     
 
   
    
  

WWI

\
1

il‘l‘ilHNHHlWll

  

L
x

L

AW

llthWWlIl

  

N
05%
(DO—3

  
  

TH

 

m ‘ LIBRARY
' (, Michigan State
9 w; _ University

This is to certify that the
thesis entitled

THE PERSISTENCE OF GUNSHOT RESIDUE IN
DECOMPOSING TISSUE AND BLOWFLY LARVAE

presented by
LISA MARIE LAGOO

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Forensic Science

 

 

"\

W

Major Professor’s Signature

ML—

Date

MSU is an afﬁrmative-action. equal-opportunity employer

.--.-.-.-.-.-.-._-.-.-._.-.-.-.-.-t-.-A,—.-

o I-.-I-O-I-

. ---- c.-.-r—4-.-.-.n.-.—.-.-----~------—-~—--ggu-c-u-n

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 KlProj/Acc8PreleIRCIDateDueindd

 

THE PERSISTENCE OF GUNSHOT RESIDUE
IN DECOMPOSING TISSUE AND BLOWFLY LARVAE

BY

Lisa Marie LaGoo

A THESIS
Submitted to
Michigan State University

in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE
Forensic Science

2008

I
winter \
(SEM E
was per
gunshot
Sb. Suc
analyze
presenct
Samplin
the fall
While it“
entire si

i

 

lm‘ﬁEti
Presenc
Charactt

to the l

 

WOunds

 

ABSTRACT

THE PERSISTENCE OF GUNSHOT RESIDUE
IN DECOMPOSING TISSUE AND BLOWFLY LARVAE

By

Lisa Marie LaGoo

The persistence of gunshot residue (GSR) in porcine tissue in the fall and in the
winter was investigated by scanning electron microscopy/energy dispersive spectroscopy
(SEM/EDS) and inductively coupled plasma-mass spectrometry (ICP-MS). SEM/EDS
was performed on adhesive carbon tabs that had been dabbed on tissue surrounding
gunshot wounds in order to look for characteristic GSR particles containing Pb, Ba, and
Sb. Such particles were located for up to one day after death. ICP-MS was used to
analyze microwave digested tissue from a shot pig and a control pig over time for the
presence of Ba, Sb, and Pb. All three elements were detected in the tissue over the entire
sampling time frames in both the fall and winter in the shot pig only. The time frame for
the fall study encompassed all stages of decomposition and lasted thirty-seven days.
while in the winter study, the pigs did not decompose due to low temperatures over the
entire sixty day time interval.

In addition, the presence and persistence of GSR was investigated in blowfly
larvae that had been feeding on shot tissue. The larvae were analyzed by ICP-MS for the
presence of Ba, Sb, and Pb. A controlled study was performed indoors and the
characteristic elements were observed for nine days following the exposure of the larvae
to the GSR containing tissue. In addition, larvae found naturally feeding in gunshot

wounds were analyzed and found to contain the elements on days 3 and 4 after death.

 

OppO'
helpe
lCP-l
taugh
of his
C ente
Ryan
with I
let me
F acilit
MSU

Also,

chemi

 

 

ACKNOWLEDGMENTS

This project was a truly collaborative effort and I would like to take this
opportunity to thank the people who have helped. Dr. Ruth Smith was my advisor and
helped so much with the entire process. Dr. Dave Szymanski helped me with all of the
ICP-MS and sat on my committee. Dr. Brian Hunter had the original idea for the project,
taught me about pathology, and sat on my committee. Dr. Steve Dow also took time out
of his schedule and sat on my committee. Ewa Danielewicz and Carol F legler from the
Center for Advanced Microscopy helped me with SEM/EDS, and Dr. Richard Merritt,
Ryan Kimbirauskas, and Kristi Zurawski helped with the indoor study and helped me
with the pigs. Dr. David Foran and the forensic biology group let me use their freezer and
let me in whenever I needed it, and Al Snedgar and Kevin Turner from the MSU Swine
Facility provided the pigs and transported them for me. The Firearms Specialist from
MSU Police shot the pigs and the steak, and Scott Smith built the cages for the pigs.
Also, I am thankful for the support of my family, friends, and the rest of the forensic

chemistry group.

iii

TABLE OF CONTENTS

List of Tables ............................................................................................... vi
List of Figures ............................................................................................. vii
Chapter 1: Introduction .............................................................................. 1
1.1 Introduction ............................................................................................. 1
1.2 Forensic analysis of gunshot residue ..................................................... 3
1.3 Pathology of gunshot wounds ................................................................. 8
1.4 Blowfly larvae .......................................................................................... 10
1.5 Research objectives ................................................................................ 12
Chapter 2: Materials and Methods ............................................................. 14
2.1 Experimental design ................................................................................ 14
2.1.1 Introduction .................................................................................... 14
2.1.2 Indoor study design ....................................................................... 14
2.1.3 Fall and winter outdoor studies day zero procedures .................... 16
2.1.4 Sample collection and storage ...................................................... 17
2.2 Scanning electron microscopy/energy dispersive spectroscopy ............. 18
2.2.1 Introduction .................................................................................... 18
2.2.2 Gunshot residue sample preparation ............................................ 18
2.2.3 Scanning electron microscopy ....................................................... 19
2.2.4 X-ray emission ............................................................................... 21
2.2.5 Energy dispersive spectroscopy .................................................... 23
2.2.6 SEM/EDS analysis of gunshot wound samples ............................ 24
2.3 Microwave digestion ................................................................................ 25
2.3.1 Background ................................................................................... 25
2.3.2 Procedure ...................................................................................... 25
2.4 Inductively coupled plasma-mass spectrometry ..................................... 26
2.4.1 Introduction ................................................................................... 26
2.4.2 Calibration standards and sample preparation ............................. 27
2.4.3 Instrumental theory ....................................................................... 27
2.4.4 lCP-MS of standards and digest solutions .................................... 30
Chapter 3: Results and Discussion ........................................................... 32
3.1 Introduction ............................................................................................. 32
3.2 Decomposition observations ................................................................... 33
3.2.1 Decomposition during fall sampling period (37 days) .................... 33
3.2.2 Decomposition during winter sampling period (60 days) ............... 37

iv

3.3 SEM/EDS results and discussion ............................................................ 40

3.3.1 Fall SEM/EDS analysis results ...................................................... 40
3.3.2 Winter SEM/EDS analysis results ................................................. 43
3.4 ICP-MS results and discussion ............................................................... 44
3.4.1 lCP-MS calibration ......................................................................... 44
3.4.2 lCP-MS precision study ................................................................. 47
3.4.3 ICP-MS analysis of procedural blanks ........................................... 48
3.4.4 ICP-MS analysis of blowﬂy larvae from indoor study .................... 49
3.4.5 ICP-MS analysis of blowﬂy larvae found feeding in
gunshot wounds (fall) .................................................................... 52
3.4.6 lCP-MS analysis of decomposing porcine tissue for
GSR components (fall) .................................................................. 55
3.4.7 lCP-MS analysis of decomposing porcine tissue for
GSR components (winter) ............................................................. 59
3.4.8 lCP-MS tissue analysis summary .................................................. 62
Chapter 4: Conclusions and Future Work ................................................ 65
4.1 Conclusions ............................................................................................. 65
4.2 Future work ............................................................................................. 68
Appendices .................................................................................................. 71
Appendix 1 .................................................................................................... 71
Appendix 2 .................................................................................................... 72
References ................................................................................................... 74

LIST OF TABLES

Chapter 3: Results and discussion

Table 3.1: lCP-MS precision study results. Each average represents ﬁve
replicates ....................................................................................................... 48

Table 3.2: Elemental distribution in gunshot wound digest samples: Fall and
Winter ............................................................................................................ 63

vi

LIST OF FIGURES

Chapter 1: Introduction

Figure 1.1: Diagram of a bullet showing a stripped side view (left) and

the breach face view (right) ........................................................................... 2
Figure 1.2: Gunshot wound stippling (left) and histology of gunshot

wound showing imbedded GSR particles (right).19 ....................................... 9
Figure 1.3: Blowﬂy larvae of various instars .................................................. 11

Chapter 2: Materials and Methods

Figure 2.1: Indoor study setup ...................................................................... 15
Figure 2.2: Fresh gunshot wounds showing time interval from death to

collection in days for the fall experiment ....................................................... 17
Figure 2.3: Diagram of an SEM ..................................................................... 19
Figure 2.4: Inelastic scatter resulting in an X-ray. An electron from the

M shell ﬁlls in a vacancy in the L shell, resulting in a La X-ray ...................... 22
Figure 2.5: Schematic of EDS detector components..................................;. 23
Figure 2.6: Schematic of ICP-MS components ............................................. 28

Chapter 3: Results and Discussion

Figure 3.1: Day 0 gunshot wound exhibiting blackened wound edges

and GSR deposition ...................................................................................... 33
Figure 3.2: Blowﬂies and blowﬂy eggs one day after the shooting ............... 34
Figure 3.3: Day 2 bloating, putrefaction, and blowﬂy larvae ......................... 34

Figure 3.4: Shot pig stages of decomposition. A) Day 2 bloating and

blowﬂies in wounds. B) Day 5 active decay, skin slippage, and blowﬂy

larvae mass. C) Day 11 beginning of desiccation and diminishing larvae
population. D) Day 37 fully desiccated .......................................................... 36

vii

Figure 3.5: Wound comparisons. A, B, and C show stab wounds on days
0, 7, and 26, where D, E, and F show gunshot wounds on the same days.. 37

Figure 3.6: Fresh gunshot wound from winter study ..................................... 38

Figure 3.7: Comparison of images of the shot pig on day 0 (left) and on
day 60 (right) ................................................................................................. 39

Figure 3.8: Gunshot wound on day 20 with milky secretion .......................... 39

Figure 3.9: Example spheroid particle from the day 1 gunshot
wound ........................................................................................................................ 41

Figure 3.10: Representative EDS spectrum of particle shown in
Figure 3.9 that indicated the presence of Pb, Ba, and Sb in the particle ..... 41

Figure 3.11: Example calibration curves showing linearity from
0 — 500 ppb for Ba, Sb, and Pb standard solutions ....................................... 45

Figure 3.12: Example calibration curves showing linearity from O - 5 ppb
for Ba, Sb, and Pb standard solutions ........................................................... 46

Figure 3.13: Graph showing average Pb, Ba, and Sb concentrations
in blowﬂy larvae and pupae ......................................................................... 50

Figure 3.14: Decreased scale version of the graph showing Pb, Ba, and
Sb concentrations in blowﬂy larvae and pupae ............................................. 51

Figure 3.15: Graph of concentrations of Ba, Sb, and Pb in blowﬂy
larvae from gunshot and stab wounds over time .......................................... 53

Figure 3.16: Decreased scale version of the graph of concentrations of
Ba, Sb, and Pb in blowﬂy larvae from gunshot and stab wounds over
time showing low concentrations at later time points .................................... 54

Figure 3.17: Graph of concentrations of Ba, Sb, and Pb in tissue
from gunshot and stab wounds over thirty-seven days in the fall ................. 56

Figure 3.18: Decreased scale version of the graph of concentrations of
Ba, Sb, and Pb in tissue from gunshot and stab wounds over thirty-seven
days in the fall ............................................................................................... 57

Figure 3.19: Graph of concentrations of Ba, Sb, and Pb in tissue
from gunshot and stab wounds over sixty days in the winter. *The
concentrations of Pb in the gunshot wound digests on saturated the
detector on day 5 and was above the upper LOQ on day 60 are thus

viii

only included as approximations of the tissue concentrations ...................... 60
Figure 3.20: Decreased scale version of the graph of concentrations of

Ba, Sb, and Pb in tissue from gunshot and stab wounds over sixty days
in the winter ................................................................................................... 61

ix

1. INTRODUCTION
1.1 FIREARMS

Firearms were ﬁrst developed in China as early as the 12008 and were introduced
to Europe by the 13005.1 Since then, they have played important roles in every military
conﬂict. Today, ﬁrearms still play an important military role, but are also commercially
available to the public for hunting, recreation, and self-defense. Unfortunately, ﬁrearms
are not only used for these purposes. Of the 14,990 murders in 2006 in the United States,
10,177 of them were ﬁrearm related.2 Handguns were the most common ﬁrearms used in
these crimes, accounting for 7,795 of the cases. Shotguns and riﬂes accounted for 917 of
the cases in roughly equal numbers and the remaining cases involved ﬁrearms of an

unknown nature.2

Handguns are small ﬁrearms that are generally of the semiautomatic or revolver
varieties. As the name suggests, they are held and ﬁred from the hand. Riﬂes are
generally larger and ﬁred from the shoulder. They generally have an accurate range of
approximately ﬁfty meters, as opposed to riﬂes, which can be accurate for several
hundred yards. Both handguns and riﬂes have riﬂed barrels. Riﬂed barrels have grooves
cut in a spiral pattern down the barrel to impart spin to the bullet and stabilize its
trajectory. Shotguns, like riﬂes, are ﬁred from the shoulder, but unlike riﬂes, have smooth
barrels. Handguns and riﬂes ﬁre bullet cartridges with a single projectile known as a

bullet, or slug, while shotguns ﬁre cartridges containing shot, which consists of hundreds

of packed lead pellets.3

Cartridges consist of a casing, primer, propellant, and a bullet3 [Figure 1.1].

