensic evidence: A study of the decomposition of buried pigs in a heavy clay soil. Forensic Sci. Int. 101, 113–122. Ubelaker, D. H. (2001). Artificial radiocarbon as an indicator of recent origin of organic remains in forensic cases. J. Forensic Sci. 46, 1285–1287. Ubelaker, D. H., Buchholz, B. A., and Stewart, J. E. B. (2006). Analysis of artificial radiocarbon in different skeletal and dental tissue types to evaluate date of death. J. Forensic Sci. 51, 484–488. Vanezis, P., Sims, B. G., and Grant, J. H. (1978). Medical and scientific investigations of an exhumation in unhallowed ground. Med. Sci. Law 18, 209–221. VanLaerhoven, S. L. and Anderson, G. S. (1999). Insect succession on buried carrion in two biogeoclimatic zones of British Columbia. J. Forensic Sci. 44, 32–43. Vass, A. A., Bass, W. M., Wolt, J. D., Foss, J. E., and Ammons, J. T. (1992). Time since death determinations of human cadavers using soil solution. J. Forensic Sci. 37, 1236–1253. Wells, J. D. and Lamotte, L. R. (2001). Estimating the postmortem interval, in Forensic Entomology: The Utility of Arthropods in Legal Investigations (J. H. Byrd and J. L. Castner, Eds.). Boca Raton, FL: CRC Press, 263–285. Wild, E. M., Arlamovsky, K. A., Golser, R., Kutschera, W., Priller, A., Puchegger, S., et al. (2000). 14C dating with the bomb peak: an application to forensic medicine. Nucl. Instrum. Meth. B 172, 944–950. Wild, E., Golser, R., Hille, P., Kutschera, W., Priller, A., Puchegger, S., et al. (1998). First 14C results from archaeological and forensic studies at the Vienna environmental research accelerator. Radiocarbon 40, 273–281. Willey, P. and Heilman, A. (1987). Estimating time since death using plant roots and stems. J. Forensic Sci. 32, 1264–1270.

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Principles and Methodologies of Measuring Microbial Activity and Biomass in Soil

10

Phil Brookes

Contents 10.1  Introduction............................................................................................. 248 10.2  Measuring Microbial Biomass and Activity........................................ 250 10.2.1  Soil Collection and Preparation.............................................. 250 10.2.2  Soil Dry Matter Content and Water-Holding Capacity....... 253 10.2.2.1  Soil Dry Matter Content........................................ 253 10.2.2.2  Water-Holding Capacity........................................ 253 10.2.3  Measuring the Soil Microbial Biomass.................................. 253 10.2.3.1  Direct Microscopic Counting............................... 253 10.2.3.2  FI Method................................................................ 254 10.2.3.3  FE Method............................................................... 254 10.2.4  Measuring Microbial Biomass C by Fumigation Extraction.................................................................................. 255 10.2.4.1  Reagents and Experimental Procedure................ 255 10.2.4.2  Analysis of Soil Extracts........................................ 256 10.2.4.3  Automated Analysis of Organic C........................ 257 10.2.5  Measuring Microbial Biomass N by Fumigation Extraction.................................................................................. 258 10.2.5.1  Reagents and Experimental Procedure................ 258 10.2.6  Measuring Microbial Biomass Ninhydrin-N by FE............. 258 10.2.6.1  Reagents and Experimental Procedure................ 259 10.2.7  Measuring Microbial Biomass P............................................. 260 10.2.7.1  Reagents for Extraction.......................................... 260 10.2.7.2   Soil Extraction........................................................ 260 10.2.7.3  Preparation of Murphy-Riley Colorimetric Reagent...................................................................... 261 10.2.7.4  Analytical Procedure.............................................. 261 10.2.8  Measuring Microbial Adenosine 5’-triphosphate................. 262 10.2.8.1  Preparation of Stock ATP Solution...................... 262 247

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10.2.8.2  Preparation of ATP Extraction Reagents............ 263 10.2.8.3  ATP Extraction....................................................... 263 10.2.8.4  Measurement of ATP (Firefly Assay)................... 263 10.2.9  Substrate Induced Respiration (SIR)....................................... 264 10.2.9.1  Procedure................................................................. 265 10.2.10  Soil CO2 Evolution and N Mineralization........................... 265 10.2.10.1  Procedure............................................................... 266 10.2.11  Arginine Ammonification...................................................... 267 References............................................................................................................. 267

10.1  Introduction The soil microbial biomass (defined as the sum of the masses of all microorganisms with a diameter less than 500 µm) can be considered as the living fraction of soil organic matter. It is the agent of mineralization of all the dead plant and animal residues that enter soil; it therefore plays a vital role in the cycling of all the major nutrients on the planet (Figure 10.1). The biomass has been eloquently described as “the eye of the needle, through which all organic matter must pass as it is broken down to the simple inorganic components that plants can use again” (Jenkinson 1977). Generally comprising from about 1% to 3% of the total soil organic matter pool, or humus as it is commonly known, the biomass might be thought to live in an environment awash with substrate. In fact, this is far from the case. Soil organic matter, for reasons that we still do not fully understand, is extremely resistant to microbial attack and does not provide enough energy for maintenance, let alone reproduction, of the microbial population. Instead it is inputs of fresh organic matter, such as plant residues, root exudates, or animal remains, that provide the energy for growth and reproduction of this population. Without them the biomass slowly declines in size and activity, although it can exist for months or longer using the slow trickle of nutrients from soil organic matter. However, it is able to rapidly metabolize fresh inputs of fresh organic materials as soon as they become available. Changes in microbial biomass occur much more rapidly in response to changing soil conditions than do those to soil organic matter as a whole. It therefore provides an early warning of changing soil conditions, due either to decreases (decline in biomass) or increases (increases in biomass) in availability of fresh substrate. This usually reflects changes in plant productivity (Powlson and Jenkinson 1976; Powlson et al. 1985). The methodology to measure the soil microbial biomass, introduced by Jenkinson and Powlson (1976), was developed to better understand and

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The Microbial Biomass Concept

Plant and Animals Residues CO2

Microbial and Genetic Diversity

Inorganic N.P.S etc.

Soil Organic Matter

Figure 10.1  The role of the soil microbial biomass in the cycling of plant nutrients.

quantify carbon (C) and nutrient cycling in agricultural soils. The methods were never intended to be used in forensic investigations, and their value in this regard awaits evaluation. However, forensic science already uses techniques from a number of scientific disciplines. For estimating time of death, medical techniques such as measurements of body temperature or rigor mortis may be used, although these measurements can only provide reliable data during the first two or three days after death (Catts and Goff 1992). However, postmortem intervals from the first few days to several weeks can be estimated by determining the age of immature necrophagous insect species present (Amendt, Krettek, and Zehner 2004). There are also different patterns of insect succession in aboveground and buried corpses. Thus, the

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presence of maggot masses may indicate delayed burial (VanLaerhoven and Anderson 1999). A wide-ranging literature survey revealed only one paper that had tried to link microbial biomass and forensic science. Hopkins, Wiltshire, and Turner (2000) measured selected microbial characteristics of soils from the 0–15 cm and 15–30 cm depths of the graves of three buried pigs at 430 days after burial. They showed that the grave soils contained larger total C, nitrogen (N), amino-N, ammonium-N (NH4+–N), and soil microbial biomass C concentrations. Alkali-soluble sulfide concentrations were also increased in the grave soils, indicating reducing conditions. Factors that need to be considered in linking microbial biomass and activity measurements to forensic science investigations include soil pH, soil type (e.g., sandy versus clay soils), drainage, and local climatic conditions. A range of methods to measure microbial biomass and activity are presented here and briefly discussed. Whether such measurements would prove a useful addition to standard forensic scientific techniques await rigorous testing. Research in this area is extremely limited and could be easily undertaken using the methods detailed here. Certainly, entomology is now a well-established forensic methodology, so there is already a successful precedent. However, in addition to possibilities for forensic science, the biomass methodologies are also proven in research into many aspects of soil ecology, including ecosystems as diverse as arable, grassland, and forest soils, so they are already relevant to scientists in many other fields.

10.2  Measuring Microbial Biomass and Activity 10.2.1  Soil Collection and Preparation It may seem almost too simplistic to spend significant time to explain how to collect and handle soils for biomass and activity measurements. Nevertheless, the correct handling of soil from the onset is vital if satisfactory results are to be obtained. Past experience has shown that this step is often done wrongly, with the inevitable result that the analysis is doomed from the start. If a soil is sampled when too wet, it may be damaged during sieving, caused by smearing and the production of anaerobic aggregates. It may also be damaged during drying, which both kills a proportion of the microbial biomass and increases the mineralization potential of nonbiomass soil organic matter. Soil for microbiological studies is normally collected from the plow depth (10–23 cm at Rothamsted, United Kingdom, but it may vary elsewhere) and 0–10 cm depth for grassland or forest soils. For forensic analysis the sample depth will be decided by the nature of the crime scene. However, maximum biological activity will be adjacent to a corpse and will decline with depth

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above and below, due to leaking of body fluids and transference of body tissues by soil fauna. There may also be considerable disturbance of the corpse due to the activities of large scavengers (e.g., foxes, badgers) (Devault, Brisbin, and Rhodes 2004; Hopkins et al. 2000). Sampling is usually with an auger or soil corer, although an ordinary spade or garden trowel may sometimes be the best tool. Every effort should be made to ensure that the pooled soil samples give as representative a bulk soil sample as is possible. Ideally, if a period of drought or heavy rainfall and a sampling date should coincide, the researcher should use his or her discretion whether to go ahead with the soil collection, depending on the reason for which it is required. Clearly, under some circumstances, sampling has to proceed irrespective of conditions. In any case, even if the soil is dry when sampled, it can normally be sieved somehow; similarly, if it is too wet, it may be carefully dried without allowing any portion to become air-dried (see following section). Once the soil has been collected and brought back to the laboratory, it should be spread out on a piece of plastic sheeting and large pieces of plant material, animal tissue, obvious living organisms, (e.g., earthworms, insect larvae) and stones removed by hand. The soil can usually be stored at 4oC for several days or even weeks until preparation. However, ideally it is best to proceed immediately. Depending on the season, soils at field moisture content may be too wet to be sieved immediately after collection without smearing or rolling of the soil occurring. The soil must therefore be dried to a moisture content where sieving is possible without these problems occurring. The soil should be spread out as thinly as possible while remaining as a continuous and even layer. This is particularly important around the edges of the soil. Larger lumps of soil should be carefully broken up by hand so that the pieces of soil are more or less of the same size. If the soil does not break apart along natural fracture lines but simply stretches or smears, then it must be left to dry intact until the moisture content is such that the lumps can be broken apart. The ideal moisture content of a soil is that which allows it to crumble easily when sieved, while still being moist enough to support the whole of its original microbial biomass. While the soil is drying it must be turned (i.e., mixed) regularly so that no part of the soil becomes too dry. The soil at the edges dries the most quickly and can become air-dry while the soil in the center is still too wet to sieve. A piece of soil that is not in contact with others will dry more quickly still— hence the importance of keeping the soil as a continuous layer at all times. If a few soil aggregates do become air-dry, they should be discarded. If, by accident, a significant proportion of the soil becomes air-dry, then it will be useless for experimental purposes, and a fresh sample of soil will have to be collected. Ideally, it is best not to process soil in a glasshouse as temperatures may rise quickly, causing rapid drying and irreparable damage.

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If the soil takes longer than one day to reach the proper moisture content (as is usually the case), it should be returned to cold storage overnight, or, if it is very wet and in a permanently shady and cool position, it may be simply covered by another sheet of plastic. Once the soil has reached the necessary moisture content indicated by the still moist soil being easily crumbled by hand, it can then be passed through a < 2 mm mesh sieve. This is best achieved with the fingers or the back of a scrubbing brush. The entire soil sample must be sieved—do not discard any soil that will not pass readily through the sieve. It may be necessary to carry out the drying and sieving process several times. For soils with a very high clay content, it may not be possible to pass more than a small proportion of the soil through the sieve, whatever its moisture content. If this is the case, then the remaining soil can be air-dried and then sieved using a mechanical roller mill. This soil should then be remoistened with distilled water using a handheld sprayer and then mixed thoroughly with the soil that was sieved by hand. Alternatively, the researcher may consider whether the soil would better be broken up by hand as much as possible and used in an unsieved state (Ocio and Brookes 1990a). Once the soil has been sieved, as much plant and animal residues must be removed as possible. This is best done with a pair of forceps, one small portion of soil at a time. The bulk soil should be covered to prevent any further drying. When the soil has been picked over, subsamples can be taken for determination of moisture content and water-holding capacity (WHC; see following section); the soil should be stored at 4°C until use. It is advisable to use the soil as quickly as possible, and if storage is unavoidable, it should not normally exceed 3 months. When the soil is to be used experimentally, it should be spread out thinly onto plastic sheeting and enough water added via a handheld sprayer to readjust the soil to 40% WHC (the moisture content of soils previously stored at 4°C should be redetermined). Rewetting is a critical process. Many soils have been rendered useless at this stage. The safest way is to first calculate the total volume of water to be added and then to add this volume to a handheld sprayer. The soil should then be evenly spread out onto a plastic sheet to a depth of 1–2 cm. The soil is then sprayed evenly with water and then covered with another plastic sheet. Ideally this should all be done at 4°C but can be done at room temperature if necessary. The soil is then left covered overnight or at least for several hours. It is very important that the soil is not disturbed during this period or lumping will occur; that is, small balls of soil will form, and the rewetting will not be uniform. The rewetted soil is then carefully placed into a labeled plastic bag with a large air space. A folded tissue is placed in the top of the bag to permit air exchange, and the bag is tied loosely with a rubber band. The bag is then placed in an airtight metal drum, together with a 100 ml jar of soda lime and several bottles containing

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distilled water. Following incubation at 25°C for 7 to 10 days, the soil is ready for use. Of course, the measurements are of biomass in the conditioned soil rather than in the soil as sampled. However, some theoretical reasons and empirical observations strongly indicate that the measurements will not be in serious error. 10.2.2  Soil Dry Matter Content and Water-Holding Capacity 10.2.2.1  Soil Dry Matter Content Soil dry matter content is calculated from the loss of weight of previously moist soil following oven drying at 105oC. All measurements should be triplicate determinations. Ideally, 10–20 g of fresh soil should be used. Dry matter (DM) content is calculated from DM% = (Dry weight of soil–fresh weight of soil) × 100%. Soil moisture content (MC) is calculated from: MC% = 100 – DM. 10.2.2.2  Water-Holding Capacity A short length of rubber tubing (~8 cm) is attached to the mouth of the stem of 5 × 100 ml glass funnels. A clip is then placed on each piece of tubing and closed. A ball of glass wool (0.25–0.30 g) is lightly compressed into the neck of each funnel. Three moist portions of 50 g soil, of which the dry matter has been previously determined, are placed in three of the funnels and glass wool only in the other two. Fifty ml of water is then gently poured into each of the funnels and left for 30 minutes to fully saturate the soils. After this time, the clips on the tubing are opened, and the water that drains from each funnel for thirty minutes is collected in a measuring cylinder held beneath the funnel stem. The final volume of water drained is recorded. Alternatively, the soils may be weighed into filter funnels containing filter papers and then water added as already described. Then, the surplus water is drained off, and the soils, still in the filter papers, and blanks may be weighed and oven-dried for 24 hours at 25oC, reweighed, and the water retained calculated by difference. Results are usually expressed as percent of WHC (ml H20 retained 100 g–1 oven dry soil). 10.2.3  Measuring the Soil Microbial Biomass 10.2.3.1  Direct Microscopic Counting Microscopic counting is still the most direct method of estimating the amount of microbial biomass in soil but is technically difficult and completely unsuitable for routine use. Thin films are prepared from an agar-soil suspension, are mounted on microscope slides, and then are treated with an appropriate stain. Phenolic aniline blue is often used as it stains protein and is thus considered to give an estimate of the entire population. The numbers

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and sizes of spherical organisms and the lengths and diameters of fungal hyphae are measured and converted to total biomass by using conversion factors for specific gravity, percentage carbon content, and percentage dry matter, obtained from microorganisms grown in vitro. Other stains, especially fluorescent ones like fluoroscein isothiocyanate or acridine orange, are much easier to count but do not stain the full range of organisms. This method is discussed in more detail by Jenkinson, Powlson, and Wedderburn (1976) and Jenkinson and Ladd (1981). Due to the time and skill required, this method is hardly suitable for routine use. Computer-aided counting procedures are available, but there is little evidence that this method is routinely used for total biomass measurements. Direct microscopy is invaluable, however, in verifying other, indirect methods (e.g., fumigation–incubation [FI]). 10.2.3.2  FI Method The first method to measure total microbial biomass directly was described by Jenkinson and Powlson (1976). They showed that more carbon dioxide (CO2) was evolved from a soil fumigated with chloroform, following fumigant removal and aerobic incubation, than from a similar nonfumigated soil. They subsequently showed that this extra CO2 (the CO2 flush) came from the microbial cells killed by chloroform (CHCl3), as they were decomposed by the subsequent recolonizing population. They suggested that measurement of the CO2 flush could provide an estimate of the amount of microbial biomass in soil. This method was the standard procedure to measure microbial biomass for many years. However, it does have several limitations, a main one being that it does not give valid results in soils that contain actively decomposing substrates—which would certainly preclude it from forensic research. It has been superseded by the fumigation–extraction (FE) method, described in the following section. 10.2.3.3  FE Method Vance, Brookes, and Jenkinson (1987) first showed a close linear relationship between biomass C measured by the FI method and Ec, where Ec= [(organic C extracted by 0.5 molar (M) potassium sulfate (K 2SO4), soil fumigated for twenty-four hours) minus (organic C extracted from a similar, nonfumigated soil)]. They proposed that biomass C can be estimated from the relationship, Biomass C = 2.64 Ec. The FE method does suffer from the disadvantage that comparatively small amounts of C have to be measured in 0.5 M K 2SO4. Vance et al. (1987) used a dichromate digestion method. However, C can be more conveniently determined by an automated system using persuflate and ultraviolet (UV) oxidation, which gives essentially the same results but more rapidly and easily (Wu et al. 1990).

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Chloroform fumigation also increases the amount of total N extractable with 0.5 M K 2SO4. Brookes et al. (1985) showed that this extra N also comes from the microbial biomass and proposed that biomass N could be estimated from the relationship, Biomass N = 2.22 EN, where EN is analogous to Ec. About 16% of the total N released by CHCl3 after twenty-four hours and extracted by K 2SO4 is in either ammonium-N or α-amino N. These forms react with ninhydrin, giving a blue-purple color, and measurement of ninhydrin-N can be used to estimate the amount of biomass C (Amato and Ladd 1988; Joergensen and Brookes 1990). Following CHCl3-fumigation there is also an increase in inorganic phosphorus (Pi) made extractable to 0.5 M sodium bicarbonate (NaHCO3). Brookes, Powlson, and Jenkinson (1982) reported that biomass P could be estimated from measurement of this increase in Pi, with a correction made to account for incomplete extraction of Pi due to fixation on, for example, soil colloids, and assuming that about 40% of the P in the soil microbial biomass is extracted by 0.5 M NaHCO3 following CHCl3-fumigation. The FE method offers some considerable advantages over FI. Biomass measurements can be made across the whole pH range in soils containing actively decomposing substrates (e.g., cereal straw), both in the laboratory (Ocio and Brookes 1990b) and in the field (Ocio, Brookes, and Jenkinson 1991) and in freshly sampled soils, all conditions in which FI is unreliable. Reliable biomass measurements by FE have also been reported in paddy (i.e., waterlogged) soils by Inubushi, Brookes, and Jenkinson (1991). Indeed, provided large soil samples are used (ca. 250 g fresh weight), the soils do not need to be sieved. Instead, the soils may be broken into 1–2 cm pieces and then extracted, as mentioned previously (Ocio and Brookes 1990a). It is worth noting that a slight modification has been introduced in the fumigation procedure. Instead of ethanol-free CHCl3, which is quite laborious to produce and rather unstable (see Jenkinson and Powlson 1976), CHCl3 stabilized with 25 ppm amylene is now commercially available. This gives identical results to those obtained using distilled CHCl3 (Kaiser et al. 1992; Mueller, Joergensen, and Meyer 1992). 10.2.4  Measuring Microbial Biomass C by Fumigation Extraction 10.2.4.1  Reagents and Experimental Procedure Ten litres 0.5 M K2SO4 are prepared by shaking on a reciprocal shaker about 9.5 l distilled H2O and K2SO4 (AR grade) (871 g) in a 10 l aspirator for about two hours. When the K2SO4 has dissolved completely, the volume is adjusted to 10 l with distilled water. Three replicate samples of each sieved (< 2 mm) soil (40%–50% WHC), usually 50 g (oven-dry basis) are weighed into glass

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screw-top jars (60 or 100 ml). They are identified with, for example, gummed labels and numbering, in pencil. The corresponding nonfumigated soils (controls) are weighed directly into 350 ml plastic screw-top bottles and closed to prevent water loss. The jars containing soils to be fumigated are placed in a desiccator containing moistened tissue paper at the base, together with a 25 ml vial of soda lime and a 50 ml beaker containing at least 30 ml CHCl3 and two to three antibumping granules. All operations involving CHCl3 must be done in an efficient fume cupboard. The desiccator is evacuated using a water pump until the CHCl3 boils vigorously for a further two minutes. The desiccator valve is then closed and detached from the pump. The desiccator is then incubated at 25°C for 24 hours in the dark. After this period, there should still be a vacuum in the desiccator. If there is not, the fumigation may be considered to have been successful provided some liquid CHCl3 still remains in the beaker. After the fumigation, the jars are removed and the CHCl3 safely disposed of. The fumigated jars of soil are placed in the newly cleaned desiccator, which is evacuated using a water pump and then using an electric vacuum pump, again three times, each for 2 minutes followed by three 2-minute evacuations with the electric pump. The soil samples are quantitatively transferred to 350 ml plastic screwtop bottles containing 0.5 M K 2SO4 in a ratio of 4:1 (i.e., 50 g oven dry soil is extracted with 200 ml K 2SO4), rinsing the glass jars with some of the K 2SO4, if necessary, and extracted as described following. Unfumigated samples are extracted at the start of the fumigation period. For some procedures (e.g., biomass measurements in soils containing significant quantities of straw), large (i.e., ≥ 250g unsieved soil portions) should be used (Ocio and Brookes 1990b). In this case, 1–2 ml liquid CHCl3 is also dribbled over the surface of the soil prior to fumigation in the usual way. The bottles are placed upright on a reciprocal shaker and are shaken for 30 minutes at approximately 200 strokes per minute. At least three blanks are included. The bottles are removed from the shaker, and the soil suspensions are filtered through Whatman No. 1 filter papers. They may be analyzed immediately or stored at –18°C until required. On thawing, there is usually a white precipitate of calcium sulfate (CaSO4) in the extracts. The bottles should be shaken thoroughly and then allowed to stand so that the precipitate settles out again before any extract is removed for analysis. The extracts are then refrozen in case repeat analyses are required. 10.2.4.2  Analysis of Soil Extracts 10.2.4.2.1  Dichromate Method  The measurement of soluble organic C following extraction with K 2SO4 is based on the reaction, 2Cr2072– + 3C + 16 H+ → 4Cr3+ + 3CO2 + 8H2O. The dichromate that has not been consumed during the reaction is then determined volumetrically following titration with

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standardized ferrous ammonium sulfate (FAS) and related to the amount of organic C oxidized. As with all back-titrations, the problem is that the volume of dichromate oxidized is very small compared with the volume consumed, so the errors may be considerable, especially in inexperienced hands. Nevertheless, excellent results can be obtained with practice. 10.2.4.2.2  Experimental Procedure  An aliquot (8 ml) of the filtered extract, 66.7 millimolar (mM) potassium dichromate (K 2Cr2O7: 2 ml), mercuric oxide (HgO: 70 mg), and a mixture (15 ml) of two parts concentrated sulfuric acid (H2SO4) and one part concentrated phosphoric acid (H3PO4) are accurately transferred to a round-bottomed flask. The mixture is then gently boiled under reflux for 30 minutes, allowed to cool, and diluted with 20–25 ml water. This water is slowly added through the top of the condenser as a rinse. The residual dichromate is measured by back-titration with acidified 0.4 M ferrous ammonium sulfate (FAS) using 25 mM 1,10 phenanthrone ferrous sulphate complex as an indicator. The color change is purple to green. The FAS is then titrated against the 66.7 mM K 2Cr2O7. This result is then the cold (unrefluxed) blank. A hot (refluxed) blank is prepared similarly. The digested soil extracts are then treated similarly (Vance et al. 1987). It is extremely important that the concentrations of the reagents are not changed from the aforementioned numbers or endpoint determinations may become more difficult or even impossible. The amount of dichromate consumed is that remaining in a blank digestion with 8 ml 0.5 M K 2SO4 (hot blank) minus what is remaining in the digest of the extract. Extractable C is calculated assuming that 1 ml 66.7 mM K2Cr2O7 is equivalent to 1200 µg C. Biomass carbon (Bc) is calculated from Bc = 2.64 Ec, where Ec = [(organic C extracted from the fumigated soil) minus (C extracted from the nonfumigated soil)]. The factor Ec is a proportionality constant to account for the fact that only about 45% of the biomass C is made extractable to K 2SO4 after fumigation (Vance et al. 1987). 10.2.4.3  Automated Analysis of Organic C Measurements of biomass C by FE were all originally done by dichromate digestion, exactly as described previously (e.g., Ocio et al. 1991; Vance et al. 1987). The method is perfectly valid and ideal where only a small number of samples are to be analyzed, and then only occasionally. However, since its publication, laboratories where biomass C measurements are routinely done normally use automated organic C analyzers in which organic C is oxidized by UV-persulfate oxidation or following combustion. These methods offer the advantages that the carbon is measured directly rather than following back-titration, increasing the accuracy and precision. The analyzers are normally fully automated, which saves much time and labor. It should

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be noted, however, that because the automated analysis is more efficient, the proportionality factor of 0.38 used following dichromate digestion should be replaced by 0.45 (Wu et al. 1990). Thus, Biomass C = 2.22 Ec, where Ec is as already mentioned. Results are expressed as microgram (µg) Biomass C per gram (g–1) soil. 10.2.5  Measuring Microbial Biomass N by Fumigation Extraction As previously described, following fumigation and extraction of soil, there is a release of organic and inorganic N, analogous to biomass C. Brookes et al. (1985) showed that this N came from the soil microorganisms and could be used to measure microbial biomass N. The total N in the extracts can be measured after Kjeldahl digestion or K 2SO4 digestion. 10.2.5.1  Reagents and Experimental Procedure Thirty ml aliquots of K 2SO4 soil extract are pipetted into digestion tubes containing a few antibumping granules. Ten ml of conc. H2SO4 is slowly and carefully added to the tubes, followed by 0.6 ml copper sulfate (CuSO4: 0.19 M). The mixture is then refluxed at 360 oC for three hours and then allowed to cool. Water (20 ml) is cautiously added to the tubes, and the mixture is cooled again. The inorganic N is then determined by steam distillation of the entire aliquot with 10 M NaOH into a titration vessel containing boric acid (5 ml 2%) until 40 ml of distillate have been collected. The distillate is titrated to pH 4.7 with H2SO4 (50 mM) using either a standard burette or an autotitrator. Biomass nitrogen, (BN; µg g–1 soil) is then is calculated from BN = 2.22 EN, where EN = [(total N extracted from fumigated soil) minus (total N extracted from nonfumigated soil)] and the factor 2.22 (analogous to FE measurements of biomass C) accounts for about 45% extraction efficiency of biomass N. Results are usually expressed as µg BN g–1 soil. 10.2.6  Measuring Microbial Biomass Ninhydrin-N by FE Hydrolytic enzymes remain active during CHCl3 fumigation (Brookes 1982). Amato and Ladd (1988) showed that the amounts of ninhydrin-reactive compounds (i.e., ammonium, amino acids, peptides, and proteins) released from the biomass during fumigation and extracted by 2 M potassium chloride (KCl) are closely correlated to the initial biomass C concentrations. Estimates of biomass from ninhydrin-reactive N measurements (Amato and Ladd 1988) have many advantages. The method is extremely quick (involving a simple colorimetric determination, thus avoiding a tedious digestion step), reliable, and accurate. Biomass measurements of ninhydrin-reactive N (BNIN) are also closely correlated with total biomass N (BN)

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measurements, so a simple conversion factor (BN = 5.0 BNIN; r2 = 0.91) can provide a rough estimate of biomass N. Amato and Ladd (1988) chose KCl as the extractant. However, chloride interferes with the dichromate digestion procedure and with the UV-persulfate methods used to measure biomass C in some automated procedures. In the original ninhydrin method, K 2SO4 precipitates during the colorimetric assay, causing erratic results. Joergensen and Brookes (1990) modified the assay to overcome this problem so that biomass C, biomass N, and biomass α-amino-N and ammonium-N can all be measured in the same extract. As with biomass C and N, biomass ninhydrin-N can also be measured in soils where substrate is decomposing (Ocio and Brookes 1990a) and in acidic (Vance et al. 1987) and waterlogged soils (Inubushi et al. 1991). 10.2.6.1  Reagents and Experimental Procedure To prepare the ninhydrin reagent, ninhydrin (2 g) and hydrindantin (300 mg) are dissolved in dimethyl sulfoxide (DMSO) (75 ml) in a foil-covered 400 ml flask (to exclude light) and are adjusted to 100 ml with 4 M lithium acetate buffer. The jar is stoppered with a rubber bung with inlet and outlet tubes, and the reagent is flushed with O2-free N2 for 30 minutes. The operations must be done in a fume hood. The final solution is then stored sealed at room temperature. Ninhydrin is toxic and stains the skin, so gloves should be worn at all times. To prepare the citric acid buffer, sodium hydroxide (NaOH) (16 g) is dissolved in a minimum amount of distilled water in a beaker, cooled, transferred to a 1 l volumetric flask, and about 900 ml water is added. Citric acid (42 g) is dissolved in this solution, the pH adjusted to 5.0 first with 10 M abd then 1 M NaOH, and the volume adjusted to 1 l with water. The mixture is then transferred to a reagent bottle and is stored at 5°C in the dark. Ethanol water is prepared by mixing 1 l water and 1 l 95% ethanol in a 2.5 l brown glass bottle. To prepare the ninhydrin-N standard, a solution containing 1,000 micromolar (μM) leucine-N is prepared by dissolving D-leucine (0.1312 g) in 1 l 0.5 M K2SO4. Standards ranging between 0 and 250 µM leucine-N are prepared and stored at –18°C. Triplicate aliquots (0.75 ml) of standard solutions, blanks, and soil extracts are pipetted into labeled 20 ml thick-wall test tubes (15 mm diameter). Citric acid buffer (1.75 ml 0.2 M) is pipetted into each tube. Ninhydrin reagent (1.25 ml) is then pipetted slowly into each tube and vortex-mixed for two seconds. The top of each test tube is loosely covered with aluminum foil and placed in a vigorously boiling water bath for 25 minutes. There should be sufficient water in the bath so that the water level is always above the level of the solutions in the test tubes. The water should return to boiling within 2 minutes of the tubes’ being placed in the bath. After 25 minutes, the test tube

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rack is removed from the water bath, cooled at room temperature in the dark for approximately 2 minutes, and then put in a tray of water, similarly in the dark, until ambient temperature is reached. Ethanol-water solution (4.5 ml 1:1) is dispensed to each tube and vortex-mixed thoroughly several times. After each rack is processed it is wrapped in aluminium foil and stored in the dark. The solutions are briefly vortex-mixed on the vortex mixer again, and the absorbencies of the solutions are measured at 570 nm against a distilled water blank. Results are expressed as µg BNIN g–1 soil. 10.2.7  Measuring Microbial Biomass P Soil microbial biomass P is measured after the method of Brookes et al. (1982). It is calculated from the difference between the P extracted by 0.5 M NaHCO3 at pH 8.5 (Olsen reagent) from unfumigated soil and soil that has been fumigated with alcohol-free chloroform for 24 hours at 25°C. Most of the biomass P release following fumigation actually occurs during extraction and not during fumigation. Some of it will be adsorbed by soil colloids, so to make an approximate allowance for this fraction a known concentration of P (usually 250 µg P 100 ml –1 Olsen reagent) is added to the Olsen reagent before extraction of a further set of soils. A further correction is also made because only about 40% of the P in the original biomass is released to Olsen reagent following extraction. Thus, three sets of soil are used to measure biomass P, as explained following. Measurement of inorganic P is by the colorimetric method of Murphy and Riley (1962). As with all biomass and activity measurements fresh soil is used, but results are reported in terms of oven dried (24 hours, 105°C) soil. 10.2.7.1  Reagents for Extraction To prepare the Olsen Reagent, 0.5 M NaHCO3 (420 g) is weighed into approximately 9 l water, NaOH pellets (7.2g) added, the pH adjusted to 8.5 with 10 M NaOH, and then the final volume adjusted to 10 l. To prepare the P spike, potassium dihydrogen phosphate (KH2PO4: 1.0975 g) is quantitatively transferred into a 1 l volumetric flask with water and adjusted to 1 l. This will provide 250 µg P ml–1. 10.2.7.2   Soil Extraction Nine portions of moist soil are weighed into three sets, each containing 10 g soil on an oven-dry basis. This provides three replicates each of (1) fumigated, (2) nonfumigated, and (3) nonfumigated P-spiked samples. The first set of samples is fumigated for 24 hours at 25oC as described previously and the other two sets at 25oC for 24 hours in a sealed desiccator containing water and soda lime. For each soil, 200 ml Olsen reagent is added to 9 × 250 ml numbered 250 ml conical flasks. To Sets 1 and 3, 1 ml of water is added. To

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Set 2, 1 m1 of KH2PO4 containing 250 µg P ml–1 is added and mixed gently. The soils are carefully added to the flasks, rinsing in any soil residues. A set of at least three blanks is included for each full run. The mixtures are shaken for 30 minutes at 25oC on an orbital shaker (30 minutes, 150 rpm) and then are filtered (fluted Whatman 42) into clean plastic bottles. The Olsen method is extremely sensitive to changes in operating conditions, and unless care is taken, reproducible results will not be obtained. It is therefore imperative that all the extractions are done at the same temperature, at the same shaking speeds, and on the same shaker each time if results are to be compared between soils. For example, it is important even to adopt a standard method of filtration. The one we use is to swirl the flask briskly, add soil extract to the filter paper, and then replace the flask on the bench. Proceed in exactly the same way with the next soil replicate. If a top up is required, all flasks should be topped up in the same way. Analyze the same day if possible, although extracts may be frozen. 10.2.7.3  Preparation of Murphy-Riley Colorimetric Reagent The following solutions are prepared: (1) Ammonium molybdate (40 g) [(NH4) 6MO7O24.4H2O] is dissolved in about 900 m1 water then adjusted to 1 l. (2) Potassium antimony tartrate (1.454 g) (KSbO.C4H4O6) is dissolved in 450 ml water. (3) L-Ascorbic acid (C6H8O6) (26.4 g) is dissolved in 1 l water and prepared freshly for each analysis. (4) About 1.5 l water is placed in a plastic beaker and 278 ml concentrated H2SO4 slowly added, mixing carefully as it is added. After cooling, the solution is added to a 2 l volumetric flask and adjusted to volume to provide 2 l 2.5 M H2SO4. To prepare the colorimetric reagent, sulfuric acid (250 ml 2.5 M) is added to ammonium molybdate (75 ml) and mixed. Then, ascorbic acid (50 ml) is added and mixed, followed by antimony potassium tartrate (25 ml) and further thorough mixing. The volume is finally adjusted to 500 ml. A standard solution containing 40 mg P l–1 prepared by dissolving potassium dihydrogen phosphate (0.1757 g) in about 900 ml water and adjusting to 1 l. This solution is then diluted to give standards of 5, 10, 25, 50, 75, 100 µg P l–1, and water blanks are also prepared. 10.2.7.4  Analytical Procedure Hydrochloric acid (1 ml 5 M) is added to 10 ml water in a 100 ml volumetric flask. Olsen soil reagent or Olsen extractant (blank) (both 10 ml) are then added and the flask swirled until all effervescence has ceased. Murphy-Riley

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reagent (8 ml) is added and mixed again. Once effervescence is over, the volume is adjusted to 100 ml, and the flasks are adjusted to volume and then are stoppered. A set of standards in water is prepared similarly but without the 5 M HCl. The absorbances are read on a spectrophotometer at a constant wave length of either 710 or 882 nm. Biomass P (BP) is calculated from: BP = (P extracted from fumigated soil minus P extracted from nonfumigated soil) × (1/kp) x (100/R), where kp is the fraction of BP released by CHCl3 (0.40) and extracted by 0.5 M NaHCO3 and R is the percentage recovery of the spike of added inorganic P. The results are expressed as µg BP g–1 soil. 10.2.8  Measuring Microbial Adenosine 5’-triphosphate Adenosine 5’ triphosphate (ATP) is an important energy compound in the metabolism of all living organisms. Soil ATP content is closely correlated with other indices of biomass (e.g., C, N) and can serve as an independent estimate of soil biomass content. Problems occur in the extraction of ATP from soil, such as ATP hydrolysis by ATPases in soil and sorption of ATP onto soil colloids. We use the method developed by Jenkinson and Oades (1979), using a trichloroacetic acid/phosphate/paraquat reagent to extract ATP and ultrasonification to disrupt the microbial cells. The anion (phosphate) and the cation (paraquat) are strongly sorbed on positive and negative soil sites, thus blocking sites that would otherwise adsorb microbial ATP. Even using these reagents some ATP may still be adsorbed onto the soil, so it is necessary to calculate the amount recovered. This is done by adding a known amount of ATP to extractant B and comparing the counts obtained with those from extractant A without added ATP. Following ultrasonics, the filtered soil extracts can be analyzed immediately or stored frozen (–18°C) for weeks or months. The ATP is assayed by the firefly luciferin-luciferase enzyme system using a bioluminometer or scintillation counter set to count out of coincidence. 10.2.8.1  Preparation of Stock ATP Solution A stock ATP solution is prepared by dissolving di-Na ATP tetrahydrate (59.9 mg) in water, adjusting to 1000 ml. It is stored frozen at –15°C. This provides a solution containing 0.1 mM ATP. This stock solution may conveniently be frozen in convenient aliquots (e.g., 150–200 ml) to facilitate thawing as required. It is stable for at least one month. The molecular weight may change from batch to batch due to variations in water content. The molarity must therefore be calculated from the molecular weight quoted on each specific bottle of ATP as supplied by the manufacturer.

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10.2.8.2  Preparation of ATP Extraction Reagents Trichloroacetic acid (TCA: [81.6 g]) and disodium hydrogen orthophosphate dodecahydrate Na2HPO4.12H2O (89.6 g) are dissolved in 600 ml water. Paraquat dichloride (1,1-dimethyl-4,4’-bipyridinium dichloride) (70 ml 36%) is then added, and the solution is adjusted to 1,000 ml with distilled water. The mixture is then cooled on ice to prevent formation of large crystals that are slow to dissolve on thawing and is stored at –15 oC. Extractant A is prepared from 5 ml distilled water adjusted to 1,000 ml with stock solution. Extractant B is prepared by adding 10 ml stock solution (0.1 mM ATP) to approximately 950 ml Extractant A and adjusting to 1 l with further Extractant A. Normally it is advisable to prepare 5 l of stock solution so that the same batch of extractant is used for the complete analysis. 10.2.8.3  ATP Extraction To determine the ATP concentration in a single soil, six portions of moist soil, each containing the equivalent of 5 g oven-dry soil, are weighed separately into 6 × 100 ml plastic centrifuge tubes and held on ice. Extractant A is added to the first tube and sonicated immediately for 2 minutes at 760 MHz. The tube is then cooled on ice for at least 5 minutes. The next two tubes are then treated in the same way followed by three similar extractions with Extractant B. Three tubes containing Extractant A and three containing Extractant B without soil serve as blanks. Normally up to 20 soils are analyzed at the same time, and two sets of blanks, each in triplicate, are prepared similarly. Following cooling, the extracts are filtered (Whatman 42) until about 5 ml extract or blank are collected, recooled on ice, and then frozen at –15°C. They are stable for at least 3 months. If paraquat cannot be used it may be omitted, with only a small loss in precision and extraction efficiency of native ATP (Inubushi, Brookes, and Jenkinson 1989). As the reagents are toxic, full safety precautions must be taken and any wastes disposed of safely depending on local regulations. 10.2.8.4  Measurement of ATP (Firefly Assay) The method originally proposed by Jenkinson and Oades (1979) is described here. It is based on extraction of ATP with the TCA reagent, with the scintillation counter set out of coincidence as described previously, followed by scintillation counting using a large dilution of the TCA reagent (50 l) in arsenate buffer (5 ml). The ATP is measured following addition of the firefly luciferin-luciferase enzyme assay. The enzyme assay system can be obtained from a number of companies supplying biochemicals for research and should be made up for use according to individual instructions. Bioluminometers can also be used to measure ATP. However, unlike arsenate buffer, they measure light released from the ATP-luciferin-luciferase reaction in a single burst

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immediately after adding the reactants. Also, bioluminometers usually use TRIS buffer, which may not be effective in removing the inhibitory effect of TCA on the enzyme system. In contrast, arsenate buffer has the very useful property that light is emitted slowly and proportionately to ATP concentration over at least 24 hours. Indeed, we generally find that it is best to wait at least 1 hour before actually using the counts obtained, and the count may be delayed even longer if required. 10.2.8.4.1  Sodium Arsenate Buffer (To Make 1 Litre)  Sodium arsenate (Na2HAsO4.7H2O: 31.2 g) is dissolved in 800 ml water; then 0.2 M EDTA (10 ml) and then magnesium sulfate (MgSO4.7H2O: 2.46 g dissolved in 100 ml water) are added; and the pH is adjusted to 7.4. The final volume is then adjusted to 1,000 ml. This provides a solution 0.1 M with respect arsenate, 0.01 M with respect to Mg2+, and 0.02 M with respect to EDTA. 10.2.8.4.2  Preparation of ATP Standard Solutions  One ml of 0.1 mM ATP is pipetted into a 50 ml volumetric flask and adjusted to volume with extractant A. This provides 100 picomolar (pM) ATP 50 μl–1 standard solution. Further dilutions may be made to provide from 0.5 pM ATP 50 μl–1. The calibration is linear over this range. 10.2.8.4.3  ATP Luciferin-Luciferase Reagent  This is prepared as per manufacturer’s instructions and then kept on ice. Arsenate buffer (5 ml) is pipetted into labeled 20 ml scintillation vials, and 50 µl TCA soil extract, standards, or blanks are added. It is best to add a set of standards and blanks at both the beginning and end of the assay to check that the counting is uniform throughout. Finally, the luciferin-luciferase reagent is added (usually 50 or 100 µl per scintillation vial); the vial is swirled gently and counted for ten seconds. It is recommended that a few test vials are prepared and counted before the main analysis to ensure that the assay is functioning correctly. Results are typically expressed as nanomolar (nM) ATP g–1 soil or µM ATP g–1 biomass C. 10.2.9  Substrate Induced Respiration (SIR) Following excess glucose addition to soil, there is a rapid increase in the rate of evolution of CO2-C. Over the first 2 hours, the increase in CO2-C is proportional to the size of the initial microbial biomass. After this time the relationship weakens as new microbial cells proliferate (Anderson and Domsch 1978). This method, usually termed substrate induced respiration, provides a rapid means of measuring the microbial biomass. Lin and Brookes (1999a) also showed that it was strongly correlated with ATP and biomass C measured by microscopy. The SIR method, in combination with the use of an antibiotic (streptomycin) and a fungicide (cyclohexamide) can also provide

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estimates of the relative proportions of fungal and bacterial biomasses in soil (Anderson and Domsch 1978; Lin and Brookes 1999a). 10.2.9.1  Procedure Three portions of moist (40% WHC) soil (equivalent to 10 g oven-dry soil) are weighed into 250 ml quick-fit flasks. Soil water may vary from 35% to 75% WHC. Then finely ground glucose-talcum powder (to provide 4–6 mg glucose-C g–1 soil) is added to the soils and mixed carefully and thoroughly, without damaging the soil. The flasks are stoppered 30 minutes after glucose addition, and the CO2 evolved is measured by gas chromatography 2 hours after glucose addition. If the soil pH is higher than 6.6, calibration for CO2 retained in soil water is required. The procedure is as follows. A portion of soil is fumigated with chloroform at 25°C for 48 hours. After removal of chloroform, the same amount of soil as used previously (Procedure 1) is weighed into flasks. The CO2 concentration in the flasks is measured, and then a series of volumes of pure CO2 is added separately to different flasks to make a calibration curve. The flasks are kept at 25°C for 2 hours, and the CO2 concentration in the flasks is measured as the final CO2 concentration. The CO2 retained in soil is calculated from the curve. If the CO2 concentration in the flask is too high (> 1.0%) a significant amount of CO2 will dissolve, even in soils of lower pH. The weight of soil used in the measurement depends on the initial soil microbial biomass concentration and the flask volume. Normally, 20–40 g moist soil is suitable for this measurement. Lin and Brookes (1999a) showed that SIR can give reliable estimates of microbial biomass in unamended soils, soils that contain actively decomposing plant residues, or soils recently treated with pesticides or fumigants. The final results are usually expressed as the SIR rate: µl CO2 evolved g–1 soil h–1. Biomass carbon (BC) is estimated by BC (µg C g–1 soil) = 15 SIR (Lin and Brookes 1999a). 10.2.10  Soil CO2 Evolution and N Mineralization Soil respiration is a very valuable measurement in soil microbial ecology, giving information about the mineralization rate of soil organic matter and, simultaneously, the energetic state of the soil microbial biomass (biomass specific respiration). It is generally measured from CO2 evolved from soil (see Tibbett et al. 2004). Carbon dioxide evolution may be determined by backtitration with hydrochloric acid (HCl) or eclectrical conductivity (Rodella and Saboya 1999) following trapping in NaOH or potassium hydroxide (KOH). In addition, CO2 evolution can be measured by gas chromatography (Putman 1976) or infrared gas analysis (Hashimoto 2002) following accumulation in a sealed vessel. Oxygen measurement is usually less convenient.

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10.2.10.1  Procedure 10.2.10.1.1  Reagents  To prepare 10 l of 1.0 M NaOH, sodium hydroxide pellets (400 g l–1) are cautiously dissolved in a minimum amount of distilled water, in a 5 l plastic beaker, using full safety precautions. Once the solution has cooled it is quantitatively transferred to a plastic aspirator and adjusted to 10 l final volume with distilled water. It may be stored in an aspirator stoppered with a rubber bung, which holds a glass tube containing soda lime. To prepare 1 M HCl, concentrated AR HCl (85 ml) is added to about 900 ml distilled water and then is adjusted to 1 l to provide a solution of approximately 1 M HCl. This is standardized against 0.5 M Na2CO3, prepared by dissolving oven-dry (24 hours, 105°C) anhydrous Na2CO3 (2.650 g) in distilled water and adjusting to 100 ml. 10.2.10.1.2  Soil Incubations  Sodium hydroxide (20 ml 1.0 M NaOH) is slowly pipetted, via an automated dispenser, into 40 ml glass vials, or similar, and then is placed in 1 l glass jars containing CO2-free distilled water (10 ml) and soil in a 60–100 ml glass bottle at 40%–50% WHC. The larger jar is stoppered securely with a clean rubber bung and is incubated at 25°C for the required period in darkness. Blank incubations are set up similarly, except that no soil is included. It is advisable to open all jars after seven days, replacing the NaOH vials with fresh NaOH and allowing air replacement. Treated thus, the incubations may be continued indefinitely. The removed NaOH vials may be stored in a desiccator or airtight glass jar for many weeks, if necessary, together with a vial of soda lime. After a few weeks, the solutions become cloudy and a white precipitate, presumably sodium silicate, forms. This does not appear to affect the results. 10.2.10.1.3  Carbon Mineralization Measured by Gas Chromatography  Carbon dioxide accumulation may also be conveniently measured by gas chromatography. In this case the incubation vessels are sealed with a Suba-Seal, and volumes of air are removed via a syringe at intervals. Again, the incubation vessels should be opened at intervals to permit aeration. 10.2.10.1.4  Nitrogen Mineralization  The mineralization of soil organic N may conveniently be measured at the same time as C mineralization. However, to provide cumulative CO2, though a single set of soils and blanks may be incubated and CO2 evolved measured, this cannot be done for inorganic N measurements unless the soils are leached at intervals. This provides its own set of problems and is not discussed further here. Instead, we set up sufficient incubations so that a new set of soils is harvested at each time interval. This is not a problem within itself, but it can make the experiment unwieldy unless it is accounted for during the experimental design. The inorganic N accumulated may be extracted with 2 M KCl if biomass C measurements

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are not required, as chloride interferes both with dichromate digestion and automated organic C analysis. Otherwise, 0.5 M K 2SO4 must be used. The extracts may be frozen at –18°C until required. Results are usually expressed as µg CO2 g–1 soil, µg NH4+-N g–1 soil, µg NO3–-N g–1 soil, or µg NH4+-N plus NO3–-N g–1 soil. 10.2.11  Arginine Ammonification Ammonium and nitrate are mineralized from arginine following addition to soil. The amounts of nitrate, ammonium, and ammonium plus nitrate are all closely correlated with biomass C measured by FI, CO2 evolved during SIR and soil ATP content (r = 0.83 to 0.91) (Lin and Brookes 1999b). Arginine ammonification appears to be a rapid method to estimate the biomass. However, it does not give valid biomass measurements in acid soils and in soils containing actively decomposing substrates. In view of these and other limitations, the method is not discussed further.