Bullets are typically lead based and can be unjacketed, semi-jacketed, or fully jacketed.

The jackets are a thin layer of another metal, often copper. Bullets can be rounded or
pointed, which generally keep their form upon impact; or hollow point, which deform
into a mushroom shape upon hitting the target. Mushroomed bullets cause more damage
than rounded or pointed bullets. The casing is typically brass and the propellant is a low
explosive, such as a smokeless powder. Smokeless powder consists of nitrocellulose (NC,
single-base), a mixture of NC and nitroglycerine (NG, double-base), or a mixture of NC,
NG, and nitroguanidine (triple-base).3 The primer compounds are located inside the
primer cup, which is positioned in the center of the breach face. The primer is stable but
explosive upon impact. It is composed of an explosive, an oxidizer, and a fuel. In the US,
the most common explosive used is lead styphnate, the most common oxidizer is barium
nitrate, and the most common fuel is antimony sulﬁde.3 Shotgun shells are constructed
similarly, but with packed shot replacing the bullet. Also, the casing is plastic rather than

metal.3

Bullet

Primer

 

Casing

 

Propellant

 

 

 

Figure 1.1 — Diagram of a bullet showing a stripped side view (leﬁ) and the breach face

view (right).

A ﬁrearm is discharged when the ﬁring pin strikes the primer. The impact causes
the primer to explode and ignite the propellant, which combusts and produces a rapidly
expanding gaseous cloud that expels the projectile. The heated primer partially vaporizes
and plumes from the gun along with unburned propellant. The vapor condenses to form
small droplets with a diameter of approximately ten microns. These droplets harden and
are deposited on the hands of the shooter, the surrounding area, and the tissue of the
shooting victim. The combination of hardened primer droplets, combustion products, and
unburned propellant is known as gunshot residue (GSR).3 GSR particles are generally
spheroid and consist primarily of lead (Pb), barium (Ba), and antimony (Sb), which, as
described above, are components of the primer.4 This information is useful in the

chemical identiﬁcation of GSR.

1.2 FORENSIC ANALYSIS OF GUNSHOT RESIDUE

Historically, GSR has been chemically detected on the hands of suspected
shooters and around possible bullet holes using a variety of methods. The most common
methods have included various color tests, neutron activation analysis (NAA), atomic
absorption spectroscopy (AAS), and scanning electron microscopy with energy
dispersive spectroscopy (SEM/EDS). The use of inductively coupled plasma-atomic
emission spectroscopy (ICP-AES) and inductively coupled plasma-mass spectrometry

(ICP-MS) to detect GSR are currently being investigated.5

The most common presumptive tests for GSR are the modiﬁed Griess test and the
sodium rhodizonate color test. The modiﬁed Griess test is a color test that identiﬁes the
presence of nitrites, which are produced in the combustion of smokeless powder, through

the use of sulfanilic acid, alpha-naphthol and dilute acetic acid. Vaporous lead deposits

are detected through the subsequent addition of sodium rhodizonate, buffer, and dilute
hydrochloric acid.6'7’25 This test is useful to identify GSR from hand swabs and on
clothing; however, the technique is limited for dark-colored clothing as the results rely on
the visual identiﬁcation of a color change. The pattern of GSR on clothing can be used to
determine the muzzle to target ﬁring distance based on comparisons with patterns made

from test ﬁres at known distances.

NAA was the ﬁrst elemental analysis technique used for GSR analysis. It was ﬁrst
published in 1964 by Ruch et al. and was shown to be sensitive enough to identify trace
amounts of Sb and Ba on hand swabs.8 The GSR was collected by swabbing shooters
hands with ﬁlter paper wetted with 1% nitric acid. This technique was widely used for
years because of its sensitivity, which is on the order of parts per billion (ppb, or ug/L),
despite the need for a nuclear reactor to generate neutrons.5 The use of AAS to detect
GSR components was introduced by Krishnan in 1971, however, the ﬂame AAS used
was only sensitive enough (on the order of parts per million, or mg/L) to detect the Pb
from hand swabs, which is typically present in higher concentrations than Sb and Ba.9
Later, ﬂameless AAS instruments achieved the appropriate sensitivity levels for Sb and
Ba analysis of hand swabs, which were approximately 0.02 to 0.06 ug/L,30 in addition to
Pb analysis.10 AAS, however, is a single element technique, which makes its use for
multiple elements quite time consuming. Each GSR sample would have to be analyzed
three times: once for Pb, once for Ba, and once for Sb. If each element is analyzed in
triplicate, a total of nine runs would be needed per sample, which is not only time

consuming but costly as well.

SEM/EDS is currently the most common technique for the detection of GSR
because it combines a morphological examination with elemental analysis. The most
extensive study on GSR analysis by SEM/EDS was published by Wolten et al. in 1979.ll
Four elemental compositions were observed in GSR only and were thus labeled
characteristic GSR. The most prominent was the combination of Pb, Ba, and Sb. The
other three combinations were as follows: 2. Ba, calcium (Ca), and silicon (Si) with
traces of sulfur (S); 3. Ba, Ca, and Si with traces of Pb if copper (Cu) and zinc (Zn) are
absent; 4. Sb and Ba. GSR sampling for SEM/EDS is most often utilized by swabbing the
hands of shooters with either dilute nitric acid or a solution of ethylene diamine
tetraacetic acid (EDTA) or by dabbing with an adhesive tab. The most common method
for collecting the particles from hands or surfaces with GSR deposits is dabbing the area
suspected to have GSR with an adhesive tab mounted on an aluminum stub.“2 This
method was shown to be the most efﬁcient collection technique by DeGaetano er al. in
1992 when compared with glue lifting and concentrating techniques.I2 SEM is then used
to locate spheroid particles of a diameter of approximately ten microns,4 and if such
particles are located, an EDS spectrum is taken in order to determine which elements are
present. The presence of Pb, Ba, and Sb is considered characteristic of the presence of
GSR on the swab.ll Torre et al. demonstrated that certain types of brake pads contain
particles with Pb, Ba, and Sb, however, these particles lack the characteristic spherical

morphology of GSR particles and would be excluded based on SEM examination. '3

Although SEM/EDS is a proven technique, there are several drawbacks for its
use. Searching large areas for very small particles by SEM can be quite time consuming,

automated programs have been developed to search for particles that ﬁt the description of

GSR. One of the major problems associated with SEM/EDS analysis of GSR is the
distinct possibility of false negative results. A lack of GSR particles could indicate that
no ﬁrearm had been discharged, that the suspected shooter did not discharge a ﬁrearm, or

that the sampling procedure had simply not collected any of the GSR particles.

In 1998 Koons demonstrated that ICP—MS could be used to detect Pb, Ba, and Sb
in extracts from hand swabs of people who had recently ﬁred a ﬁrearm.M Through the
analysis of standard solutions of Pb, Ba, and Sb, it was determined that ICP-MS was fast,
accurate, precise, with relative standard deviations (RSDs) of less than 5% for each
element. ICP-MS also exhibited very low detection limits of 0.1 ug/L for 206+207+208Pb,
0.02 ug/L for 138Ba, and 0.05 ug/L for me. In order to assess the usefulness of ICP-MS
of forensically relevant samples, it was then performed on acid extracts of swabs of the
hands of a person who had recently discharged a ﬁrearm. The GSR components were
successfully detected and the concentrations measured were between 0.004 and 0.95
jig/swab.l4 In 2007, a feasibility study was conducted by Santos et al. in order to see if
shooting distance could be determined by quantifying the amount of gunshot residue
deposited on cotton tissue.l5 Test shots of varying distances were ﬁred into the cotton and
one cm2 squares were cut at four radial distances from the hole. The squares were
extracted in 10% nitric acid with sonication. The samples were diluted and then analyzed
using ICP-MS to quantify Pb, Ba, and Sb. It was found that it was possible to estimate the
ﬁring distance when the shots were ﬁred from between twenty to eighty centimeters. The
best sampling location was determined to be approximately four centimeters away from

the bullet hole as it was the distance that provided the most linear data. '5

All of the techniques outlined have been used to detect GSR that is deposited on
the hands of a shooter or on a nearby surface. Only very recently has work been done to
identify GSR that had tattooed into the tissue of a shooting victim. The ability to
discriminate wounds from jacketed bullets and unjacketed bullets was investigated by
Klintean et al in 2007. Fresh skin from gunshot wounds of people who had been shot to
death was digested in acid and analyzed by ICP-AES,16 which has detection limits of
approximately 0.3 to 50 ug/L, depending on the instrument used.30 ICP-AES is
signiﬁcantly less sensitive than ICP-MS, which has detection limits of approximately
0.02 to 0.06 ug/L for Pb, Ba, and Sb. It was found that wounds from jacketed bullets
contained high concentrations of iron (Fe) and Zn from the jacket, while wounds from
unjacketed bullets contained high concentrations of Pb. The large amount of Pb detected
was due to the bullet, which was made of Pb. Ba and Sb were also detected in the tissue

from both bullet types,16 which indicated that they were likely from the GSR.

In 2007, the ability to detect GSR in decomposing tissue was investigated by
Schaeffer.l7 It was demonstrated that GSR could be detected in porcine tissue up to two
days after death by SEM/EDS where the GSR was collected by dabbing with carbon-
coated adhesive tape.17 The same study demonstrated that GSR could be detected by ICP-
MS for at least seven days following death by microwave digesting the skin surrounding
the wound in concentrated nitric acid.17 Schaeffer’s work was limited by the number of
samples that were tested. The gunshot wounds were only tested for seven days after death
and signiﬁcant levels of GSR components were still detected at the end of the sampling

period. Thus, it is not known how long after death GSR could be detected by ICP-MS.

The ability to chemically identify gunshot residue in the tissue of a victim would be

useful to aid in the identiﬁcation of a gunshot wound.

1.3 PATHOLOGY OF GUNSHOT WOUNDS

Gunshot wounds are typically identiﬁed by a small, round or ovoid entrance
wound and a larger, irregular exit wound. Contact wounds often show the stamp of the
muzzle in the skin. They may also exhibit star shaped tearing around the hole due to a
build up of gases under the skin that then explodes outwards. With close range gunshot
wounds, GSR will often tattoo into the tissue, which results in the phenomenon known as
stippling. Stippling is a pattern of tiny abrasions surrounding the wound [Figure 1.2].26
The pathologist will typically conduct a visual examination of the suspected gunshot
wound, often accompanied by a histological examination of the wound edge to look for
GSR particles [Figure 1.2]. Radiology is also often employed to visualize internal injuries
and any bullets, bullet fragments, or shot that may be present.18 Besides the identiﬁcation
of a gunshot wound, it is important for the pathologist to also be able to estimate the
ﬁring distance based on the amount of stippling, the presence and location of abrasions or

tears, etc.18

 

 

Figure 1.2 — Gunshot wound stippling (left) and histology of gunshot wound showing

imbedded GSR particles (right).19

Gunshot wounds can vary greatly based on the type of ﬁrearm, the distance from
the barrel to the victim, the type of ammunition, and the location of the wound. The
variability in gunshot wound appearance can make their interpretation extremely difﬁcult
for medical personnel. A study conducted by Collins found that 52% of 46 presented
gunshot wounds at a Level I trauma center were misinterpreted by trauma specialists.20
The difﬁculty in gunshot wound interpretation can be due to many different factors,
which may include the level of bodily decomposition, the distance ﬁom which the
ﬁrearm was discharged, whether a body had been buried, and any insect activity in and
around the wound tract. Decomposition and burial can obscure any obvious GSR
tattooing or stippling and the ﬁring distance affects the amount of GSR deposited. Insects
can chew new tracts, obscure existing tracts, and/or change the morphology of the
wound. They can even cause an entrance wound to resemble an exit wound.

If no bullet is recovered and the wound is obscured by any means, the ability to

chemically detect GSR around a suspected gunshot wound would be a valuable tool for

deﬁnitively identifying a, gunshot wound. In addition, a chemical means for the
identiﬁcation of gunshot wounds could also aid in the interpretation of entry wounds
versus exit wounds in situations where one of the wounds has been compromised. The
GSR content would be expected to be much higher around an entry wound than around
an exit wound. As was described in the previous section, very little work has actually

been done to chemically identify gunshot wounds in decomposing tissue.

1.4 BLOWFLY LARVAE

Blowﬂies are typically the ﬁrst to arrive at a death scene and lay eggs
immediately in mucous membranes and wounds.21 They proliferate quickly and rapidly
ﬁll any exposed oriﬁces. Because they are often present when a body is found in a
decomposed state, their interaction with gunshot wounds cannot be ignored.

Blowﬂies have been of use to forensic scientists for years, particularly in time
since death estimations. They develop in a very predictable manner according to the
ambient temperature. The eggs hatch small, off-white larvae. These larvae are known as
ﬁrst instars. As they grow, they molt to become second instar larvae. This process
happens once more to produce third instar larvae. All of the instars actively feed on dead
tissue and accelerate the decompositional process [Figure 1.3]. After a time, third instars
ﬁnish the feeding stage and leave the body in search of a cool, dry place to pupate. Their
outer layer hardens and darkens and they shrink in length. The new blowﬂy emerges

from the puparium after a time and leaves the body to begin the process again.21

10

 

 

 

Figure 1.3 — Blowﬂy larvae of various instars.

Blowﬂy larvae have been shown to be useful for more than just time since death
estimations. Since the larvae feed directly on the tissue of a corpse, they contain some
compounds found in the tissues. In 1972, Nuorteva and Hasanen showed that mercury
could be detected in blowﬂy larvae that had been feeding on ﬁsh containing high levels
of mercury.22 A year later, Beyer et al. detected phenobarbital in trace levels in larvae
that had been feeding on a suicide victim.23 Since then, various other toxicological
substances have been detected in larvae. As gunshot residue has been shown to actually
tattoo into the skin of shooting victims, the question of whether gunshot residue could be
detected in blowﬂy larvae feeding on said tissue arises. Recently, Roeterdink et al. tested
whether GSR could be detected in larvae that had been feeding on tissue that had been
shot.24 A one-kilogram block of beef was shot twice for maximum GSR deposition,
placed in a controlled environment, and colonized with Calliphora dubia larvae. The
larvae were allowed to feed on the GSR tissue for up to four days. Larvae, pupae,
puparia, and adult ﬂies were collected at various time points over the course of the

experiment. The study was conducted twice, however, adult ﬂies were only analyzed in

one study. After collection, they were rinsed, then digested by boiling in nitric acid. The
digest solutions were evaporated, reconstituted in dilute nitric acid, and analyzed by ICP-
MS for the presence of Pb, Ba, and Sb. Larvae that had been feeding for up to 84 hours
on the beef were found to consistently contain the characteristic GSR metals, whereas
larvae that had been feeding for 96 hours on the beef, post-feeding larvae, pupae, puparia,
and adult ﬂies did not exhibit signiﬁcant Sb levels and Pb and Ba were only occasionally
detected.24 The data were not normalized to the mass of the larvae and the isotopes that
were monitored were not reported, so the signiﬁcance of the results obtained is difﬁcult

to assess.

1.5 RESEARCH OBJECTIVES
Gunshot wounds in decomposing tissue have been shown to be quite difﬁcult to
identify with conﬁdence, which makes a means to chemically identify them desirable. As
very little work has been done to chemically characterize GSR content in decomposed
tissue or in blowﬂy larvae found naturally occurring in the decomposing tissue, the goals
of this research are as follows:
0 To analyze Pb, Ba, and Sb in GSR containing blowﬂy larvae by ICP-MS.
9 To investigate the persistence of GSR in decomposing porcine tissue by
SEM/EDS of tissue swabs and ICP-MS of digested tissue.
9 To determine the feasibility of detecting GSR components in blowﬂy larvae
found naturally feeding in gunshot wounds.
The research presented in this thesis will impact the forensic community by serving
as a means to chemically identify gunshot wounds on decomposing victims. This

information will be beneﬁcial, for example, in cases where no bullet was recovered and

12

the body is in a stage of decomposition that makes it difﬁcult to visualize stippling
around a wound. The ability to detect GSR in blowﬂy larvae found feeding in suspected
gunshot wounds would be useful because blowﬂy larvae are easy to collect and are

typically quite abundant on decomposing tissue.

13

2. MATERIALS AND METHODS
2.1 EXPERIMENTAL DESIGN
2.1.1 Introduction

The extraction of gunshot residue (GSR) from blowﬂy larvae was ﬁrst
investivaged in an indoor study. Blowﬂy larvae were allowed to feed on beef that had
been shot; the larvae were then collected and microwave digested in nitric acid and
hydrogen peroxide. The GSR content was subsequently determined by inductively
coupled plasma-mass spectrometry (ICP-MS). The persistence of GSR in decomposing
porcine tissue and in blowﬂy larvae found feeding in the tissue was studied in the fall and
winter. GSR was conﬁrmed on the tissue by scanning electron microscopy/energy
dispersive spectroscopy (SEM/EDS) and the characteristic elements, lead (Pb), barium

(Ba), and antimony (Sb), were quantiﬁed by ICP-MS.

2.1.2 Indoor study design

A 2.78 pound round bottom roast, purchased from a local grocery store, was shot
eight times with a 9mm GLOCKTM handgun with a 4” barrel at a distance of
approximately one foot in order to deposit a maximum amount of GSR over the entire
surface of the meat. The cartridges used were 115 grain jacketed hollow point American
Eagle Brand Federal Cartridge +p+ with 1250 ft/s muzzle velocity (Federal Cartridge
Co., Anoka, MN, Lot 430399WI40). The meat was then cut into approximately 1” cubes
and placed onto a plastic platform in the bottom of a plastic specimen cup (Premium
Plastics, Inc., Chicago, IL). Five specimen cups were prepared containing beef that had
been shot and ﬁve were prepared containing control beef that had not been shot.

Approximately twenty sterile ﬁrst instar Phaenicia sericata medicinal maggots (Monarch

14

Labs, Irvine, CA) were placed on each piece of beef. These larvae had previously been
fed a diet of soy protein and yeast. Wet paper towel was put under the platform
containing the meat in an attempt to keep the atmosphere moist and the pieces of meat
were sprinkled with water every day to prevent the meat from desiccating. The cups were
covered with two layers of Kimwipes® and secured with a rubber band [Figure 2.1].
Approximately twelve larvae from a GSR cup and twelve ﬁom a control cup were
collected on days 1, 2, 3, 4, and 5 and stored at -80°C. Additional surviving larvae and
pupae were collected and frozen on days 7 and 9. Additional larvae were allowed to feed
on the excess shot beef in case there was a problem with the larvae in the smaller cups.

Larvae and pupae from the excess beef were collected on days 4, 7, and 9.

Cube of beef

Plastic platform Blowﬂy larvae

Damp paper towel

 

Figure 2.1 - Indoor study setup.

2.1.3 Fall and winter outdoor studies day zero procedures

On September 5, 2007, two approximately 200 pound pigs were obtained from the
MSU Swine Facility and euthanized by on-site staff with FATAL-PLUS (Vortech
Pharmaceutical Ltd., Dearbom, MI), a solution of pentobarbital sodium at 390 mg/mL,
propylene glycol at 0.01 mg/mL, ethanol at 0.25 mg/mL, and benzyl alcohol at 0.2
mg/mL. The pigs were transported to the shooting range where one pig was shot eleven
times by the same ﬁrearms specialist from the local police department as the indoor
study. The spots were marked with an “X” using a Sharpie® marker. The weapon used
was a GLOCKTM model 19 with a 4” barrel and polygonal riﬂing, and was the same
weapon that had been used in the indoor study. The ammunition was American Eagle
brand 115 grain full metal jacketed cartridges (Federal Cartridge, Model AE9AP, Lot
430399WI40) with a nominal speed of 1250 ft/s at sea level. The muzzle to target
distance was consistently ﬁve centimeters and shots were spaced at least ten centimeters
apart to minimize overlap of GSR between shots. The ﬁrearm was cleaned with MP
Pro7TM gun cleaner (n-methylpyrrolidone and diethylene glycol) between shots in order
to minimize carryover and to make the shots as reproducible as possible. On the day of
the shooting, the outside temperature was approximately 80°F with high wind and
humidity.

Both pigs were transported to the MSU Entomology Research Field and the
second pig was stabbed with a clean scalpel eleven times in corresponding locations as
the wounds on the shot pig. Wire cages were placed over the pigs to prevent predation of
the carcasses by larger animals while still allowing environmental exposure. The pigs

were placed approximately two feet apart.

16

The procedure described above was repeated with two approximately 160 pound
pigs on January 11, 2008. The euthanized pigs were shot and stabbed in the same
locations, the same ofﬁcer performed the shooting using the same weapon, the
ammunition was from the same box (Lot 430399WI40), and the shooting distance and
spacing were the same as the previous study. The outside temperature was approximately

40°F with wind.

2.1.4 Sample collection and storage

Tissue samples were collected from both the shot and stabbed pigs on eleven days
over a thirty-seven day interval after death in the fall [Figure 2.2] and over a sixty day
interval in the winter. A circular tissue sample of the skin and underlying fat was excised
in an approximately four-centimeter radius around the wound, wrapped loosely in waxed

paper, sealed in a plastic freezer bag, and stored at -80°C until analysis.

 

Figure 2.2 - Fresh gunshot wounds showing time interval from death to collection in days

for the fall experiment.

Blowﬂy larvae were collected directly from the wounds on both the stabbed and
shot pigs on ten different days between days three and ﬁfteen after death. That is, blowﬂy
larvae were collected on days when no tissue was collected and larvae were present in the
wounds. The larvae were sealed in plastic freezer bags and stored in the -80°C freezer
until analysis. Blowﬂy larvae were only present in the wounds in the fall as it was too

cold for them to survive in the winter.

2.2 SCANNING ELECTRON MICROSCOPY/ENERGY DISPERSIVE
SPECTROSCOPY (SEM/EDS)
2.2.1 Introduction

Scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS) is a
versatile technique that is often used in forensic science to combine imaging with
qualitative or quantitative elemental analysis. The SEM produces a topographical image
of a sample through the use of a high power scanning electron beam and is capable of up
to 150,000x magnifying power and 3 nm resolution”. The addition of an EDS detector

allows for the concurrent imaging and elemental analysis of a sample.

2.2.2 Gunshot residue sample preparation

Gunshot wounds were removed from the -80°C freezer and allowed to thaw. The
wounds were dabbed 100 times with a 9 mm diameter carbon adhesive tab (SPI®
Supplies, West Chester, PA) on an aluminum stub. Although samples are usually coated
with gold or carbon prior to analysis to ensure conductivity, the samples in question were
not coated, as the particles of interest are metallic (Pb, Sb, and Ba) and therefore are

already conductive.