References Amato, M. and Ladd, J. N. (1988). Assay for microbial biomass based on ninhydrin-reactive nitrogen in extracts of fumigated soils. Soil Biol. Biochem. 20, 107–114. Amendt, J., Krettek, R., and Zehner R. (2004). Forensic entomology. Naturwissenschaften 91, 51–65. Anderson, J. P. E. and Domsch, K. H. (1978). A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biol. Biochem. 10, 215–221. Brookes, P. C., Landman, A., Pruden, G., and Jenkinson, D. S. (1985). Chloroform fumigation and the release of soil nitrogen: A rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 17, 837–842. Brookes, P. C., Powlson, D. S., and Jenkinson, D. S. (1982). Measurement of microbial biomass phosphorus in soil. Soil Biol. Biochem. 14, 319–321. Catts, E. P. and Goff, M. L. (1992). Forensic entomology in criminal investigations. Ann. Rev. Entomol. 37, 253–272. Devault, T. L., Brisbin, I. L. Jr., and Rhodes, O. E. (2004). Factors influencing the acquisition of rodent carrion by vertebrate scavengers and decomposers. Can. J. Zool. 82, 502–509. Hashimoto, S. (2002). A simple technique to analyze a small volume of soil CO2 gas using an infrared gas analyser. Soil Biol. Biochem. 34, 273–275. Hopkins, D. W., Wiltshire, P. E. J., and Turner, B. D. (2000). Microbial characteristics of soils from graves: An investigation at the interface of soil microbiology and forensic science. Appl. Soil Ecol. 14, 283–288. Inubushi, K., Brookes, P. C., and Jenkinson D. S. (1989). Influence of paraquat on the extraction of adenosine triphosphate from soil by trichloroacetic acid. Soil Biol. Biochem. 21, 741–742.

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268 Phil Brookes Inubushi, K., Brookes, P. C., and Jenkinson, D. S. (1991). Soil microbial biomass C, N and ninhydrin-N in aerobic and anaerobic soils measured by the fumigationextraction method. Soil Biol. Biochem. 23, 737–741. Jenkinson, D. S. (1977). The soil biomass. N. Z. Soil News 25, 213–218. Jenkinson, D. S. and Ladd, J. N. (1981). Microbial biomass in soil: measurement and turnover, in Soil Biochemistry, vol. 5 (E. A. Paul and J. N. Ladd, Eds.). New York: Marcel Dekker, 415–471. Jenkinson, D. S. and Oades, J. M. (1979). A method for measuring adenosine triphosphate in soil. Soil Biol. Biochem.11, 193–199. Jenkinson, D. S. and Powlson, D. S. (1976). The effects of biocidal treatments on metabolism in soil. V. A method for measuring soil biomass. Soil Biol. Biochem. 8, 209–213. Jenkinson, D. S., Powlson, D. S., and Wedderburn, R. W. M. (1976). The effects of biocidal treatments on metabolism in soil: III: The relationship between soil biovolume, measured by optical microscopy, and the flush of decomposition caused by fumigation. Soil Biol. Biochem. 8, 189–202. Joergensen, R. G. and Brookes, P. C. (1990). Ninhydrin-reactive nitrogen measurements of microbial biomass in 0.5 M K 2SO4 soil extracts. Soil Biol. Biochem. 22, 1023–1027. Kaiser, E.-A., Mueller, T., Joergensen, R. G., Insam, H., and Heinemeyer, O. (1992). Evaluation of methods to estimate the soil microbial biomass and the relationship with soil texture and organic matter. Soil Biol. Biochem. 24, 675–683. Lin, Q. and Brookes, P. C. (1999a). An evaluation of the substrate-induced respiration method. Soil Biol. Biochem. 31, 1969–1983. Lin, Q. and Brookes, P. C. (1999b). Arginine ammonification as a method to estimate soil microbial biomass and microbial community structure. Soil Biol. Biochem. 31, 1985–1997. Mueller, T., Joergensen, R. G., and Meyer, B. (1992). Estimation of soil microbial biomass C in the presence of living roots by fumigation-extraction. Soil Biol. Biochem. 24, 179–181. Murphy J. P. and Riley J. P. (1962). A modified single solution method for the determination of phosphate in soils: 1: Extraction Method. Anal. Chim. Acta 27, 31–36. Ocio, J. A. and Brookes, P. C. (1990a). An evaluation of methods for measuring the microbial biomass in soils following recent additions of wheat straw and the characterization of the biomass that develops. Soil Biol. Biochem. 22, 685–694. Ocio, J. A. and Brookes, P. C. (1990b). Soil microbial biomass measurements in sieved and unsieved soil. Soil Biol. Biochem. 22, 999–1000. Ocio, J. A., Brookes, P. C., and Jenkinson, D. S. (1991). Field incorporation of straw and its effects on soil microbial biomass and soil inorganic N. Soil Biol. Biochem. 23, 171–176. Powlson, D. S. and Jenkinson, D. S. (1976). The effects of biocidal treatments on metabolism in soil: II: Gamma irradiating autoclaving, air-drying and fumigation. Soil Biol. Biochem. 8, 179–188. Powlson, D. S., Jenkinson, D. S., Pruden, G., and Johnston, A. E. (1985). The effect of straw incorporation on the uptake of nitrogen by winter wheat. J. Sci. Food Agric. 36, 26–30.

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Putman, R. J. (1976). The gas chromatograph as a respirometer. J. Appl. Ecol. 13, 445–452. Rodella, A. A. and Saboya, L. V. (1999). Calibration of conductimetric determination of carbon dioxide. Soil Biol. Biochem. 31, 2059–2060. Tibbett, M., Carter, D. O., Haslam, T., Major, R., and Haslam, R. (2004). A laboratory incubation method for determining the rate of microbiological degradation of skeletal muscle tissue in soil. J. Forensic Sci. 49, 560–565. Vance, E. D., Brookes, P. C., and. Jenkinson, D. S. (1987). An extraction method for measuring soil microbial biomass C. Soil Biol. Biochem. 19, 703–707. VanLaerhoven S. L. and Anderson G. S. (1999). Insect colonisation on buried carrion in two biogeoclimatic zones of British Columbia. J. Forensic Sci. 44, 32–43. Wu, J., Joergensen, R. G., Pommerening, B., Chaussod, R., and Brookes, P. C. (1990). Measurement of soil microbial biomass C by fumigation-extraction—An automated procedure. Soil Biol. Biochem. 22, 1167–1169.

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Methods of Characterizing and Fingerprinting Soils for Forensic Application

11

Lorna A. Dawson, Colin D. Campbell, Stephen Hillier, and Mark J. Brewer

Contents 11.1  Introduction.............................................................................................. 272 11.2  Soil Evidence............................................................................................ 272 11.2.1  Background and Historical Perspective.................................. 272 11.2.2  The Evidential Value of Soils......................................................274 11.3  Methods.................................................................................................... 275 11.3.1  Sampling and Handling............................................................. 275 11.3.2  Physical Characteristics of Soil................................................. 277 11.3.2.1  Color............................................................................. 277 11.3.2.2  Particle Size Distribution Analysis.......................... 278 11.3.2.3  Microscopy.................................................................. 279 11.3.3  Chemical Analyses...................................................................... 281 11.3.3.1  Elemental Analysis, Trace Metals, Pollutants......... 281 11.3.3.2  Infrared (IR) Fingerprinting Methods.................... 284 11.3.3.3  Other Chemical Fingerprinting Methods............... 285 11.3.3.4  Mineralogy.................................................................. 285 11.3.4  Biological Analyses..................................................................... 291 11.3.4.1  Palynology................................................................... 291 11.3.4.2  Diatoms........................................................................ 295 11.3.4.3  Soil Organic Matter (SOM)....................................... 295 11.3.4.4  Botanical Fragments................................................... 296 11.3.4.5  Microbial Fingerprints............................................... 298 11.3.5  Combined Approaches............................................................... 301 11.3.6  Polyphasic Approaches and Links to Databases.................... 303 11.3.7  Statistical Considerations and Presentation of Evidence in Court......................................................................................... 304 11.4  Conclusions.............................................................................................. 306 References............................................................................................................. 307

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11.1  Introduction This chapter reviews the range of methods currently available for characterizing and fingerprinting soil for use in forensic investigations. It covers traditionally utilized methods as well as more novel techniques. It does not, however, cover areas such as indicators of the presence of cadavers (Forbes, Stuart, and Dent 2002) or means of identification such as ground penetrating radar (GPR) (Ruffell and McKinley 2005) and archaeological recovery (Hunter et al. 2001). Soil forensic analysis has been included in the wider term environmental forensics (which is often associated with contamination issues) (see Murphy and Morrison 2002) and in the term forensic geoscience (Murray and Tedrow 1975, 1992), although this often ignores the organic components found as physical evidence (see Pye and Croft 2004). In this chapter, the mineral, organic, and living components of soil are all considered as part of the wider range of potentially available evidence from soil. The chapter also looks toward future developments with an emphasis on the need to develop quantitative and statistically robust methods to avoid the pitfalls of expert judgment alone.

11.2  Soil Evidence 11.2.1  Background and Historical Perspective The concept of using soil evidence was first introduced with the writings of Sir Arthur Conan Doyle between 1887 and 1893. He was a physician who used his scientific expertise to encourage the use of scientific evidence through his Adventures of Sherlock Holmes book series. In 1893, Hans Gross wrote his book Handbook for Examining Magistrates, in which he suggested that one could tell more about where someone had last been from the material on their shoes than from inquiries. Soil evidence was, however, first formally presented in court in 1904, when a forensic scientist called Georg Popp from Frankfurt, Germany, was asked to examine the evidence in a murder case. A handkerchief left at the scene of crime contained bits of coal, particles of snuff, and grains of minerals, particularly hornblende. Popp found both coal and mineral grains, including the mineral hornblende, under the suspect’s fingernails. In addition, minerals from two layers of soil found on the suspect’s trousers matched those found in a sample collected from the place where the body had been found, and the second soil type matched samples collected from the path that led from the murder scene to the suspect’s home. From this evidence, it was concluded that the suspect picked up the lower soil layer at the scene of the crime and that this lower, thus earlier, material was covered by splashes of mica-rich mud from the path on his return

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home. When confronted with this evidence, the suspect, Laubach, admitted to the crime (Murray 2004a). Later, in 1908, Popp presented evidence from soil adhering to a suspect’s shoes. The suspect’s wife testified that she had dutifully cleaned his best shoes the day before the crime. Those shoes had three layers of soil adhering to the leather in front of the heel. Popp, using the methods available at that time, estimated that the oldest layer contained goose droppings and other soil material that compared to samples in the path outside the suspect’s home. The second layer contained red sandstone fragments and other particles that compared well to samples from the scene where the body had been found. The most recent layer contained brick, coal dust, cement, and a whole series of other materials that compared to samples from a location outside a castle where the suspect’s gun and clothing had been found. The suspect said that he had walked only in his fields (underlain by porphyry with milky quartz) on the day of the crime. No porphyry was found on the shoes (Murray 2004a). In this case, Popp had used much of the principles that are involved in present-day forensic soil examination. Popp compared two sets of samples and identified them with two of the scenes associated with the crime and confirmed a sequence of events consistent with the theory of the crime. Popp set a foundation for the science of environmental forensics, which is increasingly being used in a range of both criminal and civil cases involving comparison of samples associated with the crime scene to those associated with investigation of suspects. The French criminalist Edmond Locard established what is now known as Locard’s exchange principle: “Whenever two objects come into contact with each other, there is always a transfer of material.” (Murray 2004, p. 43a). Many of the more recent advances in the use of soil in forensic investigations occurred after the publication of the book Forensic Geology by Murray and Tedrow (1975). In 1978 the body of Aldo Moro, the kidnapped Italian prime minister, was found in a car parked in the center of Rome. In this investigation, use was made of a multiple-technique approach. Sand from the car was identified as coming from the seashore close to Rome. A section of seashore with a limited number of roads leading to the beach was identified as compatible with the textural and compositional characteristics of the sand. The study of the vegetation in the car sample suggested that they had been picked up in a period of time close to the killing. Polyester of the type used in boat manufacturing was also found under the car fenders, in the tire tread, and inside the car, as well as under Mr. Moro’s shoes. Pollen analysis showed that adhesion of volcanic soil to the car fenders antedated adhesion of the sand (Lombardi 1999). Soil evidence has also been successfully used and reported in New Mexico (Daugherty 1997), California (Lee et al. 2002), and Australia (Hanson et al. 2002). Many more examples throughout history where soil has been used as physical evidence are given in Chapter 1 of Murray and Tedrow (1992) and in Chapters 1 and 2 of Murray (2004a).

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High-profile cases such as the work of the U.S. Federal Bureau of Investigation (FBI) in the Camarena case (McPhee 1997), the laboratory of the Garda Siochana in the Lord Mountbatten case, and G. Lombardi in the Aldo Moro case (Lombardi 1999) have contributed to the general recognition that soil as physical evidence can indeed make significant and important contributions to criminal investigations. However, there is still a general lack of awareness among members of the legal profession and police force as to its true potential, although a range of exciting new approaches have recently been outlined by Pye and Croft (2004). 11.2.2  The Evidential Value of Soils Soils and related materials do have evidential value. This value lies in the many kinds of materials and the large number of measurements and observations that can be made on a soil sample. Soils contain mineral material, remnants of vegetation (both vegetative and as pollen), and animal and microbial material (living and dead). All these can be characterized using a range of increasingly sophisticated methods. There may also be rare or unique components such as fossils or artifacts, including glass or bone fragments, which are useful for unique characterization. More often, however, the soil sample does not contain any obvious unique components; consequently, its value relies on accurate characterization of the common constituents of a soil. The number of different soils and the number of observations and measurements that can be made on them are almost unlimited. There are very many kinds of rocks, minerals, fossils, plants, animals, and microbes within soils. These diverse components in a soil sample, varying both horizontally and vertically, can all be characterized. It is this diversity, combined with an increasing ability to measure and observe these different kinds of attributes, that provides the true forensic discriminating power within soil samples. Indeed, it may be the value of soil in excluding a sample from having a particular source that is important. In the past, density gradient techniques have commonly been employed, although they are now seldom used. Particle density (i.e., weight per unit volume) varies depending on the minerals present in a sample. Since the density of particles in soil can vary, this distribution of particle density can be used to determine whether a soil sample is different from another or not. Murray and Tedrow (1992) suggested that density gradient methods could be used to indicate similarity but not to demonstrate it. Currently in the United States, the commonly applied methods in forensic laboratories for the forensic analysis of soil trace evidence are color (before and after heating), macroscopic observation, low-power stereomicroscopic observation, and occasionally, the elemental composition by scanning electron microscope (SEM) energy dispersive x-ray spectrometer (Murray 2000). However, currently soil analyses

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are generally only performed in an investigation of a serious crime. There is therefore an opportunity for the development of a soil forensic approach that would permit the routine analysis of soil for forensic investigations. An outline of where these methods, as well as new and exciting approaches, have been used in a forensic context is now presented.

11.3  Methods 11.3.1  Sampling and Handling In any investigation where soil evidence may be used, it is important that the scene of crime (SOC) officer has a basic knowledge of soils. At a scene of crime, any material that looks different in any way to the main soils at or around the scene should be carefully sampled. Soil sampling has to also consider the different soil horizons present in a soil profile, and any abnormality seen may indicate recent disturbance. Hanson (2004) illustrated the importance of adopting a stratigraphic approach as it reveals the sequence of events through time reflected in surviving buried deposits and features. Soil samples from a suspect’s clothing, shoes, vehicle, or other object may link suspects to the SOC or may lead police to another location. Since a soil sample within a shoe or tire tread may consist of several layers, careful dissection through the layers can reveal several origins, and these should be sampled and archived independently. In a related field, Lioy, Freeman, and Millette (2002) discussed the status and needs for new methods for the collection of household dust, such as wipe samplers, surface samplers, and vacuum samplers. Care has to be taken to avoid contamination, particularly if other analyses are required on the same sample. For this reason, it may be useful to split samples at the site for parallel analysis of insects, pollen, and fiber. Pollen analysis is particularly sensitive to contamination (Wiltshire 2002). Links to potential locations can be achieved through a visual comparison, microscopic analysis, or any one or more combination of the methods outlined following. Information on sample sorting, fractionation, and a description of texture, color, and organic matter content and soil horizon definition were presented by Murray and Tedrow (1992), with updates by Murray (2004a). Other suggestions on separation and preparation include the use of a high-resolution electric field probe to identify the individual fibers or particles encountered in a forensic soil investigation and to separate the mixed debris into constituent components by manipulation in a precisely controlled variable frequency electric field (Hearn and Singh 1989). A systematic approach to discriminate soils using soil morphology, mineralogy, geochemistry, and wet chemical techniques has been described for Australian soils by Fitzpatrick, Raven, and McLaughlin (2006).

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Soils can have characteristics due to human activity (anthropogenic soils). The forensic examination of soil is therefore not only concerned with the analysis of naturally occurring rocks, minerals, plant, and animal matter; it also includes the detection of such manufactured materials as ions from synthetic fertilizers and from different environments (e.g., nitrate, phosphate, sulfate) and environmental artifacts (e.g., lead or objects such as glass, paint chips, asphalt, brick fragments, and cinders). Each of these materials can represent distinct soil characteristics. When unique particles are found in soil evidence, more precise and rapid discrimination can be achieved even if the amount of evidence recovered is microscopic (Sugita and Marumo 2004). For this reason, microscopy is often considered the most useful technique for the detection of such characteristic particles. Comparison aims to establish a high probability that two samples have a common source or that they do not have similar properties and are unlikely to have come from the same source. A rare constituent is particularly useful for sample comparison. Many screening and analytical methods have been used to determine the characteristics that differentiate and discriminate the forensic soil samples, but none of them are easily standardized. Soil collected from a SOC must be representative of the soil that was removed during the events under investigation. In most cases this means the surface topsoil. Consequently, care needs to be taken in avoiding contamination of the soil surface with deeper soil horizons. If soil is adhering to a shoe, then the whole shoe should be wrapped with the soil intact and carefully transported to the laboratory (Saferstein 2006). Sample preparation is also of vital importance. For example, the use of spray drying prior to x-ray powder diffraction (XRPD) (Hillier 1999) and application of pressure onto potassium bromide (KBr) disks prior to analysis using infrared spectrophotometry (Cengiz et al. 2004) is worth considering. Indeed, an emphasis on sample preparation has led to much debate in the literature on the overreliance on standardization (Bull et al. 2005; Cengiz 2005). Caution should always be exercised when extrapolating results to real forensic situations. Attention also must be given to sample representation, measurement uncertainty or error (instrument and sample preparation), and the extent of natural variation. Ultimately, the methods of choice will depend on each individual case: the size and condition of the soil sample and whether it is being studied for the purpose of a sample comparison or in providing clues in intelligence for police search and detection. Also, consideration has to be given to the destructive nature of an analysis technique and, consequently, the logical order in which a series of measurements are to be made.

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11.3.2  Physical Characteristics of Soil 11.3.2.1  Color Most examiners begin with a visual color comparison for sample matching, whether qualitatively or quantitatively, and can often eliminate a large percentage of samples that do not compare. Samples are typically dried at 100°C and viewed under natural north-facing light (Murray and Tedrow 1992). In addition, color is often characterized using the semisubjective comparison to Munsell soil color charts (Munsell Color Company Inc. 1954) because it is often determined in the field. However, extreme care must be taken in its applicability. In a study of 73 soil samples, Sugita and Marumo (1996) used multiple colorimetric features such as color after air-drying, after wetting, after organic matter decomposition, after iron oxide removal, and after ashing. Although only about 70% of the soils were differentiated by comparing the colors of air-dried samples, combining color measurements of soils after air-drying and wetting and of the clay fraction after organic matter decomposition and iron oxide removal enabled them to differentiate 97% of soil samples. When color data was combined with particle size analysis data, it was possible to discriminate 99.5% of the soil samples, even when particle size analysis of the fine particle fraction was not carried out (Sugita and Marumo 2001). Recently, instrumental methods have been applied to soil color analysis with increased resolving power, such as the Minolta CM2600d spectrophotometer. Figure 11.1 illustrates the type of quantitative output that can be used to discriminate soils based on their spectral reflectance curves using this system. Results of research and actual casework were presented by Croft and Pye (2004a) and show the spectrophotometer method to provide a precise and rapid method for soil sample comparison. In a study assessing the

% Reflectance (SCE)

35 30 25

Unimproved Improved Site A

20 15 10 5 0 360

460

560

660

760

Wavelength (nm)

Figure 11.1  Spectrophotometer reflectance charts separating two grassland

soils, mean and standard deviation of seven replicate samples taken 4 m apart (specular component excluded).

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small-scale variation and discrimination based on a range of soil attributes (Pye et al. 2006), spectral color was found to show the least variation within sample sites. Although care has to be taken in sample preparation, color has the advantage that it is one attribute commonly recorded in soil databases— although as a field moist measurement. 11.3.2.2  Particle Size Distribution Analysis Particle size is a physical property of any sediment, soil, or dust deposit that can provide important clues to the nature and provenance of a sample. The distribution of sizes of particles in a soil sample is determined to produce samples with particles in the same size range for comparison and to determine the particle size distribution of the whole sample. Historically, the main methods for determining particle size distribution use a bank of sieves and measure the rate of settling of grains in a fluid using Stokes’ Law (see Marshall and Holmes 1979). In comparative analysis it is important to make comparisons within the same size range, especially when a certain fraction may be missing from the suspect sample (e.g., sand may not be retained on a shoe) but when that fraction is present in the control sample. Soil traces adhering to boots, shoes, and tissues have been compared with control samples using particle size analysis in the past. Comparisons of percentage of particles per class interval and multivariate analyses are used to determine how the size distribution of each suspect sample varies compared with the original distribution. For example, in a study by Chazottes, Brocard, and Peyrot (2004), a loss of the coarse fraction was observed on most of the suspect samples, even though they were derived from soils having different distributional patterns. The finding of significant differences within size classes ranging from 63 µm to 1000 µm should be considered as a dissimilarity sign between trace sample and control soil (Chazottes et al. 2004). Experimental results also suggested that equal weights of soils should be compared in the determinations of both comparator and questioned samples (Wanogho et al. 1987a, 1987b). Examination of the soil science literature indicates that the field conditions at the time soil is collected (as a control or contact trace), the pretreatment of the soil prior to sieving, and the form of sieving (wet or dry) may influence the particle distribution. Robertson et al. (1984) recommended that standard procedures using wet sieving be adopted. For forensic work, the determination of particle size distribution requires precise determination using a rapid and precise high-resolution method. Recently, particle-size analyzers have been used for the fine particle fraction (Pye and Blott 2004b; Wanogho et al. 1987c). The Coulter LS230 laser granulometer, for example, offers rapid and accurate sizing of particles in the range 0.04 µm–2000 µm for a variety of sample types, including soils, unconsolidated sediments, dusts, powders, and other particulate materials. Reliable results are possible for sample weights of just 50 mg, although the minimum soil weight limit to produce

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reproducible results is strongly affected by the size distribution, with coarsergrained materials requiring a larger sample weight. Discrimination between samples is performed on the basis of the shape of the particle-size curves (Figure 11.2) and statistical measures of the size distributions. Particle size can vary significantly on a small scale, and care must be taken to base comparisons on the least variable (at a local scale) parameters, such as the modal size, which may provide a better basis for comparison than measures such as the mean size (Pye et al. 2006). 11.3.2.3  Microscopy The stereobinocular microscope can be useful and is often used after soil color has been determined and after the recording of objects in the sample such as fibers, metals, paint, glass, and plastics is made. Any individual seeds or leaves can be recorded at this time. The sample can then be soaked in water to remove organic debris, making the identification of minerals much easier. This can also identify concurrently grains of starch, ceramics, and some abrasives. In addition, some comparisons can be made through the use of thin sectioning techniques giving information on grain position in relation to other material in the soil matrix (Kubiena 1970). Electron microscopes have been used in forensic soil analysis to provide a resolution not possible with lower-power optical microscopes. The great advantage of such methods lies in their ability to demonstrate the association of grains with other material such as coatings (Figure 11.3). Figure 11.3 shows two quartz grains, which could be described as having similar textural characteristics, that is, the same size class and shape (both angular). However, they differ by the composition of their coating, which can only be identified by energy dispersive spectroscopy (EDS). There has been a great advancement in the development of microscopes and analytical microprobe 4.0

Silt Loam Sandy Loam

3.5 Volume (%)

3.0 2.5 2.0 1.5 1.0 0.5 0.0

0

20

40

60

80

100

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Particle diameter (µm)

Figure 11.2  Particle size distribution of two contrasting soils determined using a Coulter LS230 laser granulometer.