18

2.2.3 Scanning electron microscopy

A SEM consists of an electron gun, a series of electromagnetic lenses, scan coils,

a ﬁnal aperture, a scintillator, a detector, a computer, and a cathode-ray tube [Figure

2.3].28 The instrument is kept under vacuum using a series of pumps and cooling

systems.28

 

 

 

 

 

 

 

Scan coils
\.

l H l ]——E|ectron gun

—1 Condenser lens

 

 

 

 

 

 

 

 

 

 

 

Objective
lens -
Final _ _ _
aperture
Scintillator “ ‘—

 

 

 

Figure 2.3 - Diagram of an SEM

Scan

 

 

gellclaiUI

 

  

 

   

 

— Preamplifier

Cathode—ray
tube

     

Photomultiplier tube

Light pipe
Faraday cage

The electron gun is composed of a high voltage source connected to an electron

source, typically a lanthanum hexaboride (LaBé) crystal or a tungsten wire. This electron

l9

source is the cathode for the electron gun. A negatively charged shield is placed around
the cathode with a small aperture for the electron beam to focus the emitted electrons into
a beam. The anode of the electron gun is located below the shield. The voltage difference
between the cathode and anode is known as the accelerating voltage of the beam and is
typically set between 5 kV and 30 kV. After the beam is accelerated it travels through the
condenser lens, which is a ring-shaped electromagnetic lens that serves to condense the
electron beam. The electrons then travel to the objective lens, where they are focused into
a very narrow beam. The scan coils within the objective lens serve to allow the beam to
move back and forth in a raster pattern. After the electromagnetic lenses and scan coils,
the beam travels through the ﬁnal aperture, which serves to limit the number of electrons
striking the sample. The number of electrons striking the sample inﬂuences the brightness
and contrast of the ﬁnal image.

SEM samples must be free from volatile compounds, either by the nature of the
sample or by preparation; ﬁrmly mounted, and conductive. When the electron beam
strikes the sample, a series of interactions occur which has many varied effects, including
the release of secondary electrons, backscattered electrons, and x-rays. Secondary
electrons are low energy electrons, typically between 3 and 5 eV, and are released from
just under the surface of the sample.28 Because of this, secondary electrons are the most
useful for imaging. Backseattered electrons are higher in energy than secondary electrons
and are the result of elastic scattering from deeper within the sample.28 Because they
come from a deeper area of the sample, back scattered electrons provide less information
about topography. In exchange, however, they provide information about atomic weight.

Areas with a higher average atomic weight will appear brighter in backscatter mode.

20

The electrons produced are typically detected with an Everhart-Thomley detector.
The detector consists of a Faraday cage with a scintillator, followed by a light pipe, a
photomultiplier tube (PMT), and a preampliﬁer. The Faraday cage is positively charged
at approximately 300 V and thus attracts the electrons from the sample into the detector.
The electrons come in contact with the scintillator, which is a positively charged metal-
coated disc that converts the electrons into photons, which enter the light pipe and are
transmitted outside the vacuum.28 The photons enter the PMT and cause the release of
many electrons, which are converted to a voltage, which the preampliﬁer ampliﬁes.28 The
amount of voltage ultimately determines the brightness of a spot on the cathode ray tube.
which produces the ﬁnal image. Higher voltages result in brighter spots, which

correspond to raised areas of the sample.28

2.2.4 X-ray emission

X-rays are the result of an inelastic electron scatter that creates a vacancy in one
of the inner shells of a particular atom. When this vacancy is created, an electron from an
outer shell ﬁlls the vacancy [Figure 2.4].l The result is an energy difference, which can
be resolved in one of two ways: the ejection of an electron from an outer shell, known as

an Auger electron, or the production of an X-ray.28

21

  
 
  

X-ray produced

‘ ‘ ~Vacancy due to
inelastic scattering

“Electron ﬁlls in
from outer shell

Figure 2.4 - Inelastic scatter resulting in an X-ray. An electron from the M shell ﬁlls in a

vacancy in the L shell, resulting in a La X-ray.

X-rays are named according to the shell (K, L, M, or N) where the vacancy
occurred and according to how many energy level jumps were required to ﬁll the
vacancy, where a one-shell jump is denoted a, two-shells is [3, etc.28 For example, if a
vacancy occurred in the L shell and an electron jumped one shell to ﬁll it in from the M
shell, the X-ray produced would be an L0, X-ray. The critical excitation energy is the
energy required to eject an electron from a given energy level for an X-ray to be
produced.28 The accelerating voltage of the SEM can be adjusted in order to obtain the

intensity needed to produce sufﬁcient X-rays for analysis.

22

2.2.5 Energy dispersive spectroscopy

EDS uses the X-rays produced by inelastic scattering to determine qualitative and
quantitative information about the elements present in a given sample. Each X-ray of a
given type (e.g. L0, or KB: etc) is called a line. If sufficient X-rays of a speciﬁc line are
produced, they produce a peak on the EDS spectrum at the corresponding energy. The
energy, E, of the X-ray is related to its wavelength, 7», according to the equation A =
1.2398/E. 28 The resulting EDS spectrum is a plot of intensity versus X-ray energy.

The EDS detector attaches to the column of a SEM and consists of a collimator, a
window, a crystal, a ﬁeld-effect transistor, a pulse processor, an analog—to-digital

converter, the multichannel analyzer, and a computer [Figure 2.5].28

 
   
 
   

 

 

 

Field-effect
transistor

Collimator Electron beam

/
I

 

 

 

  

 

 

 

 

g I

X-ray from sample

 

 

Crystal

 

Figure 2.5 - Schematic of EDS detector components.

The collimator blocks stray X-rays and backscattered electrons from entering the

detector and producing noise in the spectrum. It is lined with carbon, which acts to absorb

23

the stray radiation. The window is a wafer that acts as a physical barrier protecting the
crystal from the column vacuum and any vapors that may be present. The detector crystal
is a silicon wafer with lithium drifted in to produce a semiconductor region. The crystal
must be kept at liquid nitrogen temperature to ensure the stability of the lithium region
When an X-ray enters the semiconductor region, a vacancy and a free electron are created
there. The release of electrons results in a voltage pulse proportional to the energy of the
X-ray.28 The ﬁeld-effect transistor ampliﬁes the voltage pulse and reduces electronic
noise. The pulse processor generates separate voltage pulses from the ramp output from
the preamp. These separate pulses are proportional to the energy of the X-rays that
produced them. The analog-to-digital converter converts the pulses to a signal that can be
recognized by the computer, and the multichannel analyzer sorts its output into channels
of varying energy and counts the X-rays that have energies in each channel. The
computer then ultimately produces a spectrum containing peaks that correspond to

different types of X-rays at the corresponding energy.28

2.2.6 SEM/EDS analysis of wound samples

A JEOL 6400V SEM (JEOL Ltd., Tokyo, Japan) with LaB(, emitter coupled with
an INCA EDS detector (Oxford Instruments, Oxfordshire, United Kingdom) located in
the Center for Advanced Microscopy at Michigan State University was used for this
study. The accelerating voltage used was 20 kV and the working distance was 15 mm.
The samples were manually scanned for spheroid particles with a diameter greater than

10 pm. If a particle was located, a qualitative EDS spectrum was collected over the entire

particle to qualitatively identify Pb, Ba, and Sb.

24

2.3 MICROWAVE DIGESTION
2.3.1 Background

Microwave digestion is a versatile technique for the digestion of complex solid
matrices into liquid form. The matrix is degraded in order to release the elements of
interest into the solution.29 Although there are many different techniques for
accomplishing this, the most common methods have employed a closed system. That is,
the sample to be digested is placed in a closed container with digestion agents, sealed
tightly, and subjected to microwave irradiation. Digestion agents typically consist of a
combination of acids and oxidizing agents, such as concentrated nitric acid, hydroﬂuoric
acid, or hydrogen peroxide. Microwaves rotate polar molecules within a sample. This
rotation causes friction between the molecules and consequently releases a signiﬁcant
amount of heat. The advantage of a closed system is that the heat generated increases the
amount of vapor in the container, which increases the pressure and raises the boiling
point of the reagents, ultimately resulting in a more efficient decomposition of the
sample.29

Commercial microwave digestion systems are capable of digesting several
samples at once while following a speciﬁc temperature program. Temperature monitoring

is typically achieved through the use of a thermocouple or ﬁber optic probe.

2.3.2 Procedure

The wounds and blowﬂy larvae were taken from the -80°C freezer and thawed.
The wound skin was separated from the underlying fat and connective tissue using a
scalpel. Between 0.30 and 0.90 grams of porcine skin or between 0.03 and 0.50 grams of

larvae were placed into acid and peroxide washed quartz vessels. 1.00 mL of 30%

25

hydrogen peroxide (H202, CCI, Columbus, WI) and 2.00 mL Optima grade nitric acid
(HNO3, Fluka, Buchs, Switzerland) were added to the vessels, which were then capped
and placed inside a larger Teﬂon® vessel containing 10.0 mL high purity water
(Honeywell Burdick & Jackson, Muskegon, MI) and 2.00 mL 30% hydrogen peroxide.
These vessels were securely closed and placed into a Milestone Ethos EZ microwave
digester (Milestone, Inc., Shelton CT). The microwave program consisted of a ﬁfteen
minute ramp to 210°C followed by a ten minute hold at that temperature. The maximum
wattage was set at 600W in order to prevent system errors. The Teﬂon® vessels were
allowed to cool to below 95°C before they were opened. Each digest run consisted of nine
samples and one procedural blank containing only 2.00 mL HNO3 and 1.00 mL H202.
Procedural blanks were run in each vial after samples had been run as a cleaning step and
then rinsed with deionized water before reuse.

After microwaving, the digest solutions were immediately diluted to 2% nitric
acid by adding 0.5 mL of the digest solution to 11.15 mL high purity water (Honeywell
Burdick & Jackson). The dilution step was necessary to prevent degradation of the
polypropylene tubes used for storage as well as for use with ICP-MS. Both the diluted
solutions and original digest solutions were stored in a refrigerator in 15 mL

polypropylene tubes (Corning Inc., Corning, NY).

2.4 INDUCTIVELY COUPLED PLASMA-MASS SPECTROMETRY

2.4.1 Introduction
Inductively coupled plasma-mass spectrometry (ICP-MS) is a fast, precise, and

sensitive mass spectral technique that allows for the multielement analysis of samples

26

over a wide range of concentrations. The technique utilizes an argon plasma which is
more than twice as hot as a combustion ﬂame to atomize and ionize a sample prior to
introduction into a mass spectrometer.30 The mass spectrometer is capable of determining
which elements are present in the sample based on mass-to-charge (m/z) ratio and

quantifying the elements using internal and external calibration.

2.4.2 Calibration Standards and Sample Preparation

Thirteen multielement calibration standards were prepared using SPEX CertiPrep
standards of 1,000,000 ppb of Pb, Ba, and Sb in 2% HNO3 (SPEX CertiPrep, Inc.,
Metuchen, NJ). The solutions were diluted with 2% HNO3 (Optima grade, F luka) to
concentrations ranging from 0.10 ppb to 500 ppb. Standards were analyzed in order of
increasing concentration to minimize error due to memory effects.

Prior to analysis, 2 mL of diluted digest or standard solution was added to 2 mL
of an internal standard solution containing 20 ppb SPEX CertiPrep indium (In) and
bismuth (Bi) in 2% HNO3. Digest samples and procedural blanks were analyzed in
triplicate and a set of standards was analyzed after every thirty to thirty-three samples in

order to minimize error due to instrumental drift.

2.4.3 Instrumental theory
ICP-MS consists of a quartz torch, a series of cones, a high-voltage extraction

lens, a hexapole collision cell, a quadrupole mass analyzer, electrostatic deﬂector plates,

and an ion detector [Figure 2.6].30

27

Sampling l th
cone Extraction lens Quadrupole mass on pa
\ analy\zer

‘   \ ll“

 

Torch

\ Plasmq

   

    

 

 

argon 1 I _ —
—
Plasma
argon \ Photon
Rf coils - ‘ / path
. Hexapole El ct t'
Sk'mmei collision cell deﬂzctiiiséiaizs

cone

Figure 2.6 - Schematic of ICP-MS components

The torch contains the argon plasma source and consists of three concentric tubes
for three ﬂows of argon (Ar), one to carry the sample, one to support the plasma, and one
to cool and lift the plasma. A spark from a Tesla coil ionizes argon gas and a radio-
frequency ﬁeld that oscillates around the coil accelerates free electrons. These electrons
collide with the argon and transfer their energy to the gas, creating and sustaining the
plasma. The sample being analyzed is carried into the plasma by a carrier gas and is
immediately atomized and ionized. The sample is then carried through the plasma and
onto a sampling cone, which allows only a fraction of sample to enter the mass
spectrometer. The sample then passes through the skimmer cone, which is similar to the
sampling cone but with a smaller aperture. Since the ICP operates at atmospheric
pressure and the mass spectrometer operates under vacuum, the sampling and skimmer
cones also act as barriers for differential vacuum zones. The extraction lens sits behind
the skimmer cone and is negatively charged in order to attract positive ions from the
sample. The ions that successfully passed through the extraction lens enter the collision

cell, which contains a gas, typically helium or hydrogen, that causes the dissociation of

28

polyatomic ions that could interfere with elemental analysis, such as 4OArz, which has an
indistinguishable m/z to 8°selenium (Se). The collision cell also reduces the spread of ion
kinetic energies before mass spectral analysis.30

The ions then enter the quadrupole mass analyzer, which consists of four
conducting rods with an oscillating voltage and radio frequency. The ratio between the
voltage and radio frequency remains constant. The resulting electric ﬁeld causes ions to
travel in a somewhat spiral path along the length of the instrument. At a given dc/RF
voltage ratio only ions within a narrow m/z range can pass through the quadrupole to the
detector. All ions with other m/z ratios become unstable and are not detected. A range of
masses can be scanned or only selected masses may be chosen to be allowed to reach the
detector.30 The latter technique is known as selected ion monitoring (SIM),30 which
increases the sensitivity of the instrument by dwelling longer at selected dc/RF ratios that
allow m/z ratios of interest to reach the detector and not spending time seaming the
entire available m/z range.

The electrostatic deﬂector plates direct ions that reach the end of the rods towards
the detector, which is not aligned with the rest of the instrument. Because only ions can
be deﬂected electrostatically, any photons from the plasma that enter the mass analyzer
travel straight through and miss the dynode detector. Photons would cause an interfering
signal if allowed to strike the detector.30 Ions strike the detector and are converted to an
electronic signal. The signal generated from the detector is proportional to the amount of

ions of a given m/z hitting the detector. The output is a spectrum with a peak at a

particular m/z, which corresponds to the element present.

2.4.4 ICP-MS of standards and digest solutions

29

The ICP-MS used was a Micromass (now Thermo Fisher Scientiﬁc, Inc.,
Waltham, MA) Platform quadrupole ICP-MS with a hexapole collision cell, a DynoliteTM
detector with a -15kV conversion dynode and photomultiplier tube, and a CECTAC
ASX-SOO autosarnpler, located in the ICP-MS Laboratory at Michigan State University.
Argon gas carried the samples into the argon plasma and hydrogen gas was used in the
collision cell. The sample scan time was 1.25 min with a 0.1 sec dwell. The cool gas ﬂow
rate was 13.00 L/min and the auxiliary gas ﬂow rate and the sample gas ﬂow rate were
0.72 L/min. The RF power was 1350W. The sample cone was nickel with a copper core
and a 1.14 mm diameter oriﬁce and the skimmer cone was nickel with a 0.89 mm
diameter oriﬁce.

After a sample injection, the injector was rinsed for three minutes with clean 2%
nitric acid. In general, the injection order was as follows: procedural blanks and control
blowﬂy digests, control tissue digests, GSR blowﬂy digests, and GSR tissue digests. The
GSR tissue digests were run from the last day of collection to the ﬁrst day of collection,
as it was hypothesized that wounds collected later would contain less GSR than wounds
collected earlier, and would thus have lower concentrations of Pb, Ba, and Sb. A set of
standards in order from low concentration to high concentration was run at the beginning
of each different set of samples (e.g. control tissue digests) and again at the end of the
rim. The order was chosen in order to minimize memory effects. Selected ion monitoring
of me, 138Ba, and 208Pb, was performed and the signal was normalized to that of ”5 In
for me and 138Ba and 209Bi for 2°°Pb. These isotopes were chosen based on relative

abundance and a lack of interference with other elements. The analysis software used was

MassLynx v. 3.4 (Theme).

30

Concentrations of Sb, Ba, and Pb were reported in parts per billion (ppb), which is
equivalent to units of micrograms of analyte per liters of solution (pg/L). The
concentrations were used to calculate the amount of each element in the original mass of
tissue or blowﬂy larvae that were digested in units of microgram of element per gram of

tissue or larvae (pg/g).

31

3. RESULTS AND DISCUSSION
3.1 INTRODUCTION

As stated in Chapter 1, the objectives of this research were to extract the
characteristic GSR components from blowﬂy larvae and analyze the extracts by ICP-MS,
to investigate the persistence of GSR in decomposing porcine tissue by SEM/EDS and
ICP-MS, and to determine the feasibility of detecting GSR elements in blowﬂy larvae
that were found naturally feeding in gunshot wounds.

In order to fulﬁll these objectives, three separate studies were conducted. The ﬁrst
study involved shooting a piece of beef and allowing blowﬂy larvae to colonize the tissue
for up to nine days. These larvae were then microwave digested and analyzed by ICP-MS
in order to determine if Ba, Sb, and Pb could be detected. To assess the persistence of
GSR in decomposing porcine tissue, one pig was shot eleven times at close range and
tissue samples were collected over the following thirty-seven days. The tissue samples
were dabbed with carbon tabs and analyzed by SEM/EDS for characteristic GSR particles
in order to conﬁrm the presence of GSR. Tissue samples were then microwave digested
and analyzed for the presence of Ba, Sb, and Pb by ICP-MS. This study was originally
conducted in the fall and then was repeated in the winter over the course of sixty days in
order to assess the effect of temperature on GSR persistence. The feasibility of detecting
GSR components in blowﬂy larvae found in gunshot wounds was also investigated by
microwave digesting larvae found in the gunshot wounds from the decomposing pig and
then analyzing the digests by ICP—MS. This study was only performed in the fall, as

blowﬂies were not present in the winter.

32

3.2 DECOMPOSITION OBSERVATIONS
3.2.1 Decomposition during fall sampling period (37 days)

The fresh gunshot wounds, inﬂicted from a ﬁring distance of 5 cm, exhibited
typical characteristics of close range gunshot wounds including blackened wound edges
and GSR particle deposition in and around the wound [Figure 3.1]. Since the pigs were
euthanized prior to the shooting, no stippling was present, as stippling is an antemortem

phenomenon that requires blood ﬂow.26

 

 

 

 

Figure 3.1 — Day 0 gunshot wound exhibiting blackened wound edges and GSR

deposition.

On day 1, both the shot pig and stabbed control pig were beginning to bloat and
blowﬂies were actively colonizing the carcasses. Blowﬂy eggs were observed in the
oriﬁces [Figure 3.2], which indicated their early arrival to the carcasses, and ﬂies were

observed walking in and around the gunshot and stab wounds.

33

 

 

 

Figure 3.2 — Blowﬂies and blowﬂy eggs one day after the shooting.

By day 2, bloating was pronounced and putrefaction was evident in the blood
vessels on the carcasses’ undersides. Blowﬂy larvae ﬁlled the mouths, snouts, and eyes

while eggs were present in both types of wounds [Figure 3.3].

 

Figure 3.3 — Day 2 bloating, putrefaction, and blowﬂy larvae.

34

Bloating continued through days 3 and 4, along with blowﬂy larvae colonization
of the wounds. Seepage of decompositional ﬂuids was evident by day 4 and pronounced
by day 5, which indicated the start of active decay. Skin slippage was also evident
beginning on day 4 and pronounced on day 5. There was a large larvae mass adjacent to
the carcasses as well as in all oriﬁces and wounds. Active decay began to taper off and
was no longer obviously happening by day 11, which indicated the start of desiccation.
Blowﬂy larvae were present in the wounds until day 15, and around the carcass until day
17. By day 37, the end of the collection period, the carcasses were almost fully

desiccated. The process is illustrated in Figure 3.4.

35

 

Figure 3.4 — Shot pig stages of decomposition. A) Day 2 bloating and blowﬂies in
wounds. B) Day 5 active decay, skin slippage, and blowﬂy larvae mass. C) Day 11

beginning of desiccation and diminishing larvae population. D) Day 37 fully desiccated.

The weather over the course of the experiment included a maximum daily high of
88.6°F on day 19 and a minimum daily low of 35.3°F on day 11. The maximum average
temperature was 75.4°F on day 0 and the minimum average temperature was 45.3°F on
day 37. Higher temperatures generally result in faster decomposition rates, although there
are many factors involved. Signiﬁcant rainfall occurred on days 2, 5, 20, 21, 26, and 35.
The details of the weather conditions are located in Appendix 1.31

Initially, the stab wounds and gunshot wounds were easily distinguishable.

However, over the course of the collection period, the wounds became much more

36

 

 

similar in appearance, as is illustrated in Figure 3.5. GSR was no longer visible on the
surface of the tissue after the area around each wound darkened. As the tissue darkened

on earlier days closer to the head, this happened later for the thigh wounds.

 

 

 

Figure 3.5 - Wound comparisons. A, B, and C show stab wounds on days 0, 7, and 26,

where D, E, and F show gunshot wounds on the same days.

3.2.2 Decomposition during winter sampling period (60 days)
As in the fall, the fresh gunshot wounds exhibited typical characteristics of close
range gunshot wounds including blackened wound edges and GSR particle deposition in

and around the wound edges [Figure 3.6].

37

 

Figure 3.6 — Fresh gunshot wound from winter study.

The primary difference between the fall study and the winter study was that the
pigs did not signiﬁcantly decompose over the course of the study. Even by day 60
bloating was not yet evident, however, the tissue was developing a light dusky green
color that was most likely the result of the beginning of putrefaction. That is to say, after
sixty days the carcasses were still less decomposed than on day 1 (75°F) of the fall
experiment. Figure 3.7 shows a side-by—side comparison of day 0 (39°F) versus day 60
(27°F) for the winter pigs.

GSR was still visible around the gunshot wounds after sixty days; however, it was
less prominent than on day 0. After the second day, many of the wounds began secreting

a milky, gelatinous substance similar in consistency to petroleum jelly [Figure 3.8].

38

 

 

 

Figure 3.8 — Gunshot wound on day 20 with milky secretion.

39

The maximum daily high over the collection period was 54.0°F, which occurred
on day 52. The minimum daily low over the collection period was -5.4°F, which occurred
on day 31. Day 52 also had the highest average temperature, which was 41.3°F, whereas
day 31 had the lowest average temperature of 3.2°F. Over the course of the experiments
the carcasses were subjected to freeze/thaw temperature conditions. The carcasses were
covered with snow on days 5, 8, 12, 16, 26, 30. Complete weather tables for the entire
course of the experiment are available in Appendix 2.31 As the temperature was below
freezing for the majority of the sampling period, the carcasses were still in the fresh stage

of decomposition after sixty days.

3.3 SEM/EDS RESULTS AND DISCUSSION
3.3.1 Fall SEM/EDS analysis results

Adhesive carbon tabs were dabbed 100 times each on gunshot wound tissue
samples from days 1,2, 5, and 8 and then viewed by SEM. Any spheroid particles that
were seen were qualitatively analyzed by EDS for the presence of Pb, Ba, and Sb. If
spheroid particles of a diameter of approximately ten microns were found to contain all
three elements of interest, the particle was determined consistent with GSR according to
parameters that were outlined in Chapter 2. All samples were manually searched and the
day 1 sample was found to contain a roughly spheroid particle with a diameter of
approximately ten microns [Figure 3.9]. The SEM was then used to zoom in on the
particle of interest and ﬁll the ﬁeld of view with the particle. An EDS spectrum was then
taken and the elements present were identiﬁed as Pb, Ba, and Sb [Figure 3.10]. Other

particles were located, however, the shapes of these particles were more crystalline in

40

nature and contained elements such as silicon, potassium, calcium, and chlorine. These

particles were not consistent with GSR.