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b

a

Si Al O Clay Coating

Control Soil

200 µm

c

Control Soil

Fe

50 µm

d

S

Ca

O Gypsum Coating Evidence A

200 µm

Evidence A

50 µm

Figure 11.3  Comparative scanning electron microscope (SEM) photomicro-

graphs of a control soil sample (a, b) and an evidence sample (c, d) taken from a piece of clothing. At low magnification (a, c) the samples differ by higher amounts of fine material in the comparator and the presence of fibers (from the clothing) in the evidence sample. At higher magnification (b, d) individual quartz grains show similar textural characteristics (size, angularity) but can positively be differentiated by their coating (clay vs. gypsum) using EDS analysis. ((Photos: Evelyne Delbois Macaulay Institute.)

instruments, which complement the basic SEM and electron microprobe analysis (EMPA), such as x-ray microscopes, electron tunneling microscopes, scanning acoustic microscopes, and transmission electron microscopes (TEMs). Although TEMs have only seldom been used in soil forensics they have the advantage that extremely small amounts of material (a few µg) can be examined in great detail. This enables particles that are beyond the resolution of other techniques to be characterized, and TEM has been widely applied in soil mineralogy (Wilson 1987). SEMs have been more commonly used for the identification of fibers, hair, paint, fossils, and any other unusual objects. In particular, SEM-EDS analysis has been used to characterize both elemental composition and particle shape (Pye 2004). Back-scattered electron (BSE) imaging combined with x-ray mapping provides a method of locating unusual particles and grain coatings and of mapping their distribution, which may be of diagnostic or of discriminatory importance (ibid.). However, since x-ray mapping is usually performed on polished thin sections, preparation is

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time consuming. Criticism has been voiced at the unrepresentative nature of many of these methods, at too few particles being examined, at its subjectivity, and at overreliance on qualitative evaluation (ibid.). Suggested improvements, such as subjecting a sample to a specific pressure prior to analysis leading to the production of more reproducible results, have been discussed (Cengiz et al. 2004). One point of caution is that with the SEM, owing to its high power of resolution, no two grains will be exactly the same, so care has to be taken in the interpretation of images and the interpretation placed into the context of the number of images interpreted. 11.3.3  Chemical Analyses 11.3.3.1  Elemental Analysis, Trace Metals, Pollutants One main criterion used for soil sample comparison is the composition of major and trace elements, either in the bulk sample or in one or more separated fractions (Pye and Blott 2004a). Analytical method development has meant that increasingly smaller sizes of sample can be analyzed in terms of elemental composition with high precision and accuracy. A range of techniques is available to determine the inorganic constituents in a soil sample: x-ray fluorescence (XRF), atomic absorption spectroscopy (AAS), inductively coupled plasma (ICP) spectrometry, neutron activation analysis (NAA), and energy and wavelength dispersive x-ray (EDX and WDX, respectively) microanalysis. When sample size is not a problem, XRF is often used (Pye and Blott 2004a). Soil samples collected from 110 different sites in the Kyoto district, Japan, were analyzed quantitatively using XRF spectroscopy to predict the origin of unknown soils. Soil samples were analyzed for silicon (Si), potassium (K), calcium (Ca), titanium (Ti), iron (Fe), rubidium (Rb), and strontium (Sr) with good reproducibility. Analytical data were normalized to a standard rock sample (JG-1) and were subjected to multivariate analysis. Trace elements, such as Sr and Rb, as well as K and Fe were able to characterize soil samples, and these soils were classified into nine types that showed good agreement with geological features. Probabilities of correct identification by comparing unknown soils with control data sets were about 71%, according to the systematic discrimination that was derived from multivariate analysis and a geochemical survey map of soils (Hiraoka 1994). Elemental ratios can also be used, such as in the study by Eckel, Rabinowitz, and Foster (2002), where data from 10 known lead smelting sites showed that the Sb:Pb ratio is most characteristic of secondary lead smelting sites. Lead contamination at the eight sites investigated in this study could be attributed at least in part to the former smelters because of the association between Sb and Pb (ibid.). ICP spectrometry has been used to measure the abundance of a broad range of elements (Duckworth et al. 2002; Pye and Blott 2004a; Shinomiya

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Phosphorus (mg/kg)

2000 1600 1200 800

Unimproved Improved Site A Improved Site B

400 0

0

2

4 6 % Carbon

8

10

Figure 11.4  Separation of three grassland soils (seven replicates, 4 m apart at

three grassland sites, unimproved and improved A and B) using bivariate plots of elements carbon (C) (by elemental analyzer) and phosphorus (P) (by ICP-OES) at three grassland sites.

et al. 1998). ICP optical emission spectrometry (ICP-OES) (sometimes called atomic emission spectrometry [ICP-AES]) and ICP mass spectrometry (ICPMS) are the main two types of analyses, providing concentration data for around 60 elements. ICP-OES analyses samples in a liquid matrix by using a high energy source, the plasma, to ionize the components of a nebulous mist from the sample. The energy source is strong enough (8,000–10,000K) to excite inner and outer electrons from an atom. On decay, these electrons will emit a quantum of energy in the form of light (UV-vis). The wavelength is as a result of the energy between two levels and can be derived from Planck’s equation, λ = hc/E, where h is Planck’s constant, c is the speed of light, and E is the energy between two levels. By selecting the wavelength and measuring the intensity relative to known standards, the concentrations of elements can be determined. ICP-OES is typically used for ranges from high parts per billion to percent. ICP-MS uses the same introduction system as the ICP-OES (i.e., nebulizes liquid samples and passes them through plasma). However, the ions pass into a mass spectrometer where they are separated according to the mass/charge ratio. The ICP-MS has the advantage of being capable of determining individual isotopes and can thus be used for isotope ratios as well as quantitative analysis. Typical range for an ICP-MS is parts per trillion to 100s parts per million. Elemental results from soil forensic studies have been presented as spider diagrams (Pye and Blott 2004a) or as bivariate plots (Figure 11.4). Important considerations in using this type of information are how the values in the suspect sample may reflect a different fraction of the whole and, indeed, how the sample compares to every other sample. In addition, uncertainty increases as concentrations approach the limit of detection. A study of three soils using small sample sizes (0.05 g) showed that between sample variability

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had a significant effect on uncertainty in the result (Jarvis, Wilson, and James 2004), with uncertainty increasing as concentrations reached the lower limit of detection. Although analysis of major and trace element data has been shown to play an important role in forensic comparison of soils, it has been suggested that it should only be used in conjunction with other methods (Pye and Blott 2004a). However, ICP can provide concentration data for a wide range of major and trace elements relatively quickly and at a reasonable cost, and with developments in miniaturization of sample processing for ICP-MS (e.g., Cairns et al. 2004) applications in forensics could increase. Comparison of measured elemental concentrations with soils database information could improve the use of such data in the future. Other commonly measured attributes such as pH, which is also held in soil databases, can be used to compare samples, provided enough sample is available. Geochemical techniques, using isotope ratios, and geochemical signatures have also been utilized in forensic work (Trueman et al. 2003). The determination of the isotopic composition of organic substances occurring at trace level in very complex mixtures such as sediments and soils has been made possible during the last twenty years due to the rapid development of molecular level isotopic techniques (Lichtfouse 2000). Consequently, isotopes can be employed to follow the fate of mineral and organic compounds during biogeochemical transformations and continuous-flow isotope-ratio mass spectrometry (CF-IRMS) can therefore be used as a tool in soil forensic analysis. The use of carbon and nitrogen isotopes has been shown to be useful in discriminating between soil types and sample locations, even when sampling occurs at a different time (which may well occur with a crime scene). Used in combination with other analytical techniques and, indeed, coupled with other elemental analyses, isotopic analysis may prove to be a useful tool in a forensic context (Croft and Pye 2003). These methods, however, do not always discriminate samples effectively (Sugita and Marumo 1996). Capillary electrophoresis can be a powerful tool for separation and quantification of ionic substances found in soil samples. Separation speed and direct injection of samples to the capillary without labor-intensive sample preparation are the major advantages of the method for a range of samples (Cengiz and Sakul 2001). The lead contents of 206 soil samples determined by AAS indicated that such determination provides a useful parameter for soil comparison and discrimination in forensic science (Chaperlin 1981). Soil investigations near a former smelter in Colorado revealed that historic use of arsenical pesticides has contributed significantly to anthropogenic background concentrations of arsenic on certain residential properties. A variety of forensic techniques including spatial analysis, arsenic speciation and calculation of metal ratios were successful in the separation of smelter impacts from pesticide impacts (Folkes, Kuehster, and Litle 2001).

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The forensic examination of soil also includes the detection of manufactured materials such as ions from synthetic fertilizers and from different environments (e.g., nitrate, phosphate, sulfate) and environmental artifacts (e.g., lead or objects as glass, paint chips, asphalt, brick fragments, and cinders) whose presence may impart soil with characteristics that will make it unique to a particular location. Modern gas chromatography-mass spectrometry (GC-MS) methods and equipment, with their high sensitivity, make GC-MS an excellent choice for field detection and identification of a range of organic chemicals. Numerous sampling techniques allow detection of GCMS analytes in environmental matrices, although multiple sample-handling steps and use of extraction solvents increase the complexity and time needed to complete analyses. Solid-phase microextraction (SPME) has been shown to be suitable for sampling environmental contaminants from air, water, and soil for GC-MS analysis (Hook et al. 2002). 11.3.3.2  Infrared (IR) Fingerprinting Methods Fourier transform infrared spectroscopy (FTIR) gives detailed soil signatures from minute samples (1 mg), making it a very useful forensic tool. FTIR gives an overall chemical fingerprint of the main organic and mineral components in soils (Figure 11.5) and can be analyzed using multivariate methods to determine even small differences between samples, although expert interpretation can provide an added dimension. This can be either in analyzing soil components or in terms of identification of nonsoil components/contaminants, which may be of crucial importance and which may not be readily identified by any other analytical technique. FTIR has been shown to distinguish soil patterns under different vegetation and soil conditions (Chapman et al. 2001). Discrimination of soils, involving minimal sample preparation, can potentially be achieved by use of a search/comparison facility that requires the compilation of a FTIR spectral soils database or by multivari0.7

Absorbance

0.6 0.5 0.4

Unimproved Improved Site A Improved Site B

0.3 0.2 0.1 0.0 4000

3600

3200

2800

2400 2000 1600 Wavenumber

1200

800

400

Figure 11.5  Fourier transform infrared spectra of three brown earth soils under different land use.

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ate methods. In addition, information from this technique can be extended by comparing the spectrum both prior to and after pyrolysis, obtaining an increased discrimination (Cox et al. 2000). In the study by Cox et al. (2000), more than 100 soil samples were analyzed using conventional analysis (i.e., color, percent organic matter, density gradient) and a FTIR technique. The FTIR technique involved collecting a spectrum of a soil sample that had been oxidatively pyrolyzed, thereby degrading all organic matter. This spectrum is subtracted from the spectrum of the same sample that contained the organic matter prior to pyrolysis. This resultant IR spectrum represents the organic portion of the sample. The use of organic components increases the discrimination in soils that are otherwise similar. For example, soils that have identical Munsell color values could be discriminated by subtractive FTIR. A new ancillary method using thermal gravimetric analysis in addition to IR analysis on samples prior to and after pyrolysis has been applied to soils and could give additional valuable information for the discrimination of soils (Thermo electron corporation application note 50862). 11.3.3.3  Other Chemical Fingerprinting Methods Nuclear magnetic resonance (NMR) may also offer new opportunities. NMR spectroscopy is a standard technique used in soil science research to study physical, chemical, and biological properties of matter and is an indispensable technique for identifying the chemical structure of simple organic molecules and complicated molecules such as protein, polysaccharides, and DNA. An NMR spectrum is a plot of the resonance frequency (chemical shift) against the intensity of absorption by the sample and indeed can give a fingerprint of whole soil, providing detailed spectra (see Wilson 1987). Although it has not yet been employed in the forensic analysis of soils, it has been used for drug identification (Aml et al. 1982) and in the characterization of carbohydrates within drug samples (Lurie, Hays, and Valentino 2006). The use of Raman spectroscopy of material can also be regarded as a fingerprint of its composition and can describe resins, waxes, and gums while not requiring detachment of the material (Edwards 2004; Kupstov 1994). However, whereas most forensic laboratories have IR facilities, Raman and NMR are not routinely available. 11.3.3.4  Mineralogy Most soils are composed predominately of minerals. A mineral is a solid material consisting of fixed proportions of various chemical elements arranged and bonded together into a regular structure, known as its crystal structure. Thus, different kinds of minerals do not just have distinctive chemical compositions but also have a great variety of distinctive physical and chemical properties determined by their different structures. There are very many methods and techniques for studying minerals and measuring

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Table 11.1  Some Common Mineral Groups and Minerals Encountered in Soils Type/Group Plagioclase feldspars Potassium feldpars Amphiboles Pyroxenes Iron oxides Aluminum oxides Other oxides Micas Sulfates Carbonates Zeolites Clay minerals Others

Mineral Albite, oligoclase, andesine, labradorite, bytownite, anorthite Orthoclase, microcline Various Various Geothite, hematite, magnetite, lepidocrocite Gibbsite Quartz, ilmenite, anatase Muscovite, biotite Gypsum, jarosite, alunite Calcite, dolomite, siderite Clinoptilolite, analcime Illite, kaolinite, halloysite, smectites, chlorites, Vermiculites, palygorskite, mixed-layer clays, etc. Allophane, imogolite

their properties. In principle, any of these methods could find application in the forensic examination of minerals in soil. Soils may be developed on either residual or transported material, but irrespective of which is involved, a soil may always be traced back to the parent rocks from which it has formed. This means that all of the minerals that occur in rocks may also occur in soils, in addition to those formed by pedogenic (i.e., soil-forming) processes. There are thousands of different minerals, but the main minerals and groups of minerals encountered in most soils form a much smaller set, numbering around twenty or thirty common types (Table 11.1). In nature this common set is typically further restricted over wide geographic areas because of the influence or otherwise of soil-forming factors, the most important of which are parent material and degree of weathering. Thus, a typical sample of soil will contain a suite of around six to ten different major minerals. A major mineral may be defined as one that is present at a concentration of a few percent or more, at which it will be readily detectable by routine techniques such as x-ray provider diffraction (XRPD). It is also known as energy-dispersive x-ray analysis (EDXA) or energy-dispersive analysis of x-ray (EDAX) or microscopic examination, either optical or electron. It is also not uncommon for several other minerals to be present in any given soil but usually in amounts that put them below the routine detection limits of many techniques. Nonetheless, these accessory, or trace, minerals can often be concentrated by some means that separates the soil sample into different physical or chemical fractions. Such procedures effectively lower

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the detection limits for trace minerals. Once identified, trace minerals are potentially more distinctive than the more common minerals that make up the bulk of the soil. Density is one of the properties that may be used to separate minerals. There are many procedures for obtaining so-called heavy mineral fractions from soils. These fractions consist of minerals with densities greater than those of the more common minerals such as quartz and feldspars. In a previous section the classical use of density gradients to compare soils was mentioned. A logical further step is the precise identification of the various minerals in the different fractions, particularly the denser fractions, since these are likely to be the most diagnostic. It should also be pointed out that the mineral groups listed in Table 11.1 are general groups. For example, feldspar is a name for a group of minerals, and plagioclase and potassium feldspars represent a further subdivision of feldspars. Similarly, where plurals are used for clay minerals (e.g., smectites), this refers to a group of minerals that may be further subdivided. This point is important because it emphasizes that minerals may be distinguished from each other at different levels. Furthermore, even examples of the same mineral species can still possess many features that distinguish one occurrence from another. In other words, different examples of the same minerals are rarely identical in detail, and the forensic scientist must be aware of the level to which minerals have been compared or differentiated when assessing the significance of mineralogical data. One of the simplest and most widely used techniques for the identification of minerals is polarized light microscopy (PLM). The technique is very versatile, and a skilled microscopist can quickly obtain a wealth of mineralogical information by this means. Petraco (1994a, 1994b) described its application in forensic science and suggested the examination of 2–5 mg of the 120–140 mesh fraction obtained by sieving. A 1 mg sample of soil of this size fraction (assuming 100 micron cubes of density 2.5 g cm-2) will contain in the region of 400 mineral grains so that in the case of the rare accessory minerals the single particle detection limit for the 2–5 mg sample is theoretically between 0.005% and 0.125% (50 to 1,250 ppm). Such low detection limits are undoubtedly one of the advantages of microscopy of any kind. Of course, any soil-size fraction could be examined, but it is important to realize that the mineralogical composition of a soil is more often than not a function of grain size so that any comparison must take this into account. A procedure of stereomicroscopic observations of color and grain morphologies, followed by particle size distribution determinations using sieves of particular mesh sizes, followed by PLM on the sand-sized fraction (whole and with heavy and light mineral separation) is often performed in soil forensic laboratories in the United States.

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Mineralogical analysis by electron microscopy is also a widely used technique and one that is increasingly being proposed for forensic soil examination (Pye 2004). There are many kinds of electron microscopes, but the most common instrument is the SEM, often equipped with facility for energy-dispersive spectroscopy (EDS) that permits chemical analysis based on the detection of characteristic x-rays emitted from a defined area probed by the focused electron beam (energy-dispersive x-ray analysis [EDXRA]). It is usually such chemical information that is necessary for the identification of different minerals in the SEM, but other properties, particularly shapes and habits (the regular forms shown by crystals), can be characteristic. The comparison of elemental peak height ratios determined by EDXRA can be a useful tool for rapid screening of soil samples, especially when combined with investigation of other attributes of the soil traces (Pye and Croft 2007). However, it was suggested that if sufficient material is available and can be separated without contamination or loss, higher resolution and more precise elemental data can be obtained by methods such as ICP-AES or ICP-MS (ibid.). One issue associated with all forms of microscopy is that much depends on the skill of the operator. This applies both in terms of recognizing the unique or unusual features of a sample and in terms of the judgments that must be made when attempting to quantify the mundane for comparative analysis. Recently a number of automated SEM-based analysis systems have been developed and used, for example, in the examination of contaminated soil (Kennedy, Walker, and Forslund 2002). McVicar and Graves (1997) described the validation of an automated SEM system whereby mineral identification was based on the acquisition of EDS spectra and the matching of spectra using a database of known mineral phases. Pirrie at al. (2004) suggested that the QEMSCAN® technology, successfully used in the mining industry, has considerable potential in forensic geoscience as it improves on the method described by McVicar and Graves (1997). This improvement is based on the acquisition of EDS spectra, which assigns a mineral or phase name to the material analyzed on a pixel-by-pixel basis. Automatic-microscope-based mineral analysis systems such as the QEMSCAN can clearly overcome many of the operator issues associated with comparative analysis. The QEMSCAN system can analyze thousands of mineral grains in a matter of hours and can produce statistically reproducible modal mineral analyses as well as recording other aspects such as grain shape. Its use, however, is optimal when the sample can be presented as a polished thin section. There are also limitations due to its inability to measure organic components and in relation to available reference database spectra. Its suggested application to samples presented as grain mounts with rough three-dimensional surfaces will also undoubtedly introduce the same uncertainties that affect accurate chemical analysis of such materials in conventional SEM and EDS

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(Potts et al. 1995). Furthermore, the analysis and precise recognition of claysized particles that are frequently smaller than the sample volume excited by the electron beam are also likely to pose some difficulties. Automatic analysis systems are clearly very attractive because of their precise quantitative output. Their utility, however, in relation to the recognition of the unique (Sugita and Marumo 2004) may still be no match to the eye of a skilled microscopist. Another factor to consider when choosing mineralogical methods is the particle-size distribution. As mentioned previously, the mineralogical composition of soil is generally a function of particle size. The smallest particles in a soil sample comprise the clay fraction. The term clay is used both in the sense of a particle-size fraction (< 2 μm) and in a mineralogical sense to refer to a specific group of minerals. The clay minerals are those minerals that give clay its properties, such as plasticity when wet. Most are hydrous phyllosilicates with structures analogous to macroscopic micas. Most soils contain clay minerals, and some consist mainly of clays. Traditionally, soil mineralogists have studied the clay minerals of soils by analysis of a clay-sized fraction (Moore and Reynolds 1997; Wilson 1987) isolated by particle-size separation from the nonclay minerals such as quartz and feldspars, which are usually much larger. Forensic scientists have also focused attention on this fraction of soil, perhaps in part because the properties of clays and clay minerals make them more likely than any other soil fraction to be transferred during contact. Various instrumental methods are used for clay mineral identification, but XRPD is of paramount importance (Isphording 2004; Murray 2004b). Clay mineralogical analysis is a very specialized discipline with most analyses conducted on a qualitative basis. The potential variability of clay mineralogy from one soil type to another is at the core of its potential application in forensic science. Furthermore, in some countries there is a stock of information on the distribution of clay minerals in soils because of the importance of clay minerals in relation to soil functions. Such information could prove particularly useful in the investigative stages of an inquiry. XRPD is also a very useful tool for the identification of all minerals present in soils, not just clay minerals. Although mineralogical evidence can be presented in inappropriate ways, jurors in the United States now accept that x-ray diffraction data are legitimate fingerprints of the provenance of samples (Isphording 2004). Recently, there have been many developments in the use of XRPD data for quantitative analyses of materials (Hillier 2003) where as well as identifying the minerals present, the amounts present are also determined (Figure 11.6). Soils are without doubt among the most difficult materials of any to analyze quantitatively, but with proper attention to sample preparation excellent results are possible. Murray (2004b, p. 8) even commented, “Quantitative x-ray diffraction could possibly revolutionize forensic soil examination.” However, those experienced in quantitative analysis by XRPD will point out immediately that the current sample size requirement

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290 3000

a

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2000

b c

1000

d 0 20

10 9 8

7

6

5

4

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d–spacing–Å

Figure 11.6  Some examples of x-ray powder diffraction (XRPD) patterns from

bulk soils prepared by spray drying; pairs are of the same soil sampled and then are resampled by returning to the same site more than 15 years later, are compared (a and b, both Ashintully; c and d, both Glenshee).

may place the most restrictive limitation on this possibility. Nonetheless, if suitable amounts of sample (1 g or more) are available, the technique may be applied and Murray’s prediction could be fulfilled. Indeed, future developments to miniaturize sample requirements will considerably expand the application of this approach. Sample preparation is of paramount importance, particularly with regard to particle-size requirements and reduction of texture, also known as preferred orientation. Inadequately controlled, either of these may cause severe imprecision in the intensity of diffraction. Sample preparation is the key to the reproducibility of the diffraction data and, hence, any measurements derived from it. If a sufficient sample is available, methods such as milling to sub 10 μm followed by spray drying (Hillier 1999) produce truly random powder samples from which diffraction patterns are highly reproducible. Reduction of particle size is necessary as it increases the number of particles that contribute to the diffraction pattern. For example, by reducing the particle size from 100 μm to 10 μm the number of particles in 1 mg of powder will increase from around 400 to 400,000. In comparative studies approaches that demonstrate reproducible sample preparation, such as spray drying, also offer the possibility that neither qualitative nor quantitative analysis of the diffraction data is entirely necessary. This is because the diffraction pattern itself may be taken as a fingerprint of the sample, irrespective of any subsequent data analysis.