#

10 um

 

Figure 3.9 — Example spheroid particle from the day 1 gunshot wound.

 

C GSR1Jan1

 

 

Pb in
@1'2'3'4'5'6'7'8'9'1'0'1'1

 

 

 

 

Figure 3.10 — Representative EDS spectrum of particle shown in Figure 3.9 that indicated

the presence of Pb, Ba, and Sb in the particle.

41

The images obtained were not of the highest quality as the samples were uncoated
and nonmetallic particles that were present exhibited signiﬁcant charging. These issues
were of little concern, however, as the morphology of the particle was still apparent.
Furthermore, determinations are based only partly on morphology, as particle appearance
can be somewhat variable, and rely more heavily on the EDS spectrum. The ability to
image the sample is not related to the ability to take an accurate EDS spectrum over the
area of interest.

No particles that were consistent with GSR were observed for samples analyzed
from subsequent time points. This lack of GSR particles could likely be due to the fact
that signiﬁcant rainfall occurred overnight between the day l and day 2 samplings. It is
thought that the rain likely washed away much of the GSR that was sitting on the surface
of the skin and in the hairs. Time points after day 8 were not sampled due to excessive
oily discharge from the wounds and the lack of GSR found on days 2, 5, and 8. The
discharge most likely prevented any particles that may have been present from sticking to
the adhesive tab. Also, for samples to be analyzed by SEM they must be free of volatiles,
and the discharge formed a ﬁlm of oily liquid on the tab; thus, the samples were not
compatible with SEM/EDS analysis.

Based on its lack of usefulness for time points after environmental exposure
washed away surface GSR or after decompositional secretions made particle collection
with an adhesive unachievable, it can be determined that SEM/EDS, while a sufﬁcient
technique for the analysis of GSR from swabs of shooters’ hands or perhaps even from
fresh gunshot wounds, is not a reliable technique for the analysis of GSR on decomposed

tissue.

42

3.3.2 Winter SEM/EDS analysis results

The procedure outlined above was repeated for several time points from the
winter study, speciﬁcally on the day 1, 2, 5, and 44 gunshot wound samples. As was
stated above, the tissue did not progress through the stages of decomposition as with the
fall pig, however, many of the wounds secreted a milky, gelatinous substance that was
possibly adipocere. This substance became smeared around many of the wounds and
prevented the sampling of those wounds for the same reasons as were listed above
concerning the oily discharge from the fall pigs.

The day 1 sample yielded several particles which were consistent with GSR
particles. The particles were spheroid in nature and the EDS spectrum showed Pb, Ba,
and Sb. Similar particles were not observed for day 2, 5, and 44 samples. It is possible
that weathering effects such as wind and a slight amount of precipitation could account
for the loss of some surface GSR. Also, the tissue was already slightly oily by day 2,
which likely inhibited particle adhesion to the carbon tab.

As was seen in the fall, SEM/EDS failed to be useful for the analysis of GSR for
time points after environmental exposure would have washed away surface GSR or after
decompositional secretions made particle collection with an adhesive unachievable. This
indicates that SEM/EDS may not be a reliable technique for the analysis of GSR from

wounds that are more than one day old.

43

3.4 ICP-MS RESULTS AND DISCUSSION
3.4.1 ICP-MS calibration

Multielement calibration standards of Ba, Sb, and Pb were prepared in 2% HNO3
as described in Chapter 2. The standards were prepared at concentrations ranging from 0
to 500 ppb of each of the elements. The peak areas were normalized to the peak areas of
internal standards. The signals from 138Ba and me were normalized to the signal from
115In, whereas the signal from 208Pb was normalized to the signal from 209Bi. The
resulting response factor was then plotted against concentration to generate a calibration
curve. All of the theoretical concentrations of the standards were within 15% of the
calculated values. Figure 3.11 demonstrates linearity for each element over the interval of
0 to 500 ppb and Figure 3.12 demonstrates linearity even at the low end of the
concentration range, from 0 to 5 ppb. The standards were prepared in exactly the same
manner and the same concentrations in the fall and winter. Each R2 value is greater than
or equal to 0.9995 for both the entire range and the low concentrations, indicating the

wide linear dynamic range of the instrument.

44

 

 

25

y = 0.043x + 0.0023

20 /

 

y = 0.0304x + 0.0072

 

 

 

 

 

 

a, 15 F

2 93a
8 le
3 no
°‘ 10

y = 0.0126x - 0.0029

5 /-

 

 

 

 

 

o ; . , . .
0 100 200 300 400 500 600
Concentration (ppb)

 

 

 

Figure 3.11 — Example calibration curves showing linearity from 0 — 500 ppb for Ba, Sb,

and Pb standard solutions.

45

 

0.25

 

 

 

 

 

 

 

 

 

 

y = 0.0435X + 0.0011
0.2
Y = 0.0306x + 0.0009
,, 0.15 L
g 0 Ba
8. ISb
3 A Pb
‘1 0.1
y = 0.0122X + 0.0009
0.05
0

 

 

 

Concentration (ppb)

 

 

 

Figure 3.12 — Example calibration curves showing linearity from 0 — 5 ppb for Ba, Sb,

and Pb standard solutions.

The limit of quantiﬁcation (LOQ) is the lowest concentration that could be
reported with conﬁdence, and was determined as the lowest standard that was part of the
linear range for each set of samples. For Ba, the LOQ ranged from 0.10 ppb to 1.0 ppb;
for Sb, the LOQ ranged from 0.10 ppb to 0.25 ppb; and for Pb, the LOQ ranged from
0.10 ppb to 1.0 ppb. For the following sections, no sample with a concentration below the
LOQ for that set of samples was reported.

The limit of detection (LOD) was calculated for each element in the same manner
as reported by Koons.l4 The LOD was determined by ﬁnding the standard deviation (0)
of the response for each element in the 0.0 ppb standards and multiplying by three. This

value was then divided by the sensitivity, which is the slope of the standard curve for

46

each element when element response is plotted against concentration. '4 The average LOD
values were calculated to be 0.074, 0.106, and 0.017 ppb for Ba, Sb, and Pb, respectively.
These values correspond somewhat to the values reported by Koons, which were 0.02,
0.05, and 0.1 ppb for Ba, Sb, and Pb, respectively.” Low LOD values combined with a
wide linear dynamic range for Ba, Sb, and Pb, the characteristic elements of GSR,
demonstrate that ICP-MS is an excellent technique for the trace elemental analysis of

GSR containing samples.

3.4.2 ICP-MS precision study

Five microwave digest solutions were prepared for ICP-MS analysis as described
in Chapter 2. The ﬁve digests were a sample of control stab wound tissue, two samples of
tissue from the fall day 12 GSR tissue samples, a sample of ten blowﬂy larvae harvested
from gunshot wounds on day 7, and a procedural blank. Each digest was analyzed by
ICP-MS in replicate (n=5) in order to assess instrumental precision. The concentrations
of Ba, Sb, and Pb were determined from the corresponding calibration curve and the
reported solution concentrations were converted to microgram of element per gram of
sample (tissue or larvae) with the exception of the procedural blank. The average value
and relative standard deviation (RSD) were computed for each digest (Table 3.1.) based

on replicate analyses.

47

Table 3.1 — ICP-MS precision study results. Each average represents ﬁve replicates.

 

 

 

 

 

 

Average Average Average
Sample [Ba] Ba RSD [Sb] Sb RSD [Pb] Pb RSD
(rials) M (HEEL
Day 12 control wound tissue 0.16 1.5% 0.04 6.0% 0.14 1.0%
Day 7 blowﬂy larvae 2.30 0.8% * * * *
Day 12 gunshot wound digest I 0.91 0.4% 0.35 1.2% 1.00 0.3%
Day 12 gunshot wound digest 2 0.80 0.6% 0.34 2.2% 1.08 0.7%

 

 

 

 

 

 

 

 

 

"‘ ICP-MS concentrations were below LOQ and therefore not reported.

The RSD values for each sample type were below 2.5% with the exception of the

RSD for Sb from the Day 12 control tissue. The high RSD for this set of replicates is due

to the fact that the concentration of Sb was very low, at 0.04 jig/g. The tissue

concentrations of Sb for the ﬁve injections were 0.05, 0.04, 0.04, 0.04, and 0.04 ug/g,
which is quite precise. Each of the element concentrations for all of the procedural blank
replicates was below their respective LOQ for the run and consequently were not
reported. Acceptable precision, good sensitivity, and a wide linear dynamic range make
ICP-MS a good technique for the detection of low concentrations of GSR components.

As this study demonstrated that ICP-MS exhibits excellent precision, the rest of
sample digests were analyzed in triplicate. If any of the three injections seemed to be an
outlying data point, a statistical Q test was performed to determine if the point was a

statistical outlier and could therefore be eliminated.30

3.4.3 ICP-MS analysis of procedural blanks

Five procedural blanks were microwave digested in the fall and ﬁve procedural
blanks were microwave digested in the winter. These blanks were analyzed by ICP-MS in
order to see if any signiﬁcant amounts of the elements of interest were present in the

solutions used in the digestion procedure. For the fall set of procedural blanks, the

48

concentrations of Ba, Sb, and Pb were all below their respective LOQ for the run. The
same was true in the winter for Sb and Pb; however, the LOQ for Ba was lower in the
winter set of samples and as a result, Ba was quantiﬁed. The average concentration was
0.18 ppb with a standard deviation of 0.087 ppb. The concentration of Ba was much
lower than any concentration reported for a digest solution of gunshot wound tissue or
blowﬂy larvae that had been feeding on GSR containing tissue. These results indicate that
the microwave digestion process and sample preparation procedure contributed no

signiﬁcant amount of Pb, Ba, or Sb to observed concentrations in tissue or larvae digests.

3.4.4 ICP-MS analysis of blowﬂy larvae from indoor study

Initially, ﬁrst instar Phoenicia sericata blowﬂy larvae were placed onto shot beef
and control beef as described in Chapter 2. By day 2, the larvae were primarily second
instars, and by day 3, most were early third instars. Pupae were ﬁrst observed on day 9.
Microwave digests of between 0.03 and 0.5 grams of blowﬂy larvae from the control beef
and from the shot beef were analyzed in triplicate by ICP-MS. The difference in weight
was due to the difference in mass of early instar larvae when compared to third instar
larvae. Approximately ten larvae were included in each sample. One sample of day 9
pupae from each of the control beef and shot beef were also microwave digested and
analyzed by ICP-MS in triplicate. Figure 3.13 shows the average concentrations of Pb,
Ba, and Sb in the larvae and pupae in micrograms of element per gram of larvae or
pupae. The Ba concentrations in the larvae from the shot beef started at approximately
10.3 ug/g for days one and two after exposure to the GSR beef and then rose to between
50 and 80 ug/g for days 3 through 9. It is possible that the larvae were bioaccumulating

Ba over the course of the experiment, as barium exhibits similar chemical properties to

49

that of calcium, which is necessary for larval development;24 however, further studies

would be needed to investigate this hypothesis.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

90
80
70 T
3 60
\
3
g 50 IGSR Ba
E a GSR Sb
E 40 GSR Pb
g IControl Ba
8 30 IContrOI Sb
IControl Pb
20
10 -
I
o -l r - "" 9 ..
1 2 3 4 5 7 9 Pupae 9
Time from exposure to collection (Days)

 

Figure 3.13 — Graph showing average Pb, Ba, and Sb concentrations in blowﬂy larvae
and pupae. “GSR” indicates blowﬂy larvae that had been feeding on shot beef. “Control”

indicates blowﬂy larvae that had been feeding on control beef.

Ba was also observed in the control larvae; however, the concentrations were much lower
and were all below 1.0 jig/g. The low concentrations are visible in Figure 3.14, which
shows the same data as Figure 3.13 on a decreased scale. As there is no apparent trend in
the control Ba concentrations, it is thought that blowﬂy larvae could contain a certain
amount of endogenous barium; however, many more blowﬂy larvae would need to be
analyze in order to test this hypothesis. Sb and Pb were not observed in any of the larvae

that had been feeding on the control beef. Sb was observed in the day 1 larvae from the

50

shot beef, as well as in the larvae from days 3, 4, 5, 7, and 9 in concentrations ranging
from 0.15 to 0.75 ug/g. Sb concentrations in the day 2 and day 9 pupae samples were not
above the LOQ and are therefore not reported here. The lack of Sb in the day 2 sample
when it was present on days 1 and 3 would need to be investigated further, however, it is
likely that the larvae collected on that day simply did not consume enough GSR particles

for Sb to be detected.

 

 

3

E

3

c 'GSR Ba

.3 a GSR so

g GSR Pb

3 IControl Ba
5 I Control Sb
U IContml Pb

 

 

 

1 2 3 4 5 7 9 Pupae
9

 

Time from exposure to collection (Days)

 

 

Figure 3.14 — Decreased scale version of the graph showing Pb, Ba, and Sb

concentrations in blowﬂy larvae and pupae.

The concentration of Pb was 6.26 ug/g on day 1 and then decreased to between 0.42 and
1.29 ug/g for subsequent time points. Further testing would be needed to explain the

large amount of lead in the day 1 sample. As this study was conducted indoors with no

51

environmental exposure, all of the larvae were feeding pieces of the same shot steak, and
all of the larvae were received in a batch ﬁ'om the same company, it can be assumed that
the Pb in the day 1 sample is indeed ﬁ'om the gunshot residue rather than external
contamination.

All of the characteristic metals of GSR were detected in P. sericata larvae that
had been feeding on shot beef on days 1, 3, 4, 5, 7, and 9, where only Pb and Ba were
detected in day 2 larvae and the day 9 pupae. Even though the concentrations of Sb in the
day 2 larvae and day 9 pupae were below the LOQ and were not reported, the larvae from
the shot beef were still signiﬁcantly different from the control larvae on the same day as
they have high Ba concentrations and they do contain Pb. Neither Pb nor Sb was detected
in any of the control samples, and while Ba was detected in all control larvae samples, it
was present at much lower concentrations than in the larvae that had been feeding on
GSR containing beef. These results indicate that the microwave digestion protocol used is
indeed an effective means for digesting blowﬂy larvae samples for elemental analysis of
GSR components. Blowﬂy larvae feeding on shot beef contain much higher
concentrations of Pb, Ba, and Sb than background levels in the same larvae, which makes

the analysis of these elements for GSR detection in blowﬂy larvae feasible.

3.4.5 ICP-MS analysis of blowﬂy larvae found feeding in gunshot wounds (Fall)
Blowﬂy larvae were harvested ﬁom the gunshot and stab wounds ﬁom the fall set
of pigs. Between 0.03 and 0.50 grams of blowﬂy larvae were digested in the same
manner as the larvae from the indoor study and were then analyzed by ICP-MS in
triplicate for the characteristic elements of GSR. Ba, Sb, and Pb were detected in day 3

and 4 larvae samples from the gunshot wounds and were not detected in the larvae

52

samples from the stab wounds [Figure 3.15]. For subsequent days, Ba was present in all
larvae and the levels were comparable between the larvae from gunshot wounds and stab
wounds. Low levels of Sb was detected in the day 9, 10, and 15 larvae from gunshot
wounds, along with the day 15 larvae from the stab wound at a similar concentration
[Figure 3.16]. Similarly, Pb was detected in the day 10 and 11 samples of larvae from
gunshot wounds and in the day 11 larvae from the stab wound [Figure 3.16]. As the
procedural blanks have shown to contain no signiﬁcant concentrations of these elements,
it can be assumed that the observed concentrations of the elements of interest are indeed

from the larvae.

 

 

12

 

10

 

 

 

 

 

 

3

g 8 IGSR Ba

2’ E GSR Sb

.9 GSR Pb

g 6 IControI Ba
[.3 a: Control Sb
F lControI Pb
8 4 j .

 

 

 

 

3 4 6 7 9 10 11 13 14 15
Time from death to collection (Days)

 

 

 

Figure 3.15 — Graph of concentrations of Ba, Sb, and Pb in blowﬂy larvae from gunshot

and stab wounds over time.

53

 

I"
o

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.8 -
1.6 -
r

a 1.4 - .
E IGSR Ba
:4 1.2 - 151 GSR Sb
0
:3 1 0 GSR Pb
g IControl Ba
§ 0.8 - JaControI Sb
5 lControI Pb

 

 

 

P
as

 

—-——
--I

,0 .-——-—
---
a-————
---
n——-—
=--
—-———
===-
aiil-_—-

 

.0
o

o o
N A
a: —-

7 10 11 13 14 15
m death to collection (Days)

3‘

‘I'lme

 

 

 

Figure 3.16 - Decreased scale version of the graph of concentrations of Ba, Sb, and Pb in
blowﬂy larvae from gunshot and stab wounds over time showing low concentrations at

later time points.

These results indicate that the blowﬂy larvae from gunshot wounds tested on days
3 and 4 contained GSR components, whereas the larvae from stab wounds did not. On
subsequent days, the larvae from gunshot wounds did not contain signiﬁcantly different
concentrations of the characteristic GSR metals than the larvae from the stab wounds. It
is possible that this is due to sampling technique. The larvae had most likely been feeding
on the tissue of the wound tract rather than the skin, where much of the GSR had
deposited. Each day the larvae were harvested from just inside the gunshot wounds. As
the larvae from days 3 and 4 were smaller and appeared to be generally less mobile, they

had most likely been feeding on the wound tract tissue the earliest, when more GSR had

54

been present. Later, the larvae were larger and could cover more area than before. Also,
the tissue containing the most GSR from the upper one to two centimeters of the wound
tract would have been consumed. Therefore, when the larvae were harvested from that
area, they had not been feeding on GSR containing tissue and would therefore not contain
the characteristic GSR elements in signiﬁcant concentrations. Further studies would need
to be performed to lend more weight to this hypothesis, but it is thought that if larvae had
been collected from further in the wound tract, larvae samples from later time points
would have yielded characteristic GSR components. Nevertheless, this study
demonstrates the feasibility of detecting characteristic GSR components in blowﬂy larvae

found feeding naturally in gunshot wounds.

3.4.6 ICP-MS analysis of decomposing porcine tissue for GSR components (Fall)

Microwave digests of tissue samples from control stab wounds and gunshot
wounds were analyzed by ICP-MS in triplicate for the presence of Ba, Sb, and Pb. All
three elements were found in digests of gunshot wound tissue [Figure 3.17]. The overall
trend shows a decrease in metal content consistent with GSR over the ﬁrst three time
points with a leveling off by day 8. The highest Ba concentration observed was on the
ﬁrst day after death, at 163.63 ug/g of tissue. Day 1 also exhibited the highest Sb and Pb
concentrations, at 56.50 and 122.56 ug/g, respectively. Between days 8 and 37 the
concentrations of Ba ranged from 1.21 ug/g on day 8 and 7.54 ug/g on day 34. Sb ranged
from 0.59 ug/g on day 37 to 3.64 jig/g on day 34, while Pb ranged from 1.09 jig/g on day
16 to 5.20 jig/g on day 34. With the exception of the lowest Sb concentrations, all of the

values were in the same order of magnitude.

55

 

 

180.0

 

 

 

 

 

 

 

160.0 -
140.0 -
§ 120.0
3 I659. Ba
5 100.0 BGSR Sb
'3 GSR Pb
75 80.0 IControI Ba
g Control Sb
8 60.0 IContnol Pb

 

 

 

 

 

 

 

 

1 2 5 8 12 16 20 26 30 34 37
Time Interval from death to collection (Days)

 

 

 

Figure 3.17 — Graph of concentrations of Ba, Sb, and Pb in tissue from gunshot and stab

wounds over thirty-seven days in the fall.

Even the lowest Sb concentrations are almost an order of magnitude higher than
the highest Sb concentrations observed in the control wound digests [Figure 3.18]. Sb

was only observed in the day 16 and 30 control wound digests at concentrations of 0.08
ug/g and 0.05 ug/g, respectively. Pb was observed in the control wound digests for all
time points with the exception of days 1 and 2. The concentrations ranged from 0.04 jig/g
on day 5 to 0.28 pig/g on day 20. These values are at least an order of magnitude lower
than the amount of Pb found in all time points of the gunshot wound digests. Ba was
observed in the control wound digests for all time points with the exception of days 2 and

5. The concentrations ranged from 0.12 jig/g on day 1 to 1.99 jig/g on day 34. With the

56

exception of day 34, the concentration of Ba in the control wound digests is almost an

order of magnitude lower than in the gunshot wound digests.

I GSR Ba

22-: GSR Sb

GSR Pb
IControl Ba
' Control Sb
Pb

E
3
r:
.9
E
r:
8
c
8

5 8 12 16 20 26 30 34 37
Tune interval from death to collection (Days)

 

Figure 3.18 — Decreased scale version of the graph of concentrations of Ba, Sb, and Pb in

tissue from gunshot and stab wounds over thirty-seven days in the fall.

Although the difference between the control wound and gunshot wound Ba
concentration on day 34 may not be signiﬁcant, the difference in Sb and Pb is signiﬁcant.
This indicates that there is still a pronounced difference between the GSR components in
the gunshot wound digest versus the control wound digest.

The decrease in Ba, Sb, and Pb in the gunshot wound tissue after day 1 is likely
due to a partial loss of surface GSR. Signiﬁcant rainfall occurred between the collection

time on days 1 and 2. The particles that had lightly adhered to the hairs and skin may

57

have been washed away in the rain. Between the second and ﬁfth days, additional surface
GSR likely was lost due to wind and decompositional ﬂuid seepage. Additional
signiﬁcant rainfall occurred on the evening of the ﬁfth day. Between the wind, rain, skin
slippage, and active decay processes, it can be assumed that only the GSR that had
tattooed into the skin remained by the eighth day. This would account for the apparent
leveling off of GSR component levels in the tissue.

The slight day-to-day variations of the element concentrations in the gunshot
wound tissue samples are most likely the result of the sample collection procedure. As
can be seen in Figures 3.1, 3.6, and 3.7, the distribution of GSR around the bullet hole
was not homogenous. As it was not visually apparent where most of the GSR had
deposited, sampling was simply conducted approximately one centimeter away from the
bullet hole. If it happened to be on the side of the wound that contained the most GSR, a
higher metal content would be observed than if the sampling happened to occur on the
opposite side. By the end of the collection the skin had leathered to the point that tissue
excision by scalpel had become extremely difﬁcult. Also, despite every precaution taken
to ensure that the gunshot wounds were as similar as possible, the wounds most likely
differed somewhat due to slight variations in wind speed, angle of shot, and individual
bullet cartridge characteristics. Quantiﬁcation of element concentrations spatially was not
a goal of this project, however, as GSR determination is primarily based on a qualitative
identiﬁcation of the elements of interest. The quantitative aspect was employed to
distinguish the amount of Pb, Ba, and Sb in the gunshot wound tissue from the control

wound tissue.

58

Overall, these results indicate that microwave digestion of small samples of
wound tissue, coupled with ICP-MS analysis of Ba, Sb, and Pb, is an effective means for
determining GSR in decomposing tissue for at least thirty-seven days after death, even
after several signiﬁcant rainfalls. The time frame studied encompassed many stages of

decomposition and the skin was quite leathered by the end of the sampling process.