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Thus far, most applications of soil mineralogy in forensics have largely been on a qualitative basis, most usually by XRPD with expert judgment. Recently, Ruffell and Wiltshire (2004) compared conventional XRD analyses with quantitative XRD (QXRD) as a determinant of mineral abundance. They claimed that the combined use of both XRD approaches allowed the specific mineral abundances to be matched between a scene of crime and a suspect. In this study, samples from potential SOC locations were taken from car tire tracks. An independent person drove a car to one location, and the car was submitted for sampling. The tire tread sample alone showed potential correlation to two locations: one correct and the other incorrect. QXRD showed the mineral proportions of the tire tread sample matched only the correct location. However, QXRD did fail to discriminate two locations that XRD showed to be different. The most surprising thing about this study is that although the authors advocate the use of two methods, both methods are based on exactly the same primary XRPD data. Thus, they are really comparing two different interpretations of the same basic XRPD data. This brings us back to the point that comparison of the uninterpreted XRPD data itself might in fact be a more promising and fundamental approach in forensic investigations since it requires no secondary level of interpretation. In many instances the amount of soil available for study in a forensic investigation may preclude the use of conventional powder diffraction sample preparation. In such instances one can resort to an x-ray diffractometer fitted with a system for analysis of samples loaded into thin glass capillaries (Figure 11.7). The amount of material needed for analysis by XRPD in a capillary is of the order of a few to a few tens of milligrams. The capillary is usually spun around its axis during analysis and is second only to spray drying as a method of producing near random powders and hence reproducible diffraction data. Combined with new position sensitive detectors such as the X’celerator from Panalytical or Brukers Vantec, such systems will probably become the configuration of choice for forensic XRPD work. 11.3.4  Biological Analyses 11.3.4.1  Palynology Palynology is a subdiscipline of botanical ecology, and its potential use to the criminal investigator is presented in Wiltshire (2006). Pollen grains are produced in the anthers of flowers and can be characterized using microscopic techniques. They can differ in many ways (Figure 11.8). The main forensic application of palynology (spore/pollen analysis) is in providing associative evidence, assisting to prove or disprove a link between people and objects with places or with other people.

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Figure 11.7  X-ray diffraction equipment in capillary configuration showing

from left to right: Cu x-ray tube, monochromator to select Cu K-alpha radiation, sample mounted in capillary tube, beam tunnel, and X’celerator position sensitive detector. The tube and the detector are scanned through a range of angles (theta) by the goniometer (the device in the background on which they are both mounted), and the XRPD is recorded and stored on a computer for subsequent analysis and processing.

As in all forensic analyses, the importance of minimizing risks of laboratory and cross-sample contamination during subsampling and preparation is recognized. An outline of the considerations for sampling is outlined in Wiltshire (2002), whereas a detailed procedure for the preparation of samples for pollen analysis was presented by Horrocks (2004). Pollen composition in a trace sample can be used to disassociate origin. For example, an investigation of a small amount of biological material isolated from a tubular component of the fuel assembly of a private plane that had crashed in New Mexico in 1989 showed that, consistent with other biological, chemical, and other soil evidence, the biological material was a postcrash accumulation and was unrelated to the accident (Graham 1997). Some cases in New Zealand with ropes, soil samples, and illicit drugs have received wide publicity and have helped increase its profile as a suitable tool (Mildenhall 1990). Sampling from a range of communities within Queensland, Australia (Bruce and Dettmann 1996) showed that pollen/spore types discriminated between vegetation communities. Results indicated that specific palynomorph types can be used as indicators of the communities within which they were found. Fungal spores were found to also discriminate between community types. Carbon isotope signatures also assisted in community discrimination (less negative ratios for C4 plant-dominated and more negative ratios for C3 plant-dominated) and have considerable potential as a linked methodology.

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Alnus Glutinosa

Calluna Vulgaris

Vaccinium-type

Cichorium Intybus-type

Corylus Avellana-type

Succisa

Exotic Spore (Lycopodium)

Plantago Lanceolata

Poaceae Undifferentiated

Figure 11.8  Images of fossil pollen grains found in organic soil horizons from a

range of plant species differentiated by shape and form using a high power microscope. Note an exotic spore is added to calculate the pollen concentration.

It has been suggested that a database of pollen/spore types be initiated as a reference collection to be used for expert witness evidence in this field (Bruce and Dettmann 1996). Increasingly, statistical approaches are linked to palynological evidence—for example, by using the likelihood ratio and considering how common the pollen assemblage is (Horrocks and Walsh 1998). However, results showed that localized areas of similar vegetation type, even within the same geographic region, have significantly different pollen assemblages (Horrocks, Coulson, and Walsh 199), which can indeed be used to improve discrimination. Horrocks et al. (1999) found that the pollen content in soil samples from shoes showed a close similarity to the soil samples from and between those shoeprints, indicating that pollen assemblages from soil on shoes do not differ significantly from assemblages in shoeprints in soil made by those shoes. In a case of an alleged sexual assault, the pollen content

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of samples of grass clippings and soil from the suspect’s clothing and shoes was compared to that of a sample of grass clippings from the alleged crime scene (a grassy area) to determine whether the suspect had been at the scene. The clothing and shoe samples showed a very strong correlation with each other and with the sample from the alleged crime scene in the combination of the different types of pollen present, very strongly supporting the contention that the suspect had been at the scene (Horrocks and Walsh 2001). In an alleged rape case, the pollen content of soil samples from the suspect’s clothing was compared to that of soil samples from the alleged crime scene (an alleyway) and from the alibi scene (next to a driveway) to determine whether or not the suspect had been at the alleged crime scene. These two scenes had significantly different soil pollen representations, due to different vegetation, although they were only 7 m apart. Because of this proximity, however, these differences in pollen representation were in the amounts of the same pollen types rather than in the numbers of different pollen types. The clothing samples showed a very strong correlation with each other and with the sample from the alleged crime scene in the amounts of pollen types present, very strongly supporting the contention that the suspect had been at the alleged crime scene (Horrocks and Walsh 1999). Much of the information published on forensic palynology has been based on small data sets and demonstrates general principles (Bryant and Mildenhall 1990, 1998), and it has proved impossible to obtain palynological population data because of the uniqueness of every site; expectations of any palynological profile can only be crude (Wiltshire 2006). Although palynology is unlikely to prove that a particular individual committed a crime, in certain circumstances it can connect a suspect with the scene of a crime or disprove an alibi. Pollen analysis was successfully used in an investigation of major war crimes (Brown 2006), in which it was identified that bodies may have entered primary mass graves with an associated plant and pollen assemblage from an execution site, showing that it is therefore essential that in the field as much effort as possible is made to differentiate between local matrix and imported body part matrix. Palynological profiles obtained from an individual site are unpredictable. Indeed, each forensic case is unique, making it very difficult to prescribe a detailed procedural approach. Furthermore, some plant families such as the Poaceae and the Rosaceae are difficult to differentiate using this technique. Nevertheless, pollen profiles can provide a high-resolution spatial fingerprint, particularly when coupled with other trace evidence such as mineralogy, through XRD (Horrocks and Walsh 1999) and with automated mineral phase analysis (Pirrie et al. 2004).

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11.3.4.2  Diatoms Due to their persistent silica skeletons and their diversity, diatom remains provide a good record of past and present environmental conditions. Cameron (2004) recently showed that they could be used to compare samples that had been in contact with water and for the investigation of time of death in drowning. Through the recent advances in analytical quality control and use of multivariate statistics, their use in forensics is likely to develop further. In a similar way, phytoliths (the plant opal silica structure that accumulates in some plants) have been used to differentiate soils with otherwise similar mineralogy (Marumo and Yanai 1986). 11.3.4.3  Soil Organic Matter (SOM) Although the organic matter content in most soils is often < 5%, it has an important impact on most soil functions, and the information contained in this fraction can help identify plant communities. Soil organic matter consists of the living microbiota and plant roots, dead and decomposing plant, animal and microbial remains, and humus. Fats, waxes, proteins, cellulose, hemicellulose, and lignin are part of the colloidal fraction of soil (although not exclusively so). Coupled with oxidative pyrolysis, FTIR can be used to characterize these soil organic constituents (Cox et al. 2000). Discrimination of soil samples in forensic science using organic components in the soil was investigated by Curie-point pyrolysis gas chromatography (PyGC). Pyrograms of soils under the conditions of pyrolysis temperature and time showed various contrasting patterns. In addition, 15 constituents of phenolic aromatics in pyrolysis products were identified by GC-MS (PyGC-MS). The amounts of toluene and phenol, derived from lignin, and those of 2-methylfuran and furfural derived from polysaccharide in the soil showed quantitative differences. Consequently, the comparison of these pyrolysis products may be useful for the structural analysis of organic matter in the soil and can be useful for the discrimination of soils in forensic science (Nakayama et al. 1992). Thanasoulias et al. (2002) used the soil UV-Vis absorbance spectrum of the acid fraction of soil humus to discriminate soils and concluded that this method achieved good discrimination (85% correct classification), provided that multivariate statistical techniques were also applied. Lignin, the aromatic biopolymer found exclusively in vascular plants, has been used by geochemists to indicate vegetation source because of its moderate reactivity, wide spatial and temporal distribution, and phenolic constituents (Chefetz et al. 2000). Thermochemolysis and tetramethylammonium hydroxide combined with GC-MS has been applied to the structural elucidation of fresh and altered lignin at the molecular level (Vane 2003). This procedure is suited to the forensic analysis of soil organic matter, requiring only 0.5–5 mg of

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sample, while providing specific information at the molecular level on lignin (Rawlins et al. 2006). 11.3.4.3.1  Plant Wax Markers Analysis  Patterns of organic compounds in soil, originating from plant waxes (such as n-alkanes and long-chain fatty alcohols), closely match the patterns found in associated vegetation (Almendros, Sanz, and Velasco 1996). The lipid profile of a soil largely represents the product of the synthesis, polymeric, and degradative processes on the vegetation, all of which are determined by the soil environment. Wax compounds, although only around 1% of the soil organic matter, are useful markers due to their persistence. N-alkane patterns have been used in the quantitative and semiquantitative apportioning of sources of hydrocarbons found in recent aquatic sediments polluted with fossil fuels (Volkman, Holdsworth, and Bavor 1992). This method has been validated by conventional pollen analysis (Dawson et al. 2004) but could also overcome some of its limitations (e.g., wind drift and poor identification of certain species, such as grasses). The patterns found in soil can reflect both the current (Figure 11.9) and past vegetation at that site (ibid.). Soil information pertaining to vegetation history at a given location can identify individual agricultural fields, and analyses can be performed on samples containing less than 5 mg of organic matter. In addition, such fingerprints have been used to help ascertain the extent of soil contamination by pollutant hydrocarbons (Stout et al. 2002). The soil fingerprint can be further enhanced through the use of other biomarkers such as plant wax alcohols persisting in soil. The inclusion of other organic compounds for SOM characterization can be of use, and indeed comparison of the un-interpreted spectra might be promising as a fundamental approach in forensic investigations since it requires no secondary level of interpretation. 11.3.4.4  Botanical Fragments Botanic evidence is useful in part due to the diversity of plant species (Coyle 2005). Forensic soil samples can contain plant fragments, which until now have received little attention (Block and Norris 1997). The species identification of plant fragments present in a sample can be performed using traditional microscope methods or the DNA sequencing of specific genes. Though DNA sequence databases for many mitochondrial loci have been established for the identification of animal species, less is known regarding the genomes of plants. Forensic botanical comparison can be hampered by the lack of appropriate DNA databases, and Tsai et al. (2006) reported on the use of the trnL intron and the trnL-trnF intergenic spacer (IGS) in the chloroplast genome and established a DNA sequence database for plant species identification, comprising 373 individual sequences representing 80 families, 206 genera, and 269 species.

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It has now been demonstrated that the technique of plant DNA analysis is achievable for tracing of illegal drugs such as marijuana (Coyle et al. 2003) and has now been accepted in the British courts (Linacre, Hsieh, and Lee 2005). However, it is more difficult to determine whether or not a plant sample has come from a specific plant (individualization) or group of clonal plants, although recent research on pine and silver fir has utilized microsatellite fingerprinting to match fine roots collected from soil samples to individual trees in woodland (Brunner et al. 2004; Saari et al. 2005). Microsatellites are tandem repeats of a few nucleotides (e.g., CATCATCAT) with these regions common in most genomes (Tautz and Renz 1984). Microsatellites

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are regions of high variability in the DNA and can help distinguish between individuals of the same species within a population. In both of the aforementioned studies, a fingerprint for each of the individual trees was first created by polymerase chain reaction (PCR) amplifying DNA collected from foliar tissue using primers targeting specific microsatellite regions in either plastid or genomic DNA, respectively. When combined, PCR products from two or more such microsatellite loci per tree result in a unique set of DNA fragments amplified for each individual tree. DNA extracted from fine roots was analyzed in the same way as with the foliar tissue, and the PCR products were compared against the fingerprints derived from the foliar tissue of individual trees. A similar approach may allow matching of plant fragments at a SOC with an object or suspect to allow confirmation of alibi or linking to the SOC. 11.3.4.5  Microbial Fingerprints Characterizing the microbial component of soils is another possible way of fingerprinting soils for forensic purposes, although it has not yet been established as routine. A fundamental problem to date with many traditional physiological and biochemical methods of measuring soil microorganisms was that they depended on the cultivation of the microorganisms. Such methods require that fresh soil is used, and the culturable portion is often only a very small proportion of the whole soil community (Torsvik, Goksoyr, and Daae 1990). The development of molecular methods such as the nucleic acid technologies as well as the use of signature lipid biomarkers, however, have overcome the problems associated with culturing, and there are now a variety of techniques being used extensively in soil to examine the diversity and ecology of soil microorganisms (Torsvik et al. 1998). Nucleic acid techniques are those that target analysis of the genetic information of organisms encoded in their DNA and RNA. The highest precision of these techniques is the complete analysis of DNA sequences, but this is invariably slow. A lower level of resolution can be obtained by PCR fingerprinting techniques that amplify the small amounts of DNA and RNA into more measurable quantities. General procedures for investigating microbial communities involve the extraction and purification of the RNA and DNA from the sample and then amplification using the PCR followed by analysis of the nucleotide sequence. From this point, many alternative strategies and methods can then be followed (Vallaeys, Courde, and Chaussod 1997). PCR is central to most nucleic acid techniques, and numerous variations and modifications can alter the targeting of the probes and selectivity of the amplification. The choice of primers at this stage is therefore crucial. The most widely targeted molecule is RNA and, specifically, the small subunit ribosomal RNA (SSU rRNA). rRNA sequences must be verified by hybridization techniques to ensure that naked DNA or contamination has not occurred.

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Figure 11.10  Denaturing gradient gel electrophoresis (DGGE) showing fingerprint patterns for thirteen different soils in Lanes 3–10 and 13–17. Lanes 1, 2, 11, 12, and 18 are marker lanes. Dark bands show presence of different bacterial genotypes.

Fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) (Muyzer 1999) and, more recently, single strand conformation polymorphism (SSCP) (Lee, Zo, and Kim 1996) use the PCR product to look at microbial communities. After amplification the PCR products can be separated as bands by DGGE or temperature gradient gel electrophoresis (TGGE). The banding pattern itself is a genetic fingerprint of the microbial community (Figure 11.10) and can be analyzed using multivariate methods using software that converts the image into a numerical form. From a soil forensic point of view the nature of the microbial communities is often irrelevant and is simply another type of fingerprint. Nevertheless, methods such as DGGE involve significant operator skill, and variation in the gradient of gels can influence the results. The use of TGGE somewhat overcomes some of the latter problems (Chen and Feng 1999) by using a temperature gradient to denature the DNA instead of a chemical gradient. SSCP is also a gel-based system but is simpler and faster with high reliability and can have higher sensitivity; however, it is only just starting to be applied to environmental studies (Miethling, Ahrends, and Tebbe 2003). The method is based on the property that the electrophoretic mobility of single-stranded nucleic acids depends not only on its size but also

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on its sequence. A third popular method is terminal restriction fragment length polymorphism (TRFLP), which uses restriction enzymes to cut the PCR products into fragments that can be analyzed on a sequencer to obtain an electrophoregram (Osborn, Moore, and Timmis 2000). During the PCR, fluorescent primers are used so that the terminal end of the cut fragment is labeled and can be detected in the DNA sequencer. The ability to determine the fingerprint in this way does offer greater opportunities for standardization, may have greater sensitivity, and more easily produces numerical output for ease of statistical analysis. The interpretation of fragment patterns can be more problematic, but for exercises as stated earlier, this is somewhat irrelevant. TRFLP has been used in at least one preliminary forensic study (Horswell, Cordiner, Maas, et al. 2002) and indeed is subject to international patent applications for different forensic applications (Horswell et al. 2001; Horswell, Cordiner, Sutherland, et al. 2002). The study by Horswell, Cordiner, Maas, et al. (2002) showed that a soil bacterial community DNA profile could be obtained from a small sample of soil recovered from both the sole of a shoe and from soil stains on clothing, with the profiles being representative of the site of collection. A study was carried out by Lerner et al. (2006) to test soil DNA extraction protocols on trace samples of various soil types and to perform a feasibility study of the PCR-DGGE approach as a forensic tool for analyzing the microbial diversity existing in soils collected from a crime scene. They found that samples from the suspect’s home and from the crime scene could not be separated based on cluster analysis and that DGGE reflected a large bacterial diversity, which, in most cases clustered correctly according to soil type and location. It was suggested that still further research is required to characterize a wider range of soil and vegetation type combinations while using an increased number of primer sets. Consideration should be given to the importance of sample condition and the appropriate microbial group. Soil fungal DNA profiles may offer an alternative and more robust target of microbial target taxa. New methods, such as the multiplex-TRFLP method, have the ability to simultaneously characterize multiple taxa while providing high-resolution analysis of the microbial community in a rapid and economical manner (Singh et al. 2006). The specificity of all these techniques depends still on the PCR conditions and primers used and in many cases may detect only the dominant members of the microbial community. Consequently, for forensic purposes there is a significant amount of work to be done to ensure the right balance between resolution and sensitivity to small-scale variation in soil is achieved. This approach has been reviewed in a comprehensive paper by Petrisor et al. (2006). Signature lipid biomarkers (SLB), or fatty acid profiling, is another molecular approach that has become widely used to study microbial communities (White and Ringelberg 1998) because, like nucleic acid methods, it is not dependent on the growth or morphology of organisms but relies on the direct

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extraction of lipids from cells in the soil. The most common extraction procedure for fatty acid profiling of soil communities is a modified version of the method proposed by Bligh and Dyer (1958), which extracts ester-linked fatty acids; the phospholipid fraction is then separated by fractionation and the fatty acids measured by gas chromatography (White and Ringelberg 1998). This simple method is considered to be a sensitive and reliable measure of microbial communities, but it is possible to fractionate and analyze further fatty acid classes (Zelles 1999). Zelles (1999) described an extended extraction procedure that also analyzes hydroxy-substituted and ether-linked phospholipid fatty-acids (PLFAs) and can quantify between 200 and 400 PLFAs in soil as opposed to less than 50 PLFAs found in the simple extraction. PLFA analysis has been used to study land use and management effects (Frostegård, Bååth, and Tunlid 1993; Frostegård, Tunlid, and Bååth 1996; Lundquist et al. 1999; Schloter et al. 1998; Wander et al. 1995; Yao et al. 2000; Zelles 1999) and vegetation cover (Borga, Nilsson, and Tunlid 1994; Sundh, Nilsson, and Borga 1997; Zelles 1999). PLFA analysis has also been used to biologically fingerprint windblown soil that had contaminated adjacent areas (Kennedy and Busaca 1995), and a U.S. patent exists that covers its use for locating the origin of soils (Kennedy 1998). Measuring the concentrations of different PLFAs extracted from soils can, therefore, provide a biochemical fingerprint of the soil microbial community. These PLFA profiles can be analyzed by multivariate methods to compare differences between soils. 11.3.5  Combined Approaches It has been suggested that forensic comparisons should involve the use of several techniques in combination. A wide range of analytical techniques can be used, the choice being dependent on many factors including sample size character, condition, and time and cost constraints. Junger (1996) performed an evaluation of techniques used in the forensic analysis of soils and geologic evidence. Samples were collected from a beach, an island isolated by a river, and a bus park. The samples were analyzed using color determination, particle-size distribution analysis, and mineralogical profiles of the 25 most common soil minerals. Of the 300 samples examined, more than 50% were discriminated by color alone, the remainder requiring only particle-size distribution analysis for differentiation and negating the need for lengthy mineralogical examinations. However, the sites chosen were very contrasting in type. In a study to compare single-source and primary-transfer soil samples, four analytical techniques (spectro-photometric color determination, laser diffraction particle-size analysis, stable isotope analysis, and chemical element analysis) were used (Croft and Pye 2004b). Four soil types and five footwear types were used. All four techniques showed excellent precision and good resolving power between soil types. Only relatively small differences

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were obtained between source and transferred soil samples in terms of color, carbon, and nitrogen isotope ratios and elemental chemistry, whereas significant differences were found in grain size, indicating that the primary transfer process is to some extent grain-size selective. More research must be done to establish in what instances these techniques and others provide reliable, reproducible, representative, and accurate results and in what capacity such results can be presented (Bull et al. 2004). Care should be taken in extrapolation to other casework until greater reliability of these and other techniques has been established. A study testing the independent and collective interpretation of four experts with contrasting analytical skills (XRD, SEM, palynology, and organic matter characterization) in the prediction of sample provenance was carried out (Rawlins et al. 2006). Although only investigating the provenance of three sites, this study demonstrated improved prediction when multiple techniques were combined. However, it also showed for one of the sites that although the correct parent material was identified, difficulties were encountered in identifying the garden setting. Soil characteristics have been used in conjunction with pollen and vegetation analysis to improve an association. Brown, Smith, and Elmhurst (2002) successfully used palynology along with petrology in a search in a murder investigation. Soil samples from a car believed to have been used by the suspect in a missing persons case was subjected to soil and palynological analyses. The soil characteristics and petrology were used to redefine the search area using geology and soils maps; the pollen and vegetative remains were used to target woodlands with a particular species mix. As a result, two bodies were located, and the environmental evidence was used in the subsequent trial. In this case the history of the vehicle was well known, and the wheel arches and footwells acted as reliable soil traps. The advantage of combining the techniques is that soil evidence (both mineralogy and other inclusions) provides a geological and soils comparison whereas the pollen provides independent evidence of vegetation type providing a combination that may be rare or unique. Pirrie et al. (2004) suggested that when automated mineral phase analysis of trace evidence is coupled with the examination of soil palynology, it can provide a trace evidence fingerprint, which could link a suspect or victim to a crime scene. Recently the combined use of pollen, sediment descriptions and mineralogical information (using XRD) was used successfully to provide matches between primary and secondary burial sites in the investigation of war crimes in Bosnia (Brown 2006). One other type of combination is where two aspects of evidence are present in crime samples. For example, in a rape case in the United States, three flowerpots had been tipped over and spilled on the floor during the struggle. It was shown that potting soil on the suspect’s shoe had a high degree of similarity with a sample collected from the floor and represented soil from

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one of the pots. In addition, small clippings of blue thread were identified to be present in both the flowerpot sample and on the shoe of the suspect. The thread provided additional trace evidence that supplemented the soil evidence (Murray 2000). In a similar way, soil analytical characteristics, when considered along with footwear print information, can increase the association or otherwise of a shoe or boot with a location. The use of compositional analysis should assist in the differential separation of several sources of soil, such as the soil that builds up on shoes, vehicle tires, and implements used to bury objects or bodies. This, in addition to identifying locations of mixed soil, can potentially separate out different soil horizons. This method has been applied in both sourcing vegetation and solutes and can tease apart several sources when present in a mixture (Brewer et al. 2005), with much potential in the application of mixed soil origins. However, the robustness of any unmixing solution depends on the number of samples, number of source groups, and the variance of the source group properties (Small et al. 2004). 11.3.6  Polyphasic Approaches and Links to Databases The use of a direct combined (or polyphasic) approach can create highly specific soil fingerprints from normal constituents. This, in addition to the application of appropriate statistical analysis, would make soil analysis a more effective tool for routine forensic work, thus considerably extending its applicability. Indeed, combinations of different data each with its own discriminatory potential may result in probabilities of association or disassociation that even surpass those of techniques such as human DNA. Initial work using a canonical variate analysis has shown discrimination between soil types can be improved by including more analytical data. Figure 11.11 illustrates

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among three visually similar topsoils as soil attributes (analyses) are combined. A is a cultivated podzol/improved pasture; B is a brown earth/improved pasture; and C is a brown earth/seminatural grassland. Four replicate values sampled 4 m apart plus 90% confidence ellipses.

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this point using three similar soils; type separation is gradually improved by adding in results from different analytical methods, namely color (by spectrophotometer), n-alkane values, and XRPD. However, consideration of the appropriateness of data distributions prior to any data combination method is required. Indeed, an informal layering of information can be an equally valid approach. Many of the methods described in this chapter provide digital signatures that can be analyzed by multivariate methods, and crime samples can be compared to reference databases built up using reference soils. The database comparison would allow probabilities of an association to be calculated and, with the associated metadata for the reference soil (land use, geology, and location), would provide additional clues to soil origin. However, in many forensic cases not enough soil material may be available to permit a representative sample to be measured, which could limit the confidence in comparing test samples with samples from a regional soil survey. Unknown soils in Japan have been compared with control data sets, giving a 71% “match,” according to the systematic discrimination that was derived from multivariate analysis of soil elements, including trace elements, and a geochemical survey map of soils (Hiraoka 1994). Rawlins and Cave (2004) examined data from a regional soil survey in eastern England to examine whether samples over the same parent material could be discriminated on the basis of multielement chemistry and found that although 99% could be discriminated from each other there were limitations due to the sample size required for elemental analysis. A database of particle-size characteristics in combination with elemental analysis has also been developed for coastal dune sediments in England and Wales (Saye and Pye 2004). Based on these variables, they found that not all dune fields were unique, but they suggested it would be more useful if it had been used in combination with mineralogical and biological data. They did illustrate how the database could be used in actual forensic investigations in excluding a sand dune origin and in determining the origin of sand (e.g., on a glove). Such geographical information may indeed act more as providing intelligence in suggesting likely locations, or exclusion of locations rather than providing evidence. In the future, extended types of analyses could be incorporated in database comparisons. 11.3.7  Statistical Considerations and Presentation of Evidence in Court When someone at a crime scene comes into contact with soil, transference takes place from the soil to that person. If the soil is transferred in the commission of a crime, then this soil may be used as evidence. Interpretation of this evidence relies on, among other things, the difference in the mean measured value of the soil recovered from the suspect and a control sample

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taken from the crime scene. A true Bayesian analysis would include estimation of the difference in the distributions of the measured soil value of the recovered fragments and of the “control” or “comparator” sample. Since this task is onerous, the difference in the means may be examined instead. However, just as the true distribution of the values is not known, neither is the true distribution of the difference in the means. There are also reasons to believe that the sample recovered from the clothing, shoes, or tires may have different distributions due to a preponderance of finer-sized particles in the recovered samples. It is important to quantify the likely distribution of the difference between the control and recovered samples to improve sample comparison. Also, the analyst should try to ensure that analyses are performed on similar size classes of particles and with a similar amount of material. An understanding of variability that can occur in the results from analysis of a soil sample is important to help compare soils samples, and the use of many characteristics together can improve this discrimination. However, when considering many variables, some form of dimension reduction is necessary. To this end, principal components analysis has been used in chemometrics (Thanasoulias et al. 2002). Additionally, there may be a high degree of colinearity between variables; hence, partial least squares should also be considered. In addition, exploratory statistical analysis of data using non parametric, multi dimensional scaling procedures can be used to examine similarity. Choice of the most appropriate statistic. Choice of the most appropriate statistical approach is essential to stand up in the context of court. Through increasing use of appropriate statistics the high spatial variability of soil attributes can be a positive aspect of soil as physical evidence. Choice of analytical method depends on the likely evidential value of results obtained, which depends often on the existence of a sufficient body of contextual database information (Croft and Pye 2004a). Any analysis carried out for courts must be of a high standard, and levels of uncertainty must be minimized. Providing robust analysis with known confidence levels is essential when supporting a legal argument (Small et al. 2004). If results are to be used as evidence, then analyses should be carried out by an accredited laboratory, and the use of statistical tests should be carried out by those who not only have a proper understanding of statistical analytical procedures but who also understand when the resulting numbers have true statistical significance (Isphording 2004). Isphording (2004) also stated that although the statistical results are useful—indeed, essential— for scientists, they should generally be avoided in presentation to the jury. In addition, to provide accurate evidence, it is important that forensic soil specialists interpret the data.

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11.4  Conclusions Forensic examinations involve identification of soil materials and comparison of samples to determine a likely common source and to provide clues to aid investigations in intelligence gathering. The future will also see increased use of soil as evidence, more new automated methods of examination, increased resolution and miniaturization of techniques, in situ sampling and analysis, improved training of those who collect samples, and research on the diversity and variability of soils and on how, when, and what parts of soils are transferred during various types of contact. In court, quantitative methodologies will increasingly be required as evidence, as will reference to reliable databases. In an analogy to the use of human DNA database material, when similar links are established for soil material, it will provide good and reliable estimates of probability. Consequently, the use of soil as physical evidence in sample matching and as a search tool should escalate. There is a continuum of development in techniques, and new opportunities will arise in parallel with new scientific developments in research, ensuring that scientists keep ahead of the criminal mind. The suite of techniques reviewed here, which includes the chemical, mineralogical, and molecular fingerprinting of soils, can both complement conventional forensic methods and provide new investigative or comparison tools where previously none existed. Soil is clearly a complex material, and analyses of these different components provide different types of information about its geological origins, dominant vegetation, and management history. Each forensic case is different; consequently, the individual analytical techniques will have different degrees of importance and relevance depending on the nature of the criminal case in question. Each method has its strengths for different situations, and there is great need to give more guidance on how to deploy the appropriate techniques for a given situation. As many more methods become quantitative, their use in combination will help to characterize the soil more broadly and thus will help to refine and narrow its probable origin as well as to give increasingly robust sample comparison with probabilities that can be quantified. The complexity and variability of soil properties work as both an advantage and a hindrance. Complexity means that many different characterizations can be used to provide high-resolution fingerprints, but equally the variability in this complexity creates a problem of ensuring that reference samples are representative and that sampling of real cases accounts for the expected variation. One crucial component of the forensic application of soil science is the development of a set of reference soils and databases, which would enable the estimation of the probability of obtaining accurate soil comparisons.

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This approach is analogous to the DNA fingerprinting of human tissues, which relies on knowledge of the frequency of the recorded attributes within a sample of the population. At present there is no reference population for soils against which to judge any soil analysis. There are, however, significant sources of data and archived soils around the world that have been gathered by agricultural and environmental institutes for other purposes. It seems obvious that there is an opportunity to use some of these sources to generate the population data needed to test existing and new methods for their accuracy and resolution and to determine the probability and certainty for the most promising methods. Another major challenge for soil forensics is that the methods are rigorously tested and standardized. This is important for agricultural and environmental research, but in the forensic arena the validity and rigor can be subjected to unprecedented scrutiny. DNA fingerprint of human specimens is widely perceived as an accepted technology but has been through some difficult times where for every expert supporting it there was another who was prepared to attack the methods used (Roberts 1992). This was only overcome by concerted efforts across the forensic and science communities, and this will certainly also be the case for soil fingerprinting methods.

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Index

Note: Page numbers appearing in boldface indicate placement in a table; in italic, a figure.

A AAS, lead contents of soil samples and, 283 abiotic agents, decomposition of bone and, 214 acari, 37 accelerator mass spectrometry (AMS), 234 accumulated degree days (ADDs), 34 acetate, 161 Achromobacter, 208 acidic environment, decomposing bodies and, 208 acrilan, 161 acrylic fabrics, 170, 242 resistance to chemical degradation, 162 acrylic yarn, 161 adipocere, 64, 78, 185 constituents of, 210 decomposition of, 212 formation of, 39, 42 gram positive bacteria, 212 lipids and, 210–212 persistence of in burial environment, 212 adipocere, formation of, 209–210, 211, 215, 216 anaerobic environments and, 210 body buried in soil vs. in coffin, 211 burial environments and, 210 extraction of moisture and, 212 hydrogenation, 210 hydrolysis, 210 lead-lined coffins and, 210 peat bogs and, 210 preservation of remains by, 212 subcutaneous fat and, 210 submerged location, 210 adipocere, odor of cadaver dogs and, 210

clandestine burials and, 210 adipocere formation, lime and, 211 adipose tissue, 78 lipids and, 208–209 postmortem changes in, 209 Adventures of Sherlock Holmes, 3 air, 209 Aldo Moro case, soil as physical evidence in, 274 algae, 238, 241 algor mortis, 227 normal body cooling, 204 used to establish time since death within early postmortem period, 204 alibis, 45 alternative electron acceptors (redux couple) used by bacteria and associated microbial processes, 58 Amblyosporium botrytis, 72, 87 amines, putrefaction and, 86 amino acid identification, 232 ammonia, 37, 84 alkalinity in soil and, 69 as trigger material, 72 ammonia fungi (AF), 69–75, 87, 91, 93 amino acids and, 86, 92 basidiospore germination, 84 changes in the soil, 74 early phase (EP), 72, 74–75, 78, 85, 87 early phase (EP) of succession, 92 ectomycorrhizal, 86 environmental conditions and, 75 experimental grouping of fungi, 69–72 fungal succession and mycorrhizal relations, 72–73 growth of in liquid medium at different pH, 86 growth of in liquid medium containing different nitrogen sources, 85 initial conditions and, 74 late phase (LP), 72, 74, 78 nitrogen utilization and, 86 rearing in pure culture, 93 responses of other organisms, 74

317

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318 Index succession of, 72, 78 ammonium, mineralized from arginine, 267 Andes, archeological textile remains, 165 angora, 158 Anillinus fortis Horn, 119 antemortem bone trauma, 214 anthropogenic activities, 13 anthropological remains, dating of, 232 Anthropological Research Facility, University of Tennessee, Knoxville, 139, 186 long-term research using donated human cadavers, 180 anthropology, forensic, 226, 231–235, 237, 242 analysis of skeletal remains in context of a legal investigation, 231 morphological, chemical, and immunological studies, 232–233 postmortem investigation and, 227 radioisotope studies, 234–236 antibiotic (streptomycin), 264 ants (hymenoptera), carrion and, 116 arachnida, class millipedes, 115 mites (acari), 114 spiders (arachneae), 115 archaeological textile remains preserved in iron corrosion, 155 archaeology, forensic, 226, 237, 238 postmortem investigation and, 227 archeological casework, hair survival and, 139 arginine ammonification, 267 arthropods, 110, 113 broad markers for estimating time frames, 114 in burial environment, 230 buried remains and, 229 succession of insects on a corpse, 113 succession on buried corpses, 231 urea treatment in soil and, 76 Ascobolus denudatus, 72, 74, 87 Ascobolus sp., 80 Aspergillus fumigatus, 137 atomic absorption spectroscopy (AAS), 281 ATP Luciferin-Luciferase Reagent, 264 ATP reaction agents, preparation of, 263 ATP standard solutions, preparation of, 264 Australia, 235, 273

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soil analysis and, 275 Australia, Perth mites, gasamid, 115 tyroglyphid mites, 115 Australia, Queensland, pollen analysis in, 292 autolysis, 31, 216 digestion of proteins, carbohydrates, and lipids in the body, 205 digestive enzymes and, 205 hydrolytic enzymes and, 205 marbling on body’s surface, 205 postmortem disintegration of cells, 205 skin slippage of extremities, 205 automated SEM-based analysis systems, 288

B Bacillus spp., 208, 212 back-scattering electron (BSE) imaging, 280 bacteria, 109, 209, 241 aerobes, 164 Bacillus spp., 42 breakdown of buried textiles, 164 Cellulomonas spp., 42 decomposition of bone and, 214 faculta, 164–165 Nocardia spp., 42 bacterial activity, slowed by cold and, 165 bacterial biomasses in soil, estimates of, 265 Bacteroides, 31 putrefaction and, 205 banding pattern analysis by software converting image into numerical form, 299 as genetic fingerprint of the microbial community, 299 basidiospores, 84 Bayesian analysis, 305 beetles (Coleoptera), 117, 229 buried carrion and, 119 corpses and, 112 forensic research and applications, 112 predators of dipteran larvae, 117 usefulness in estimating PMI, 117 beetles, scarab or dung (Scarabaeidae), 118 Beeton’s Christmas Annual of London, 5 belts, crime scene and, 170 benzidine testing, 232 of bone surface, 233 biogenic amines, 206, 216

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Index biological conditions, 240–241 biomass autochthonous component of, 61 zymogenous component of, 61 biomass C, 254 ammonium and, 267 nitrate and, 267 biomass in soil, principles and methodologies of measuring, 247–271 biomaterials, differential survival of, 139 biomolecules, 164 biotic agents, decomposition of bone and, 214 blowflies (Calliphoridae), 35, 112, 119 biomass reduction and, 118 larvae, 33 larvae and, 112 bodies, see cadavers bodies, identification of clothing and, 154 dental records and, 154 DNA and, 154 personal effect, 154 bog bodies, hair of red-brown color, 129 bomb pulse, the, 234 distinguishing between modern and ancient remains, 234 bone decomposition of, 214 inorganic phase of, 213 organic collagen phase of, 213 weakening of structure, 214 weathering of, 237 botanic evidence, 296 botanical comparison, forensic, lack of DNA bases for plant identification, 296 botany, forensic, 238–240, 242 estimating postburial interval (PBI), 238 British Columbia, 115–116 burial decomposition and, 40 mass graves, 216 burial, depth of, 189 insect activity and, 229 burial environment, 226 ammonia and, 207 decomposition chemistry in, 203–224 decomposition rate and, 217 burial method, decomposition rate and, 217

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319 burial silhouettes, 216 burial site locations, 23 buried cadavers decomposition rate and, 215–216 temperature and, 40 buried metals, stability of, 175 buried remains insect fauna associated with, 229 postburial interval of, 225–247 postmortem interval of, 225–247 burnt ground fungi, 91, 93 burrowers, decomposition of bone and, 214

C cadaver bloating of, 64 dermal microbial communities, 30 enteric microbial communities, 30 natural preservation of, 42 cadaver, elephant, 37 cadaver decomposition, 29, 38, 39 active decay, 33, 35 advanced decay, 33 animals and, 228 autolysis, 31, 166 bloated, 33 clothing and, 43 decay, 31 decomposer organisms, 62 dry, 33 ecology of, 30 environmental conditions, 62 factors affecting, 62 factors influencing, 38–44 fatty acids and, 209 forensic applications and, 30 fresh, 33 gravesoils and, 30 on the ground and, 76 insect succession and, 229 insects and, 30, 228 microorganisms, 228 odor and, 206 plastic bags and, 172 processes, 29–53 processes in, 31 putrefaction, 31, 166 remains, 33 resource quality, 62 retarded by clothing or covering, 212

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320 Index scavengers and, 30, 228 soil and, 29–53 soil pH and, 42 stages of, 33 studies of, 30, 33 succession data, 228 temperature and, 34 wrapped in plastic, 216 cadaver decomposition, aboveground, 38–40 associated materials, 40 moisture and, 38–40 temperature and, 38–40 trauma, 38–40 cadaver decomposition, belowground associated materials, 43 decomposer adaptation, 43–44 moisture and soil texture, 41–42 soil pH, 42 temperature and, 40–44 cadaver decomposition island (CDI), 33, 35, 36, 38, 40 active decay, 35 advanced decay, dry, and remains, 36 ammonium, 33 biophysicochemical characteristics of, 33 concentration of chemical compounds, 33 formation of, 33–38 fresh and bloated cadavers, 35 nitrate, 33 succession of fungal communities, 33 succession of insect communities and, 33 succession of plant communities, 33 cadaver dogs, 206, 207 cadaveric decay, stages of, 63 cadaveric decomposition, shift of pH and redox, 163 cadaveric fluids, 40 cadaverine, 206 cadavers blowflies (Calliphoridae) and, 35 buried, 63 clandestine disposal of, 154 clothing and, 43, 154 composition and decomposition, 31–32 flesh flies (Sarcophagidae) and, 35 fresh and bloated, 35 indicators of presence of, 272 jewelry and, 43

69918.indb 320

mass, 33 metal artefacts and, 43 naked, 154 plant material and, 43 skeletonized, 226 unburied, 63 unearthed in excavations, 231 wrapped in carpet, 200 calcium carbonate, 20 the fizz test and, 21 California, 273 Calliphoridae (blowflies), 230, 231 Camarena case, soil as physical evidence in, 274 Canadian artic, exhumation of three seamen, 165 canonical variate plots showing increases in discrimination among three visually similar topsoils, 303 capillary electrophoresis, separation of ionic substances in soil samples, 283 carbohydrates, breakdown of, 216 carbon dioxide flush, measuring, 254 carbon isotope signatures, 292 carbon mineralization, measured by gas chromatography, 266 carcass decomposition, prairie grass and, 75 carnivore chewing, 214 carnivores, decomposition of bone and, 214 carnivorous animals, 215 carpet, 200 burial conditions and, 200 partially burned, 200 carpet and carpet fibers, carpet pile fabric compositions, 200 carrion invertebrate colonization, 110 carrion beetles (Silphidae), 110, 112, 117 carrion crow carcass and Rhopalomyces strangulatus, an early colonizer, 77 carrion flies (Diptera: Muscidae), 110, 112 cashmere, 158 catchment, 10 cause of death, 29 Cellulomonas spp., 212 cellulose, 164, 170 changes, hydrolytic, 209 changes, oxidative, 209

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Index changes in the concentrations of ammonia and the pH value in urea-treated soil, 73 checkered beetles (Cleridae), 112 chemical breakdown of body’s main constituents, 216 chemical composition of mammalian cadavers during life, 32 chemical conditions, 240 chemical element analysis, 301 chemical investigations, volatile fatty acids (VFAs), 241 chemical studies, dating of remains and, 232 chemistry, decomposition, in a burial environment, 203–224 chemometrics, 21, 305 chloroform fumigation, 255 chrome tanned leather from Overton Down experimental earthwork after 32 years of burial in a chalk environment, 172 chronological time, 226 Cladorrhinum foecundissimum, 74 clay, 289 clay mineral identification, XRPD and, 289 clay minerals, 289 Clorpt equation, 8 Clostidia perfringens (welchii), 209 Clostridia, putrefaction and, 205 Clostridium, 31 clothes moth larvae (Tineola bissiella), hair and, 135 clothing, 154 degradation of, 168 effect on decomposition of bodies and, 154 matching briefs, thongs for bras in table 7.5, 198 men’s, 196–197 range of fabrics used in some bras currently available in U.K., 198 rate of cadaver decomposition, 40 some current sock yarn mixtures (U.K.), 197 typical yarn composition in currently available men’s pullovers (U.K.), 189 women’s, 197–198 yarns used in women’s hose (UK), 199

69918.indb 321

321 clothing, loss of differential decay, 168 offender actions, 168 significance of, 168 clothing, differential decay of, example of, 174 clothing material corroding metal and, 154 modification o burial environment by cadaveric decomposition, 154 soil burial and, 154 clown beetles (Histeridae), 112 fly larvae and, 117 coffin fly, see Conicera tibialis coffins disintegration of, 213 retention of water in, 216 collagen, 164, 206 Collembola (springtails), 37, 164 Colorado, soil investigations in smelter, 283 colors of leachate from humus soaked in buffer solutions with pH 11-3, 73 combining analytical methods, 303 compare, 11 comparison of soils, forensic, 3, 5 analytical, 12 color and, 13 descriptive (morphological), 12 in fiction of Sir Arthur Conan Doyle, 5–6 history of, 5–6 murder of Eva Disch and, 6 compositional analysis, 302 condition of textile samples after 24 months burial at the pasture site, 188 Conicera tibialis, 229 conidium production of Doratomyces putredinis on urea plot, 69 contamination, importance of minimizing risks of in forensic analyses, 292 continuous-flow isotope-ratio mass spectrometry (CF-IRMS), tool in forensic analysis, 283 Coprinus neolagopus, 74 Coprinus spp., 84, 87 Coprinus tuberosus, 87 Coprinus tuberosus fruiting on the ground after decomposition and disappearance of human feces, 81 cotton, 158, 185, 188, 196–198 cellulose and, 160

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322 Index Europe and, 158 preservation by corroding metal, 177 treated, 242 untreated, 242 cotton, long-term preservation of desiccation, 160 freezing, 160 cotton polyester, degraded, scanning electron microscope (SEM), 167 cotton/lycra, degraded, scanning electron microscope (SEM), 169 cotton-polyester, 242 Coulter LS230 laser granulometer, 278 counting, microscopic, 253 staining and, 253 counting procedures, computer-aided, 253 courtelle, 161 crime scene, 226 bed linens, 198 investigations of, 23 recovery of hair from, 139 soil and, 3, 304 soil samples and, 21 towels and, 198 crime soil samples, reference data bases and, 304 criminal activity establishing time of, 44 physical evidence of, 45 Croatia, exhumations in, 40 cup-fungi, 72 Curie-point pyrolysis gas chromatography (PyGC), 295 Cynomyopsis cadaverina, 118 cystine, hair and, 124

D Dacron, 161 database of pollen/spore types, 293 dating skeletal remains of forensic interest, 236 death, mycorrhizal symbiosis and, 84 decalcification, decomposition of bone and, 214 decay, 31 decay of organic matter (residue) and synthesis of decomposer biomass and carbon dioxide accumulation, 56 decay rates, seasonal fluctuation and, 165

69918.indb 322

deceased, identification of, 29 decomposed porcine remains with partial mummification and adipocere formation, 213 decomposer adaptation, 43 decomposer organisms, 55 decomposition, 53, 203, 208, 227 black putrefaction stage, 113 of buried cadavers, 62 butyric fermentation stages, 113 degree of physical protection, 57 dry stage of, 114 effect of burial on, 118 environmental factors influencing, 57 fermentative products and, 208 human process, 3 impact of clothing and, 212 lime and, 211 of materials associated with buried cadavers, 153–202 oxygen availability, 57 pH, 57 redox conditions, 57 temperature, 57 terrestrial, 53 turnover and, 54 of unburied cadavers, 62 water availability, 57 decomposition, aboveground, 38 decomposition, chemical process of, 105–213 in a burial environment, 203–225 decomposition products of carbohydrates, 208 decomposition products of lipids, 208–209 decomposition products of protein, 205–207 decomposition, factors affecting, 55–61 environmental factors, 57–61 presence and activity of organisms, 61 resource quality, 56 decomposition, stages of, 112 bloat, 112 decay, 112 deflation and decomposition, 230 disintegration, 230 dry, 112 flesh, 112 fresh, 230 inflation, 230

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Index skeletal, 112 skeletonization, 230 decomposition, terrestrial invertebrates and, 109–122 soil organisms and, 53–66 decomposition and carbon turnover in soil, 54 decomposition chemistry liquefaction and skeletonization, 213–215 rate of decomposition of buried bodies, 215–216 decomposition fluids, 36 decomposition of animal bodies, factors affecting, 63 decomposition rates, 55, 226 body fat and, 216 burial in soil vs. exposure to air, 138, 215 clothing and, 216 coffins, 216 depth of burial and, 217 forensic situations and, 216 plastic, 216 soil environment and, 215 soil particle size, 215 thin bodies and, 216 decomposition site, ecology of, 29 decompositional environment, 113 decompositional process, invertebrate fauna and, 110 denaturing gradient gel electrophoreses (DGGE) showing fingerprint patterns for thirteen different soils, 299 dendrochronology, 238 estimating date of a grave and, 238–239 denim, 189 clothing, 181 test fabric buried at 30 cm at location 1, 182, 183 test fabric buried at 30 cm in garden soil location 2., 183 test fabric buried at 60cm in clay at location 1., 182 test fabric laid on soil surface under conifer, 184 denim, blue, 185, 188 zippers and studs, results summary table, 182 denitrification, 208 density gradient techniques, 274

69918.indb 323

323 dental identification of human remains, 236 dermatophytes, pathogenic, 132 Dermestid beetles (Anthrenus spp.), hair and, 135 desiccation, 39, 42, 211, 212 deterioration of synthetic fibers buried in well-watered soil, 171 diagenesis, 237 diagram showing relationship of abandoned cadaver or excreta, fungi growing after decomposition, and trees hosting fungi in the mycorrhizal symbiosis, 84 diatoms, 295 diffraction pattern, as fingerprint of soil sample, 290 diffuse reflectance infrared Fourier transform spectra (DRIFTS), 21, 22, 23 Diptera, 229 larvae, 113 species, 230 disarticulation, 227 Disch, Eva, forensic comparison of soils in murder of, 6 discrimination of soils, forensic science and, 295 dissolution, decomposition of bone and, 214 DNA, 285, 298 fingerprinting of human tissues, 306 sequencer, 300 sequencing, 296 variability of, 298 Dokuchaev, V.V., factors of soil formation and, 8 Donated Body Program at the University of California-Davis, 181 Doratomyces putredinis:Deuteromycetes, 72, 74 Doyle, Sir Arthur Conan, 272 “A Study in Scarlet,” 5 Adventures of Sherlock Holmes, 272 awareness of forensic soil comparisons, 6 forensic soil comparisons used in fiction of, 5 Sherlock Holmes and, 5 “The Five Orange Pips,” 5 DuPont, 161 spandex and, 162

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324 Index

E earthworms, 61 ecology, 227 ecosystems, 250 terrestrial decomposition and, 53 ectomycorrhizas, 74 EDS spectra, 288 Egypt hair of coptic mummies and, 131 natural mummies in, 39 natural preservation of cadaver in, 42 Elastane (Spandex or Lycra), 162 electron microprobe analysis (EMPA), 280 electron microscopes, forensic soil analysis and, 279 electron tunneling microscopes, 280 energy and wavelength dispersive x-ray (EDX and WDX) microanalysis, 281 energy-dispersive spectroscopy (EDS), 279, 288 energy-dispersive x-ray analysis (EDXA), 286, 288 engineering and, soil classification systems and, 3 England, 172 regional soil survey and, 304 entomology, forensic, 111–112, 164, 226–229, 237, 242, 250 aboveground successional studies, 112 applications of, 111 buried remains and, 229–230 in cases of neglect of elderly or very young, 111 circumstances surrounding a death, 111 current research, 111 current research in, 112 DNA studies for identification of insects, 112 estimating of postmortem interval (PMI), 111 homicide investigations and, 110 insect succession and, 229 investigation of unexplained deaths, 111 medicolegal aspect, 111 in popular culture, 111 postmortem interval estimation, 112 postmortem interval (PMI), 231 succession, 111 urban applications, 111 environmental forensics, 272

69918.indb 324

ethanol antemortem blood alcohol concentrations and, 208 postmortem bacterial production of, 208 evidence, botanical analysis of stomach contents, 238 determining cause of death, 238 determining geographic location, 238 determining time of death or deposition, 238 fungi and, 238 placing person at crime scene, 238 evidential material, 156 excrement, 69 excrement, belowground, 80 excretion, mycorrhizal symbiosis and, 84 exhumations, 10, 119 decomposition of bone and, 214 exhumations, cemetery, 229 fly species and, 229 in Lille, France, 229

F fabrics, household, 198 fauna, invertebrate, 109 feldspar, 287 fermentation, 109 ferrous corrosion products, not biocidal, 178 fibers, natural, 158, 185 animal (protein) and, 158 plant material and, 158 fibers, semisynthetic, 161 fibers, synthetic, 200 degradation of, 170 resistance to degradation, 170 twentieth century, 158 Flanders, boot recovered from, 172 flesh flies (Sarcophagidae), 35, 112, 118 flies (Diptera) corpses and, 112 forensic research and applications, 112 larvae important in estimating postmortem interval (PMI), 116 flies (Muscina spp.), 119 footwear, 154 crime scene and, 170 modern, 172 sandals recovered after 26 years, 171 synthetic materials in, 172

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Index forensic fingerprinting of soil, methods of biological analysis, 291–300 chemical analysis, 281–290 combined approaches, 301–302 physical characteristics of soil, 277–280 polyphasic approaches and links to databases, 303 sampling and handling, 275–276 statistical considerations and presentation of evidence in court, 304–305 Forensic Geology by Murray and Tedrow, 273 forensic investigations, 230 antemortem period, 226 application of ecological principles to, 62–64 clues and, 306 comparison of samples, 306 decomposition of hair, safeguarding as evidence, 139–140 diffuse reflectance infrared Fourier transform spectra (DRIFTS), 190 geochemical technique, 283 hair survival and, 139 identification of soil materials, 306 inhibition of biodeterioration of cadaver and associated clothing, 190 insect succession and, 231 insects in decomposition process, 110 measurement of soil microbial biomass, 249 methods of characterizing and fingerprinting soils, 271–316 microbial biomass and, 250 perimortem period, 226 postmortem period, 226 role of invertebrates in terrestrial decomposition, 109–122 shallow burials, 230 use of several comparison techniques in combination, 301 forensic science, 3, 30 discrimination of soil samples in, 295 hair and, 123 fossil preservation, 225 fossilization, 237 Fourier Transform Infrared Spectroscopy (FTIR), 23, 285, 295 spectral soils database, 284

69918.indb 325

325

of three brown earth soils under different land use, 284 fumigation-extraction (FE) method, 255 fumigation-incubation (FI) method, 255 fungal biomasses in soil, estimates of, 265 fungal growth as corpse funder, 78 detection of through fungi and, 90 fungal succession, 76, 78 estimation of time since cadaver was deposited, 91 fungal succession on urea plot, example of, 71 fungi, 57, 209, 238, 241 breakdown of buried textiles, 164 cadavers and, 68 decomposition of bone and, 214 ectomycorrhizal, 80 ectomycorrhizal fungi, 85 excreta and, 68 forensic application of, 93 forensic potential of, 94 in forensic taphonomy, 68 reproduction of, 93 saprotrophic, 85 in soils with addition of urea or nitrogen, 68 succession change from EP to LP in urea plot, 90 in urea-treated soil, 72 fungi, coprophilous, 68 fungi, keratinolytic, 132, 133, 137 anthropophilic, 132 geophilic, 132 morphological changes in, 134 pathogenic, 137 in soil, 133 zoophilic, 132 fungi, keratinophilic, soft tissues and, 68 fungi, postputrefaction ammonia and, 241 early phase (EP), 241 late phase (LP), 241 succession sequence of, 37, 241 fungicide (cyclohexamide), 264

G Garda Siochana laboratory, soil as physical evidence in, 274

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326 Index garments. see clothing garments made from cotton and cotton mix fabrics, 160 gas chromatogram traces from Calluna vulgaris (heather) shoots and from the underlying moor topsoil, 293 general trends for textile preservation from cadaver and control graves, 189 geochemical techniques, 283 geochemical signatures, 283 isotope ratios, 283 geochemistry, 12 geographic information systems (GIS), 2 geology, forensic, 2, 6 geophysics, 23 geoscience, forensic, 272 Geotrupidae (dung beetles), 118 German Democratic Republic, 240 Germany, exhumations in, 40 Gestapo, 240 Glomus pubescens, 76 gnawing of bone, decomposition of bone and, 214 grave soils, 36, 57 research, 37 soil microbial biomass C concentrations, 250 graves black precipitate and, 206 microorganisms in, 61 sulfides and, 206 graves, clandestine, 163, 207, 226 excavation of, 174 locating, 29, 44 modern, 190 Gross, Hans, Handbook for Examining magistrates, 272 guinea pig, decomposition of, 37 gut flora, putrefactive change, 180 gypsum, 20, 21

H habitat-cleaning symbiosis, 83 hair, 125 Anglo-Saxon graves and, 125 archeological studies, 134 bulk amino acid analysis and, 131 buried body and, 125, 137–139 contaminants in, 129

69918.indb 326



cosmetic treatment, 128 cosmetic treatment of, 130 cuspate lesions to shaft characteristic of insect damage, 136 degradation of, 134 dermatophytes and, 136 determining drug use, 123–124 diagram of main structural components, 126 epicuticle, 127 exhumed cemetery remains, 125 exocuticle, 127 exposure to environmental contaminants, 128 extent of damage in long-buried hair samples, 131 as forensic evidence, 123, 124, 130, 134 Fourier transform infrared spectroscopy, 131 FT-Raman spectroscopy, 131 fungal damage to hair recovered from soil, 135 genetic information in, 123 grooming practice, 128 growth cycle of, 125 homicide victims and, 125 human remains and, 125 individuation and, 124 keratins and keratin associated proteins, 124, 128 oxidative damage to, 130 particulate evidence and, 123 physical and chemical alteration, 128 physical strength of, 130 postmortem exposure to changes, 128 postmortem exposure to contaminants, 128 reaction with heavy metals, 130 safeguarding sample evidence from, 129, 139 structural stability of, 128 structure, growth, and function of, 125–128 surface-exposed environments, 125 survival of, 124 toxic substances, 123, 124, 125 in various depositional environments, 131 victims of accidental death, 125 weathering of, 129, 130 wool and, 134

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Index hair, decomposition of, 139 appropriate measures for safeguarding evidence and, 139–140 burial environment, 138 burial environment and, 139 in buried body environment, 123–152 buried body environment and, 123–153 degradation of clothing and, 134 experimental studies, 134–135 experiments using pigs as body analogues, 139 field experiments and, 139 histological alteration to the hair shaft, 134–137 keratinophilic fungi and, 131–133 level of keratinization and, 137 microbial degradation of hair keratin, 133–134 progress of, 137 scanning electron microscopes (SEMS), 135 structural components of hair attacked in sequence, 137 variability of, 139 hair, long-buried histological assessment, 136 medieval deposits in Hythe, United Kingdom, 136 scanning electron microscopes (SEMS), 136 transmission electron microscopes (TEM), 136 hair, preservation of aridity, 137 DNA and, 138 hypersalinity, 137 permafrost, 137 as selective, 138 use of metal or metal-shell coffins, 137 waterlogged peat conditions, 137 hair color, fading of caused by exposure to sunlight, 130, 130 hair curl, 127 hair fibers structural alteration of, 130–131 weathering, contaminants, and color change, 128–130 hair form, assessment of to determine race in anthropology, 136 hair growth anagen (growth phase), 125

69918.indb 327

327 catagen (regression phase), 126 telogen (resting phase), 126 hair keratin, mechanism of microbial degradation, 133–134 hair root postmortem changes in, 134 postmortem root banding, 134 hair samples individualization using DNA, 123 morphological characterization, 123 hair shaft, 126 central medulla, 128 cortex, 126, 127 histological alteration to, 134–137 keratin intermediate filaments (microfibrils), 127 outer cuticle, 126 porous medulla, 126 as source of DNA, 124 trichohyalin, 128 Hebeloma, 80 Hebeloma danicum, 82, 90, 93 and H. radicosoides fruiting on buried uric acid, 89 Hebeloma radicosoides and, 74, 78 and Laccaria bicolor fruiting beside remains of paper, 91 Hebeloma radicosum, 88, 93 flush of marking a late phase of fungus succession on urea plot, 71 fruiting out of a human latrine, shown in soil profile, 82 fruiting out of the deserted latrines near the nest of a mole, shown in soil profile, 83 and H. danicum fruiting on buried uric acid, 89 Hebeloma danicum and, 78 small mammal latrines and, 82 Hebeloma spp., 72, 87 Hebeloma vinosophyllum (arrowhead), 87 fruiting beside the skull of an abandoned domestic cat body, 78, 84 herbivore chewing, 214 Hertfordshire, experimental project with decomposition, 186 hide beetles (Trogidae), 112 high standards of analysis, importance of in court cases, 306 high-resolution electric field probe, 275

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328 Index Hilgard, E. W., factors of soil formation and, 8 homicide victims, forensic investigations and, 232 horizons, 10 human bone samples modern period, 234 nonmodern period, 234 premodern period, 234 human DNA, 303 human DNA database material, courts and, 306 human hair, 242 human latrines, buried, 80 Humaria velenovskyi fruiting on the ground after decomposition and disappearance of dumped night soil, 81 humus, 74, 78 color change to black with ammonia, 76 urea in, 75 Hydrophilidae, 118

I ICDD Powder Diffraction FIle, 22 Iceman, hair and, 131 ICP mass spectrometry (ICP-MS), 282 ICP optical emission spectrometry (ICPOES), 282 ICP spectrometry, 281 ICP-AES, 288 ICP-MS, 288 images of fossil pollen grains found in organic soil horizons, 293 immature necrophagous insect species, estimating postmortem interval (PMI) and, 249 immunological techniques estimating postmortem bone age, 233 residual serological activity of bone protein as indicator of bone age, 233 Imperial Chemical Industries (ICI), 161 increased vegetative growth above a grave site, tool for locating buried remains, 207 inductively coupled plasma (ICP) spectrometry, 281 infrared (IR) fingerprinting methods, 284–285

69918.indb 328

infrared spectrometry, 275 Infrared spectrum, 285 inorganic nutrients, 60 insect activity, exclusion of, 228 insect fauna associated with bodies buried in a shallow grave, 230 in graves, 229 insect succession buried vs. above ground cadaver, 230 estimation of postmortem interval, 229 postmortem interval (PMI) and, 249 insects, 31, 33 above ground, 30 ants (hymenoptera), 116 beetles (coleoptera), 117 cockroaches (blattodea), 116 decomposition process and, 110 earwigs (dermaptera), 116 effects of on manmade structures, 111 infestation in stored commodities, 111 sarcophagous, 63 silverfish (Thysanura), 116 springtails (collembola), 115 insects, succession of, estimating postmortem interval (PMI), 228 invertebrates, buried remains and, 118–120 invertebrates in decomposition, forensic applications, 109–122 invertebrates in terrestrial decomposition, 109–122 forensic applications and, 109–122 iron oxides, 20 isotopes, 283

J Japan, 72, 76 soil data base and, 304

K Karluk archeological site in Alaska, hair and, 130 keratin, 164, 206 hair and, 165 microorganisms and, 132, 133 wool and, 165

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Index keratin degradation, mechanisms of by a dermatophyte fungus with that of an Actinomycete, 139 keratin fibers, 135 keratin proteins, hair and, 124 keratinolytic activity, ecological factors influencing, 131–133 keratinophilic fungi, geographic distribution of, 131–133 keratinophytes, saprophytic, 132 keratins, 129 knitted cotton/Lycra fabric, scanning electron microscope (SEM), 163 Kyoto, Japan, 281

L Laccaria bicolor, 90, 93 fruiting and detection of buried mammal carcus from under the fungus, 79 and Hebeloma danicum fruiting beside remains of paper, 91 Laccaria spp., 72 Lactarius chrysorrheus, 93 laser diffraction particle-size analysis, 301 latrine, 69 Laubach, 272–273 leather, 154, 242 crime scene and, 170 decomposition of, 166–174 leather, modern tanned, highly resistant to decay, 170 leather, tanned, recovered from Western Front of World War I, 171 legal profession, awareness of soil as physical evidence, 274 Leiodid beetles, 118 Leptocera caenosa, 229 Leptodiridae species, 118 light, physico-chemical properties of hair and, 129 lignin, 164, 165 lime, 211 linen, 158 Europe and, 158 from Overton Down experimental earthwork, recovered after 32 years of burial in a chalk environment, 171 linking of suspect to crime scene, 302

69918.indb 329

329 lipids breakdown of, 216 decomposition products of adipocere and, 210–212 liquefaction, 213–215, 216 soft tissue and, 216 lividity, 204 lividity, fixation of, 204 used to establish postmortem reposition of body, 204 used to estimate time since death, 204 livor mortis, 203–204, 227 Locard, Edmond, 3, 273 Locard Exchange Principle, 3, 273 Lombardi, G., soil as physical evidence and, 274 Lord Mountbatten case, soil as physical evidence in, 274 luminol testing, determining age of bone and, 233 lycra, 198 Lyophyllum ambustum: Basidiomycetes, 72 Lyophyllum tylicolor, 74, 87, 88–89 flush of marking an early phase of fungus succession on urea plot, 70 fruit bodies collected from urea plot and from human urination sites, 88 growth of under defined conditions using humus, 92 in slide culture, 88

M Macrochelidae, 114 Madgeburg, Germany, mass grave and, 240 maggots, 31, 35, 36, 180 activity, 39 as indicator of delayed burial, 250 major and trace element date, role in forensic comparison of soils, 283 major postputrefaction fungi and data of their appearance that may have forensic implications, 102–107 maps, distribution of soils and, 11 materials, decomposition of cadavers and, 154–202 casework examples, 172–175 household fabrics, 199 men’s clothes, 196 metals, 175–180

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330 Index

review of clothing based on current U.K experience, 195 women’s clothes, 197 measurement of ATP (Firefly Assay), 263 Mégnin, theory of, insect succession and, 229 melanin, 129 biochemistry of, 129 hair and, 126 mesofauna, 164 metal clothing fasteners, 154, 174 durability of, 181 jean rivets or zippers, 156 jeans and, 181 metal objects, dry corrosion, 176 metal preserved organics (MPOs), 154, 178 archeological literature and, 154 forensic investigations, 156 relevance to investigation of criminal cases, 190 metals, 154 electrochemical corrosion, 176 tools, 154 weapons, 154 metals, corrosion of, 175–180 aqueous corrosion, 176 biocidal effect of, 178 case study: the mummified body of a woman, 179 casework examples, 179 chemical nature of burial environment, 175 corrosion, 176 dry corrosion, 176 immunity, 176 interval of burial, 175 metal-preserved organics, 178 passivation, 176 rate of, 175 metapedogenesis, 10 microbes, 33, 164 microbial activity and biomass in soil, measuring, 248–270 principles and methodologies of, 248–270 soil collection and preparation, 250–252 soil dry matter content, 253 water-holding capacity, 253 microbial activity in soil, principles and methodologies of measuring, 247–271

69918.indb 330

microbial Adenosine 5’-triphosphate (ATP), 262–264 microbial adenosine 5’-triphosphate, measuring, 262–263 ATP extraction, 263 measurement of ATP (Firefly Assay), 263 preparation of ATP extraction reagents, 263 preparation of stock ATP solution, 262 microbial biomass, 255 forensic science and, 250 turnover of, 57 microbial biomass C, 258 automated organic C analyzers, 257 fumigation-extraction (FE) method, 257 measuring by fumigation extraction, 255 microbial biomass C, measuring by fumigation extract, 255–257 analysis of soil extracts, 256 automated analysis of organic C, 257 reagents and experimental procedure, 255 microbial biomass, measuring, substrate induced respiration (SIR), 264 microbial biomass N, 258–259 microbial biomass N, measuring by fumigation extract, 258 reagents and experimental procedure, 258 microbial biomass Ninhydrin-N, measuring by FE, 258 reagents and experimental procedure, 259 microbial biomass of soil changes in the concentrations of ammonia and the pH value in urea-treated soil, 248 definition of, 248 methodology of measuring, 248 microbial biomass of soil, estimating, 253–254 carbon dioxide flush and, 254 microbial biomass of soil, measuring direct microscopic counting, 253–254 FE method, 254 FI method, 254 fumigation-extraction (FE) method, 254 fumigation-incubation (FI) method, 254 microbial biomass P, measuring, 260–262 analytic procedure, 261

2/6/08 12:21:57 PM

Index

preparation of Murphy Riley Colorimetric reagent, 261 reagents for extraction, 260 soil extraction, 260 microbial diversity, in soils collected from a crime scene, 300 Micrococcus, 208 microfleece, 197 microorganisms keratinolytic, 132, 138 keratinophilic, 132 shift in populations as indicator of grave sites, 62 microorganisms, anaerobic, 31 microscope, petrographic, 21 mineral exploration, soil classification systems and, 3 mineralogical analysis, by electron microscope, 288 mineralogical evidence, U.S. jurors, 289 mineralogy, 12, 237, 285–286 X-ray diffraction (XRD) methods, 23 minerals, 10 clay, 287 density and, 287 minesoils and, soil classification systems and, 3 Minolta CM2600d spectrophotometer, 277 mites (acari), late decomposition stage and, 114 mites (Acaridae), 114 mites (Cunaxidae), 114 mites (Winterschmidtiidae), 114 modern gas chromatography-mass spectrometry (GC-MS) methods, 284 moisture level, 217 molds, 72, 238 molecular level isotopic techniques, 283 Moro, Aldo, environmental forensics in case of murder of, 273 Morpholeria kertesczi, 119 Mucor spp., 76, 80, 93 mucus shield, 213 mulberry silkworm (Bombyx mori), 159 mummies, red-brown hair and, 129 mummification, 39, 211, 212 artificial, 39 intentional, 39 natural, 39

69918.indb 331

331

of woman found dumped in northern England, 179 Munsell Color Company, 277 Munsell Soil Color Charts, 13, 22, 277 Murphy-Riley Colorimetric reagent, preparing, 261 mushrooms, 72, 89. see also fungi mycology, field application to forensic taphonomic investigations, 241 postburial interval (PBI), 241 mycology, forensic, 94 mycophagy (consumption of fungi), 83 mycorrhizal symbiosis, 83

N National and International Soil Classifications Systems, 2 natural fibers, degradation of, 169 Nechrophorus orbicollis, rodent carcasses and, 117 necrophages, 112 nematodes, 61, 76 neutron activation analysis (NAA), 281 New Mexico, 273 pollen analysis in, 292 New Zealand, pollen analysis in, 292 ninhydrin reagent, preparing, 259 nitrate, 37 mineralized from arginine, 267 nitrification, 208 Nitrobacter spp., 208 nitrogen content, 232 nitrogen loss from bone, independence from environmental factors, 232–233 nitrogen mineralization, 266 Nitrosomonas spp., 208 Nocardia, 212 nonectomycorrhizal vegetation, 78 nuclear magnetic resonance (NMR) spectroscopy, 285 Nuclear Test Ban Treaty, 234 nuclear weapons testing in the 1950s, 234 increased levels of radionuclides, 235 nylon (polyamide), 170, 198, 200, 242 degradation of, 134 development in 1930’s, 161 resistance to microbial attack, 161

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332 Index nylon , use of for military purposes, deposition of a body and, 30

O odontology, forensic, 242 identification of deceased individuals, 236 postmortem tooth loss as an indicator of PMI, 236 Ophyra capensis, 229 organic residues in soils, decomposition of, 55 organisms, noncadaveric, 31 orlon, 161 Osen Reagent, preparing, 260 Overton Downs Experimental Earthworks Project, 170, 171–172 Oxenhope cadavers, 185

P palaeoecology, 238 palaeontology, 225, 237, 238 palynological evidence mass graves and, 294 statistical approaches to, 293 use of to connect suspect with scene of crime, 294 use of to disprove an alibi, 294 used in investigation of major war crimes, 294 palynology (spore/pollen analysis) botanical ecology and, 291 petrology and, 302 palynology (spore/pollen analysis), forensic, 240, 242 associative evidence and, 291 determining time of year of a burial, 240 pollen and spores, 240 provides associative evidence, 240 skeletons in Magdeburg, Germany, 240 use to criminal investigator, 291 pantyhose, 198 woman buried in, 174 paper, 242 paper products, 154 Paris Exposition of 1889, 160 particle density, 274

69918.indb 332

particle size distribution of two contrasting soils determined using a Coulter LS230 laser granulometer, 279 pasture site, 185 pathology, forensic, 227, 237 PCR fingerprinting techniques, 298 pedogenesis, 7, 8 anthropogenic conditions, 10 speed of, 10 pedogenic horizons, 9 horizon with weathered rock or underlying sediment, 9 indurated rock layer, 9 subsurface horizon, 9 surface horizon, 9 water layers within or beneath the soil, 9 pedogenic processes, 286 spatial scale, 10 pedology, 2, 3 classification systems, 3 purposes of, 6 pedon, 10 peds, 20 Peru, natural preservation of cadaver in, 42 pesticides, arsenal, 283 petrography, 21 Peziza moravecii:Duscintcetes, 72 Ascobolus denudatus, 87 PFLA analysis biochemical fingerprint of soil microbial community, 301 biological fingerprint windblown soil, 301 pH, decomposition of bone and, 214 phospho-lipid fatty-acids (PLFAs), 301 physicochemistry, diffuse reflectance infrared Fourier transform spectra (DRIFTS), 23 physiology of the AF and PPF, 84–89 formation of reproductive structures, 87–88 physiology and succession, 89 spore germination, 84 vegetative growth, 84–87 physiology of the AF and PPF, vegetative growth enzymatic activity, 87 hydrogen ion concentration, 86–87 nitrogen utilization, 84–85 phytoliths, 295

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Index pig cadavers as analogues for human bodies, 180 in bloated and advanced decay stages of decomposition, 34 cadaver decomposition island (CDI) and, 37 experiments with, 163 studies of decomposition using, 230 Planck’s equation, 282 plant DNA analysis accepted in British courts, 297 tracing of illegal drugs such as marijuana, 297 plant evidence, at crime scene, 238 plant fragments, matching to link suspect to scene of crime or to confirm alibi, 298 plant roots, decomposition of bone and, 214 plant wax markers analysis, 296 plant waxes, 296 plastic coverings decomposition retarded by, 212 used to wrap body before burial, 212 plastics, 154, 242 Poaceae, 294 polarized light microscopy (PLM), forensic science and, 287 Polartec, 161 police, awareness of soil as physical evidence, 274 pollen, 238 pollen analysis, 292 forensic investigations and, 275 pollen grains, 291 pollen samples in case of alleged sexual assault, 293–294 on shoes and shoe prints, 293 pollen/spore types, discrimination between vegetative communities, 292 polyester, 170, 185, 197 polypedon, 10 polyphasic approach, 303 Popp, George first formal presentation of soil evidence in court, 272 forensic soil comparisons and, 6 murder case and, 272–273 science of environmental forensics, 273 postburial interval (PBI), 225–247 burial environment and, 227

69918.indb 333

333

the burial environment as a PBI determinant, 237–242 dendrochronology and, 240 estimating, 29, 227, 242 growth patterns of plants and, 226 postmortem interval (PMI) and, 226 potential determinants of, 225–247 postmortem changes algor mortis, 203 livor mortis, 203 rigor mortis, 203 skin color, 203 postmortem changes, early, 216 postmortem hydrolysis, 209 postmortem identification and, 212 postmortem interval (PMI), 225–247, 249 the body as a PMI determinant, 227–236 enzymatic changes an, 227 estimating, 29, 44, 110, 119, 226–227, 230, 242 potential determinants of, 225–247 soil and, 120 succession of insects on a corpse, 111 postmortem period, early, 227 postputrefaction, 86 postputrefaction fungi (PFF), 87, 88, 93 identification of clandestine graves and, 226 rearing in pure culture, 93 postputrefaction fungi (PFF) and AF, 76–83 fungal growth following cadaver decomposition below ground, 78–79 fungal growth following cadaver decomposition on the ground, 76–77 fungal growth following excreta decomposition below ground, 80–81 fungal growth following excreta decomposition on the ground, 80 habitat-cleaning symbiosis, 82–84 postputrefaction organisms, 83 Pourbaix, Marcel, 176 Pourbaix diagram, 177 Pourbaix diagram for copper/water (simplified), 187 prediction of sample provenance organic matter characterization, 302 palynology and, 302 SEM and, 302

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334 Index XRD and, 302 proteins, breakdown of, 216 proteolysis body’s proteins and, 205 breakdown of proteins into amino acids, 206 color changes of body and, 206 resistance of connective tissues and cartilage, 206 proteolytic enzymes, 206 protozoa, 241 Pseudombrophila petrakii, 74, 87 Pseudomonas, 208 pseudomorphs, 216 pseudorhizas, 78 pupae dating of burials and, 231 putative postputrefaction fungi in association with a pig cadaver in advanced decay stage, 38 putrefaction, 31, 35, 109, 216 bacteria from intestinal tract, 205 bloating and, 205 brain and, 206 color changes and, 205 epithelial cells and, 205 initiation by autolytic process, 205 kidney and, 206 lining of gastrointestinal tract, 206 liver and, 206 microbial activity and, 205 neuronal cells and, 205 odor caused by bacteria, 205 putrefaction fungi (PPF), 69 putrescine, 206

Q QEMSCAN technology, 288

R raccoon dogs (Nyctereutes procyonoides), latrines of, 80 radiocarbon dating, 235 of bone material from an archeological era, 234 forensic investigations and, 236 of skeletal remains of forensic interest, 234

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radionuclides, as potential indicators of postmortem interval (PMI), 235 Raman spectroscopy, 285 range of fabrics used in some bras currently available in U.K., 197 rayon, 242 regenerated cellulose, 160 rayon, viscose, wood pulp and, 160 reagents, 266 reagents and experimental procedure, 255–256 reference soils, 304 relative time, archeology and, 226 remains. see cadavers Rhopalomyces elegans var. minor, 74, 76 Rhopalomyces strangulatus, 76, 93 rigor mortis, 203–204, 227, 249 onset and duration affected by environmental conditions, 204 used to establish postmortem interval (PMI), 204 RNA, 298 Rosaceae, 294 Rothamsted, U.K., 250 Rothamsted C model, 55 rove beetles (Coleoptera: Staphylinidae, 112 maggot predators, 117

S saline and acid sulfate soils (links to policy and jurisdiction), soil classification systems and, 3 Sarcophagidae (flesh flies), 231 scanning acoustic microscopes, 280 scanning electron microscope (SEM) scanning electrophotomicrographs comparing a control soil sample and an evidence sample, 280 scanning electron microscopes (SEM), 281, 288 analysis, 280 scanning electron microscopes (SEMS), 22 scavengers, 31, 33, 63, 217 animals, 109 disturbance of corpse and, 251 foxes and, 185 scene of crime officer, 274–275 scene of crimes, 2 science, forensic, 249 Scutellinia scutellata, 76

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Index season of burial, insect material and, 231 SEM-ED analysis, 280 separation of three grassland soils using bivariate plots of elements carbon and phosphorus, 282 shallow burials, 215 plants and, 238 vegetative growth above a buried corpse, 207 shoes, leather, 158 shrew latrine, 80 Siberia, natural preservation of cadaver in, 42 sigmoidal decomposition curves associated with cadaver decomposition on soil surface and following burial in soil, 35 signature lipid biomarkers (SLD), 298 fatty acid profiling, 300 silk, 158, 159, 242 addition of metals to, 160 in burial environment, 159 protein sericin, 159 survival from 19th century burial contexts, 160 survival in anaerobic conditions, 160 silverfish (Thysanura), dry stages of decomposition and, 116 size and turnover times of different soil organic matter fractions, 55 skeletal remains, 232 skeletonization, 76, 203–204, 213–215, 216, 227 aerobic environment, 213 anaerobic environments and, 213 skin beetles (Dermestidae), 112 slickensides, 20 small mammals, decomposition of bone and, 214 sodium arsenate buffer (To make 1 litre), 264 soft tissue decomposition, 203 autolysis, 204 putrefaction, 204 soil, biological analysis of, 291–300 botanical fragments, 296–297 diatoms, 295 microbial fingerprints, 298–300 palynology, 291–294 soil organic matter (SOM), 295–296 soil, characteristics of, 12

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335

abundance of roots, 12 color, 12, 277 depth changes in consistence, 12 microscopy, 279 particle size distribution analysis, 278 segregations/coarse fragments (carbonates and ironstone), 12 structure, 12 texture, 12 soil, chemical analysis of, 281–290 elemental analysis, trace metals, pollutants, 281–283 infrared (IR) fingerprinting methods, 284 mineralogy, 285 other chemical fingerprinting methods, 285 soil, depression in, indication of shallow grave, 207 soil, forensic examination of detection of manufactured materials, 284 detection of materials from different environments, 284 environmental artifacts, 284 soil, forensic fingerprinting of, 272–315 methods, 275–305 soil evidence, 272–274 soil, properties of climate and, 8 factors in, 8 organisms and, 8 parent material and, 8 time and, 8 topography and, 8 soil analyses, used only in investigation of serious crime, 274–275 soil analysis, methods of, 22 soil analytical characteristics, footwear print information, 302 soil animals, 61 soil as a burial environment, 163–166 agents of decomposition, 164–165 degradation of the body and its effect on associated materials, 166 soil assessment, 10 soil biology locating clandestine graves, 30 postmortem interval, 30 soil biota, involved in degradation, 164 soil C, inert, 57

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336 Index soil C turnover, 55 soil carbon dioxide, 265–266 soil characteristics, 302 soil chemistry locating clandestine graves, 30 postmortem interval, 30 soil classification, 6–10 systems of, 3 soil CO2, evolution and N mineralization, 265–266 procedure, 266 soil color content of organic matter and, 14 hue, 13 intensity, 13 iron oxides and, 14 value, 13 soil compactness, insect activity and, 229 soil consistence, interpreting, 16–17 soil databases, color recorded in, 277 soil discrimination, forensic, 3 soil distribution, 6–10 soil dry matter content, calculating, 253 soil evidence background and historical perspective, 272–274 in case of murder of Aldo Moro, 273 the evidential value of soils, 274 first formal presentation in court, 272 soil examinations, forensic applications, 23 soil fingerprint biomarkers and, 296 plant wax alcohols persisting in soil, 296 soil fingerprinting methods, 306 denaturing gradient gel electrophoresis (DGGE), 299 PFLA analysis, 301 single strand conformation polymorphism (SSCP, 299 temperature gradient gel electrophoresis (TGGE), 299 terminal restriction fragment length polymorphism (TRFLP), 300 soil forensic analysis, 272 soil forensic laboratories, in U.S., 287 soil formation climate and, 8 factors in, 8 organisms and, 8 parent material and, 8 time and, 8

69918.indb 336

topography and, 8 soil fungal DNA profiles, 300 soil fungi ammonia fungi, 60–75 associated with graves and latrines, 99–101, 102 graves and latrines, 62–64 towards a forensic mycology, 67–108 soil fungi, forensic applications of, 90 assessment of initial conditions and postdeposition interval, 90 fungal growth as evidence of human behavior or activities, 90 limitations of, 91–92 simulation experiments, 91–92 soil horizons, 8 soil incubations, 266 soil information, vegetation history, 296 soil investigation, forensic, 275 soil materials, 1–29 soil matter, water-holding capacity, 253 soil microbial biomass, role of in cycling of plant nutrients, 249 soil microorganisms, 61, 64, 241 inhibited by cold and freezing, 165 measuring, 298 soil mineralogists, 289 soil mineralogy application in forensics, 291 connection between scene of crime an suspect, 291 soil morphology, 23 soil N mineralization, 265–266 soil organic matter (SOM), 295–296 soil organisms, terrestrial decomposition and, 53–66 soil origin, 6–10 soil pH, decomposition of cadavers and, 42 soil profiles, 8 soil sample comparison, composition of major and trace elements, 281 soil samples collecting, 251, 276 comparisons between x-ray diffraction (XRD) patterns of soil samples from shoe and riverbank, 23 crime scene and, 250, 276 depth of, 250 drying, 251 forensic discriminating power and, 274 leaking of body fluids and, 251

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Index

linking of suspect to scene of the crime, 275 links to potential locations, 275 preparation for experimental use, 252 rape case in United States, 302 removing plant and animal residues, 252 sieving, 252 from suspects clothing, shoes, or vehicle, 275 used in investigation of war crimes in Bosnia, 302 soil samples, forensic comparisons of advanced techniques and instruments, 21–25 petrographic techniques and instruments, 21–25 segregations and coarse fragments, 20 soil color, 13 soil consistence, 14 soil structure, 20 soil texture, 15 soil samples, forensic, plant fragments in, 296 soil science, forensic, 3, 6 approaches for comparing soil samples, 12–20 burglary case example and, 22 history of, 4–6 need for reference soils and databases, 306 soil on footwear, 22 techniques of, 11–20 theory of comparing soil samples, 12 X-ray diffraction (XRD) methods, 22 soil scientists, 1 forensic, 2 soil structure peds, 20 slickensides, 20 soil studies, forensic elemental results as spider diagrams, 282 uncertainty in, 282 soil temperatures, 217 soil texture compaction and, 15 erodibility, 15 grades of, 15 leaching capacity and, 15 moisture and, 41 proportion of sand and clay in, 15 soil texture, interpreting, 18–19

69918.indb 337

337 soil trace evidence, commonly applied methods, 274 color, 274 low-power stereomicroscopic observation, 274 macroscopic observation, 274 scanning electron microscopes (SEMS), 274 soil trace evidence, forensic analysis of, 274 soil traces comparison using particle size, 277 in forensic investigations, 277 soil type, relationship to scale, regional and global, 11 soil-corpse interface class arachnida and, 114–115 class insecta and, 115 soils ammonia in, 75 anthropogenic, 276 assessing for forensic comparisons, 10 cadaver decomposition and, 29–53, 76 chemistry of, 14 common mineral groups and minerals encountered in, 286 complexity and variability of, 306 contact traces on suspect shoes, 13 criminal activity and, 30 data and archived soils gathered for other purposes, 306 diffuse reflectance infrared Fourier transform spectral (DRIFTS) patterns, comparison of shoe and riverbank samples, 24 as evidence in forensic investigations, 2, 30, 273–275, 304 fingerprinting for forensic purposes, 298 forensic fingerprinting of, 272–315 methods of characterizing and fingerprinting, 271–316 national standard systems, 9 organic matter in, 53 organic residue in, 55 selected properties of soils from an experimental pig grave, 59 subsoil and, 9 summary of mineralogical composition from X-ray diffraction, 24 systematic approach to discriminating for forensic soil examinations, 5 topsoil and, 9

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338 Index treated with urea, 68, 75, 76 used as physical evidence, 273 value of in death investigation, 30 waterlogged, 165 soils, discriminating geochemistry and, 275 mineralogy and, 275 soil morphology and, 275 wet chemical techniques, 275 soils, forensic comparison of, 1–28, 25 introduction, 1–2 soils, forensic examination of, 276 soils, nature of relevant to forensic soil science, 3 relevant to human decomposition processes, 3 solid-phase microextraction (SPME), 284 some current sock yarn mixtures (U.K.), 197 Somme, France, brass buttons and webbing fittings recovered from, 179 South Carolina, 114 Soviet Secret Police, 240 Soviet soldiers, 240 spatial scale, pedogenic processes and, 10 spectral reflectance curves, 277 spectrophotometer, reflectance charts separating two grassland soils, 277 spectrophotometer method, soil sample comparison and, 277 spectro-photometric color determination, 301 spiders (araneae), arthropods ad, 115 stable isotope analysis, 301 stereobinocular microscope, forensic soil analysis and, 279 stock ATP solution, preparation of, 262 Stokes’ Law, 277 Streptococci, putrefaction and, 205 strontium 90, 235 subsoil, 9 substrate induced respiration (SIR), 264–265 procedure, 265 Suillus bovinus, 93 sylphid Necrophorus , 114 synthetic polymers, 164

69918.indb 338

T taphonic processes involving human cadavers, 138 taphonic time, 226 taphonomy, 227 decomposition and, 227 relation to traditional determinations of postmortem interval, 228 traditional determinations of postmortem interval, 237 taphonomy, early, palaeontology and, 225 taphonomy, forensic, 29, 43, 139, 180–181, 225, 238, 240–242 anthropology, 226 bone, 124 bone changes and, 226 botany, 226 determining cause and manner of death, 226 entomology, 226 estimating postmortem interval (PMI), 226 experiments in, 180 forensic investigations and, 226 hair, 124 mycology, 226 postburial interval (PBI), 226 soft tissue changes and, 226 soils and, 44 teeth, 124 taphonomy, modern, 225 archaeology, 225 microbiology, 225 palaeoanthropology, 225 Terelene, 161 textile biodegradation, rate affected by dyes and surface finishes, 167 textile decomposition, 168 burial in biologically active soils and, 168 example of forensic use of, 173 scanning electron microscopes (SEMS), 168 time interval between burial by perpetrator and excavation by forensics team, 168 textile remains archeological, 165 forensic applications, 167 preserved enough for identification, 167 systematic condition score, 168

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Index textiles, 158–162, 241 archeological woolen textile preserved in anoxic waterlogged conditions, 157 assessment of deterioration, 167 breakdown of buried, 164 classified by vulnerability to composition after burial, 169 decay rates for buried, 242 decomposition of, 154, 166–174 deterioration sequence of, 242 European Middle Ages and, 158 history of, 158 mixed, 166 modern, 166 natural, 154 natural cellulose fibers: cotton, 160 natural protein fibers: wool and silk, 158–159 recovered from a mass grave at Kasir-elyahud in the Jordan valley, 156 regenerated cellulose: viscose and viscose rayon, 160 synthetic, 154, 166 synthetic fibers: nylon, polyesters, acrylics, elastane, 161–162 textiles, degradation of cadaver decomposition products and, 190 case study: differential decay of clothing on a skeletonized body, 174 case study: duvet cover in woods, 172–173 case study: woman buried in pantyhose, 174 experiments, 180–189 experiments: blue denim textiles and metal zippers, rivets and fasteners, 181–183 experiments: effect of cadaveric decomposition on differential degradation of textile materials: Bradford Pig Experiments, 184–190 experiments in forensic taphonomy, 180 natural fibers, 169 synthetic fibers, 170 textiles, household, some typical fabric compositions of, 199 textiles, long-term preservation of, 156 desiccation, 156

69918.indb 339

339 freezing, 156 inhibition of microbial act, 156 textiles and leather, decomposition of, casework examples, 172–175 the body as a PMI determinant forensic anthropology, 231–235 forensic entomology and, 228–229 forensic odontology, 236 the burial environment as a PBI determinant forensic botany and, 238–240 forensic palynology, 240 forensic taphonomy, 240–242 Thiobacillus bacteria, 207 tie anaerobes, obligate aenaerobes, 164 time of death, estimating, 249 tissues hard, 68 soft, 68 tooth enamel, resistance to postdepositional decay, 236 tooth loss, postmortem, 236 as indicator of postmortem interval, 236 postmortem interval (PMI), 237 rate of, 237 topdressing materials, soil classification systems and, 3 toposequence, 10 topsoil, 9 Torrington, Petty Officer John, grave of in permafrost, 135 Torrington, Petty Officer John Franklin expedition, 135 hair and, 135 transmission electron microscopes (TEM), 22, 280 triacetate, 242 Trichophyton mentagrophytes, 137 hair melanin and, 137 Trichophyton terrestre, 137 triglicerides, hydrolysis of, 210 Trogidae species, 118 turnover times, 55

U ultraviolet (UV) oxidation, 254 undegraded cotton polyester fabric, scanning electron microscope (SEM), 162 United Kingdom, 72, 74, 161

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340 Index shallowness of clandestine burials, 163 United States, 161 forensic analysis of soil trace evidence in, 274 varieties of soil in, 11 urban planning, soil classification systems and, 3 urea treatment in soil, destroys indigenous community of soil organisms, 75 U.S. Department of Agriculture (USDA), 11 field book for describing and sampling soils, 9 U.S. Federal Bureau of Investigation, soil as physical evidence and, 274

V van t’Hoff rule, 60 vegetative growth, increased above a grave site, 207 viticulture and forestry, soil classification systems and, 3

W wasp (Vespula flavicepts), 80 waterlogging, 39 weapons, buried, wrapped in cloth, 156 West Yorkshire, U.K., experiments on differential degradation, 181 woodland site, 185 wood-mouse latrine, 80 wool, 158, 170, 188, 189, 242 bulk amino acid analysis and, 131 degradation of, 158, 159 fibers colonized by fungal hyphae, 159 fungi and, 159 hair and, 159 human hair and, 127 keratinolytic microorganisms, 158 from Overton Down experimental earthwork, recovered after 32 years of burial in a chalk environment, 170

69918.indb 340



scanning electron microscopes (SEMS), 159 Wool-synthetic mix fabrics, typical uses, 159 World War I, 172, 179 leather recovered from skeletonized bodies, 171 World War I British brass buttons and webbing fittings from plowed soil in Somme, France, 179 World War I leather boot and woolen sock from waterlogged trench in Hooge, Flanders, 173 World War II, 240 use of nylon for military purposes, 161

X x-ray diffraction data, accepted by U.S. jurors as legitimate fingerprints of the provenance of samples, 289 x-ray diffraction equipment in capillary configuration, 292 x-ray diffraction (XRD) methods, 21, 22, 23 x-ray fluorescence (XRF), 22, 281 x-ray mapping, 280 x-ray microscopes, 280 x-ray powder diffraction (XRPD), 286 application in forensics, 291 patterns from bulk soils prepared by spray drying, 290

Y yarn composition, in currently available pullovers (U.K), 196 Yorkshire, U.K, 171

Z zygomycete phase, 76

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Figure 1.2  Contact traces of yellowish-gray soil on the suspect’s shoes (left) and the control soil specimen from the bank of a river (right), which comprises a mixture of 95% coarse gravel and rock fragments and only 5% clay and silt (< 50 µm fraction).

Comparison of LRJ-1(Red) with Clay from Sole of Shoe (Black) 24 22 Intensity (Counts) × 100

20 18 16 14

46-1045 9-466 31-966 36-426 6-263 29-701 5-586 41-1366 14-164

Quartz, SYN Albite, Ordered Orthoclase Dolomite Muscovite-2M1 Clinochlore-1MIIB, FE-RICI Calcite, SYN Actinolite Kaolinite-1A

12 10 8 6 4 2 10.00

20.00 30.00 2-Theta Angle (deg)

40.00

Figure 1.3  Comparisons between x-ray diffraction (XRD) patterns of soil samples from the shoe and riverbank (< 50 µm fraction) shown in Figure 1.2. The < 50 µm fraction was separated from the stony riverbank soil by sieving through a 50 µm sieve. Shoe and riverbank samples were both ground using an agate mortar and pestle before being lightly pressed into aluminum sample holders for XRD analysis. XRD patterns were recorded with a Philips PW1800 microprocessor-controlled diffractometer using Co Kα radiation, variable divergence slit, and graphite monochromator. (From Fitzpatrick, R. W., Raven, M. D., and Forrester, S. T., CSIRO Land and Water Client Report CAFSS_027, 2007. With permission.)

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2.0

1.5

1.0

0.5 499.99988 cm–1

Variables 1.37600e+03 cm–1 2.25200e+03 cm–1 3.12800e+03 cm–1

Figure 1.4  Comparison of diffuse reflectance infrared Fourier transform spectral (DRIFTS) patterns between the yellow-brown soil on the shoe (red) and the < 50 µm fraction in the stony soil from the riverbank (blue). Shoe and riverbank samples were both ground using an agate mortar and pestle. (From Fitzpatrick, R. W., Raven, M. D., and Forrester, S. T., CSIRO Land and Water Client Report CAFSS_027, 2007. With permission.)

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a

b

Figure 2.1  Pig (Sus scrofa L.) cadaver in the bloated (a) and advanced decay (b) stage of decomposition on the soil surface of a pasture near Mead, Nebraska. Cadavers were 8 weeks old and approximately 40 kg at the time of death. Cadavers were placed on the soil surface within 30 minutes of death. Arrow indicates location and direction of maggot migration.

Figure 2.3  Pig (Sus scrofa L.) cadaver and cadaver decomposition island (CDI) on the soil surface of a pasture near Mead, Nebraska, at 70 days postmortem. Note area of dead plant material that defines the lateral extent of the CDI, which is bordered by plants that have undergone enhanced growth.

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pH11

pH10

pH9

pH8

pH7

pH6

pH5

pH4

pH3

Figure 4.5a  Colors of the leachate from the humus soaked in the buffer solutions with pH 11–3 at one-unit intervals (left to right), showing the importance of alkalinity in this color change and the sensitivity of the soil to treatment. The humus was collected from a PinusQuercus forest, Ibaraki, in April 1997. (From Yamanaka, T., Ph.D. diss., Kyoto University, 2002. With permission.)

Figure 4.9a  Coprinus tuberosus (arrows) fruiting on the ground after decomposition and disappearance of human feces (Appendix 4.2). Note the black color of the humus that indicates the flow-in of ammonia in the past. For scale see pine needles.

Figure 4.16  Hebeloma danicum (arrows) and Laccaria bicolor (arrowheads) fruiting beside remains of paper, which indicates not only antecedent human excreta but also a squatting pose (see text). Photograph was taken in a Pinus forest, Shiga, Japan, on October 20, 1981.

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(a)

(b) Figure 7.1  Archaeological textile remains preserved in iron corrosion, Macro photograph (a) with scanning electron micrograph (b) (original magnification 2000×). The iron corrosion products have formed a negative cast around the wool fibers prior to their degradation many centuries before examination. (Photo: R. C. Janaway.)

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Figure 7.2  Textiles recovered from a mass grave at Kasr-el-yahud in the Jordan valley. This was the result of an act of intercommunal violence in 614 AD. The bodies were skeletonized with surviving cotton and linen clothing. (Photo: R. C. Janaway.)

Figure 7.9  Wool from Overton Down experimental earthwork recovered after 32 years of burial in a chalk environment; inset SEM micrograph showing fungal attack. (Photo: Experimental Earthworks Committee/R. C. Janaway. With permission.)

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Figure 7.10  Linen from Overton Down experimental earthwork recovered after 32 years of burial in a chalk environment. (Photo: Experimental Earthworks Committee/R. C. Janaway. With permission.)

Figure 7.11  Chrome tanned leather from Overton Down experimental earthwork after 32 years of burial in a chalk environment. (Photo: Experimental Earthworks Committee/R. C. Janaway. With permission.)

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Figure 7.12  World War I leather boot and woolen sock from waterlogged trench in Hooge, Flanders. (Photo: R. C. Janaway.)

Figure 7.14  World War I British brass buttons and webbing fittings from plowed soil in Somme, France. (Photo: D. Charlton. With permission.)

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Figure 7.15  Denim test fabric buried at 60 cm in clay at location 1. (Photo: D. Charlton. With permission.)

Figure 7.16  Denim test fabric buried at 30 cm at location 1. (Photo: D. Charlton. With permission.)

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Figure 7.17  Denim test fabric buried at 30 cm in garden soil location 2. (Photo: D. Charlton. With permission.)

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Figure 7.18  Denim test fabric laid on soil surface under conifer hedge. (Photo: D. Charlton. With permission.)

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Figure 8.1  Increased vegetative growth above a grave site.

Figure 8.2  Decomposed porcine remains with partial mummification and adipocere formation.

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