3.4.7 ICP-MS analysis of decomposing porcine tissue for GSR components

(Winter)

The fall study described above was repeated in the winter with tissue samples
taken at the same time points until thirty days after death, with the ﬁnal two time points
taking the study out farther, to days 44 and 60 after death. Microwave digests of tissue
samples from control stab wounds and gunshot wounds were analyzed by ICP-MS in
triplicate for the presence of Ba, Sb, and Pb. All three elements were found in digests of
gunshot wound tissue [Figure 3.19]. The highest Ba concentration observed in gunshot
wound tissue was on day 44, at 125.90 ug/g of tissue, whereas the lowest concentration

observed was on day 16, at 34.91 ug/g. Day 5 exhibited the highest Sb concentration, at

35.6 ug/g, while the lowest concentration was observed on day 12, at 5.23 pg/g. The
amount of Pb in the day 5 digest solution saturated the detector, while the concentration
of Pb in the day 60 digest was signiﬁcantly above the range of standards. Therefore,
while the actual values of the tissue concentrations in these samples are not reliable, their
approximate values are represented in Figure 3.19 in order to illustrate their
concentrations relative to the other concentrations. Regardless of their actual values, the

Pb concentrations on days 5 and 60 were the highest observed over the course of the

59

experiment. The lowest Pb tissue concentration of the gunshot wound samples was on

day 30, at 81.27 jig/g.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

600

500 i‘
A *
Q 400 r —
g .659. Ba
E are GSR Sb
.9 GSR Pb
H 300 —
B lControl Ba
§ lContnoI Sb
g 200 I _ lContnol Pb

I
100 ' - I 8 I . _—
6 20 26 30 44 60

 

S 8 12 1
Time from death to collection (Days)

 

 

 

Figure 3.19 -- Graph of concentrations of Ba, Sb, and Pb in tissue from gunshot and stab
wounds over sixty days in the winter. *The concentration of Pb in the gunshot wound
digest saturated the detector on days 5 and was above the upper LOQ on day 60. These

concentrations are thus only included as approximations of the tissue concentrations.

Ba was present in all control wound samples in concentrations below 0.50 ug/g,
which is two orders of magnitude lower than the lowest concentrations observed in the
gunshot wound tissue samples. Sb was detected in all control tissue samples with the
exception of the day 26 and 60 samples. The concentrations reported were all below 0.60

ug/g, which is at least one order of magnitude lower than the tissue concentrations

60

reported for the gunshot wound tissue samples. Pb was reported in the day 1, 2, and 16
samples at concentrations of 1.20, 0.27, and 0.07 jig/g, respectively. Again, these
concentrations are one to two orders of magnitude lower than the Pb levels observed in
the gunshot wound samples. The low concentrations are visible graphically in Figure

3.20, which is the decreased scale version of Figure 3.19.

 

 

 

 

O
or

.—

.0
A

O
N

 

2.0 l a} —

1.8 I _

1.6 . é , , _

l .

A 1.4 , . I _
0'
E I I IGSR Ba
‘5 1'2 . '— assaso
7‘3 1 o _ GSR Pb
S I I I lControl Ba
2:3 0.8 _ aContnol Sb
c ‘ t ‘4 IControl Pb
8 I ll ~ .

P
o

1 2 5 812 16 20 26 304460
Time from death to collection (Days)

 

 

 

Figure 3.20 — Decreased scale version of the graph of concentrations of Ba, Sb, and Pb in

tissue from gunshot and stab wounds over sixty days in the winter.

The consistent results over the entire range of collection time points indicate that
any signiﬁcant weathering effects on the surface GSR likely occurred before the ﬁrst
collection point. However, the high concentrations compared to the concentrations

measured in the fall study indicate that a less signiﬁcant amount of weathering occurred.

61

The amount of GSR that was visible on the tissue originally was slightly reduced by day
l but appeared consistent throughout the rest of the collection period despite the snow
and sleet. As was likely the case for the later time points in the fall study, the daily
variation in metal concentration is likely due to the non-homogenous distribution of GSR
around the bullet wound. These results indicate that the concentrations of Ba, Sb, and Pb
remain somewhat stable over time when tissue decomposition does not occur, even when

exposed to snow and sleet.

3.4.8 ICP-MS tissue analysis summary

The results of the fall study show a decrease in metal content consistent with GSR
over the course of thirty-seven days, from the fresh stage to desiccation, whereas the
results of the winter study yielded a relatively constant amount of metal content
consistent with GSR over the course of sixty days when the tissue was prevented from
decomposing by freezing temperatures. The results of both studies indicate that the
persistence of GSR depends more on the stage of decomposition than on the amount of
time that has passed from wound inﬂiction to tissue harvesting.

It is interesting to note that the general elemental distribution were quite variable.
The elemental distributions were calculated in the form of percentages and compared

between the studies. These results are shown in Table 3.2.

62

Table 3.2 — Elemental distribution in gunshot wound digest samples: Fall and Winter.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fall Mass Percent (pg/g) Winter Mass Percent (jig/g)
Day [Ba] [Sb] [Pb] Day [Ba] [so] [Pbl
l 47.75% 16.49% 35.76% I 41.27% 5.83% 52.91%
38.91% 11.12% 49.96% 2 25.71% 4.53% 69.77%
5 33.04% 12.37% 54.58% 5 "' * "‘
8 20.52% 47.97% 31.51% 8 38.51% 6.61% 54.88%
12 35.61% 33.94% 30.45% 12 32.60% 3.48% 63.92%
16 48.90% 22.78% 28.32% 16 26.06% 6.52% 67.42%
20 59.32% 14.13% 26.54% 20 22.42% 3.59% 73.99%
26 50.33% 30.05% 19.62% 26 28.87% 5.86% 65.27%
30 42.01% 18.09% 39.90% 30 34.58% 5.45% 59.97%
34 45.57% 22.41% 32.03% 44 39.91% 3.48% 56.61%
3 7 61.45% 1 1.61% 26.93% 60 "' * "'
Average: 43.95% 21.91% 34.15% Average: 32.21% 5.04% 62.75%
RSD: 27% 52% 30% RSD: 21% 26% 11%

 

 

 

 

 

 

*These data were not reported because the Pb concentrations were above the upper LOQ.

The distributions were not reported for days 5 and 60 of the winter study as the Pb
saturated the detector on day 5 and the concentration was signiﬁcantly above the highest
calibration standard on day 60. The data for Ba and Sb were within the standard range for
those time points, but without reliable data for all three elements, the ratios are not
meaningful. In the winter, the concentration of Pb was always the highest, followed by
Ba. Sb always accounted for less than 10% of the total metal detected. The fall studies
were much more variable. Since ICP-MS has shown to be a very precise technique, the
high RSD values are most likely the result of a heterogeneous elemental distribution in
the tissue or cartridge-to-cartridge differences. Further studies would be needed to
determine the origins of these differences.

Haag et a1.27 has demonstrated that porcine tissue is a suitable model for human
skin. The correlation suggests that the technique of microwave digesting tissue and

subsequent ICP-MS analysis that has been shown to be useful in the identiﬁcation of

63

GSR components in porcine skin would likely also be useful for the chemical
identiﬁcation of gunshot wounds in decomposing human individuals. The ability to use
these techniques to identify human gunshot wounds could be useful to pathologists in
determining the cause of death when decomposition, insect predation, or other

circumstances have precluded conventional means of identifying the wounds.

64

4. CONCLUSIONS AND FUTURE WORK
4.1 CONCLUSIONS

The results of this study indicated that the extraction of Pb, Ba, and Sb from
Phoenicia sericata larvae by microwave digestion was successfully achieved from larvae
that had been feeding on beef containing GSR. The digests were analyzed by ICP-MS
and the concentrations of GSR components, Ba, Sb, and Pb, were determined. It was
found that the larvae that had been feeding on the shot beef contained each of the
characteristic GSR components. The concentration of Ba was approximately 10 ug/ g for
the ﬁrst two days after exposure, then rose to between 50 and 80 ug/g for days 3-9. These
results indicated a possible bioaccumulation of Ba in blowﬂy larvae, although the reason
for this was not investigated ﬁirther during this research. Sb was observed for all time
points except for day 2 and the day 9 pupae. The concentrations of Sb ranged from 0.15
to 0.75 ug/g when it was observed. Pb was observed for all time points, beginning at 6.26
jig/g on day 1 and ranging between 0.42 and 1.29 ug/g for later time points. A sample of
pupae was found to contain Ba and Pb, but no Sb was detected. More pupae samples
would be needed to investigate the statistical signiﬁcance of these ﬁndings. These results
were compared to larvae that had been feeding on beef that was not shot. It was found
that although the elements were detected for some time points, their levels were
signiﬁcantly lower than in the blowﬂy larvae that had been feeding on shot beef.

The method developed using P. sericata larvae was used to analyze blowﬂy
larvae found feeding in decomposing porcine gunshot wounds for the presence of GSR

components. It was found that the larvae contained these components on days 3 and 4

after death. The concentrations of Ba were between 5.0 and 10.0 ug/g, where the

65

concentrations of Sb were between 0.2 and 1.2 jig/g and the concentrations of Pb were

between 1.4 and 4.0 ug/g. For all subsequent time points there was no difference in the
element content of the larvae from gunshot wounds versus the control larvae from stab
wounds. It is suspected that more time points could have been found to contain GSR if
larvae had been sampled from deeper within the wound tract.

SEM/EDS and ICP-MS of the decomposing porcine tissue digests were also
employed to determine the persistence of GSR in decomposing tissue in both fall and
winter climates. For SEM/EDS analysis, potential GSR particles were collected by
dabbing the wound 100 times with a carbon adhesive tab mounted on an aluminum stub.
In the fall, it was found that SEM/EDS was useful to detect characteristic GSR particles
from the area near the gunshot wounds for up to one day after death. The particles were
collected by dabbing the wound 100 times with a carbon adhesive tab mounted on an
aluminum stub. It is thought that the weather played a role in washing away GSR
particles that were deposited on the surface of the skin between the day 1 and day 2
collection times. No GSR was detected on wounds from later time points. In the winter,
characteristic GSR particles were also detected for up to one day after death. No GSR
particles were detected on wounds from later time points. It is possible that the lack of
GSR for later time points was due to a combination of wind, light precipitation, and the
slightly oily nature of the tissue.

SEM/EDS has not been shown to be a reliable technique for the detection of GSR
on decomposing tissue as many factors such as rain and decompositional secretions can

make its collection with adhesive tabs extremely impractical if not impossible. It may be

66

a viable technique for fresh gunshot wounds, however, it is likely that the wound would
be identiﬁable by an expert performing a visual and histological examination at that time.

ICP-MS was shown to be a precise multielement technique with a wide linear
dynamic range through linearity and precision studies. Multielement calibration standards
indicated that the upper LOQ was at or above 500 ppb for each of Ba, Sb, and Pb and that
the lower LOQ was between 0.1 and 1.0 ppb for each element. The average 30 LOD
values were calculated to be 0.074, 0.106, and 0.017 ppb for Ba, Sb, and Pb, respectively.
Relative standard deviations for replicate injections were less than 3% for each element.

The fall set of pigs decomposed to a desiccated state over the course of the thirty-
seven day collection interval. Tissue samples were microwave digested and analyzed by
ICP-MS for the presence of GSR components. It was found that all three characteristic
elements could be detected in the tissue digests over the course of the entire collection
interval. The tissue concentrations of each element decreased over the ﬁrst three
collection points and then leveled off for the rest of the time points. The Ba, Sb, and Pb
concentrations were 163.63, 56.50, and 122.56 ug/g on day 1, respectively. By day 8, the
concentrations had decreased to between 0.5 and 8.0 jig/g for all elements and remained
in this range for the duration of the experiments.

In the winter, the pigs did not decompose over the sixty day collection interval.
ICP-MS analysis of tissue digests revealed that all three characteristic elements could be
detected for each time point over the sixty days. Moreover, the concentrations of the
elements remained relatively constant over the course of the collection. The

concentrations of the elements were between 35 and 126 ug/g for Ba and between 5 and

36 jig/g for Sb. The lowest Pb concentration observed was 81.27 ug/g and the highest

67

concentrations could not be quantitated as they were above the upper LOQ. These results
indicate that ICP-MS of tissue digests can provide a chemical means of identifying
gunshot wounds and that the concentrations of GSR components are more reliant on the
stage of decomposition rather than the amount of time between death and analysis.
Unlike SEM/EDS, which proved unreliable after more than a day after death, the ability
to detect GSR components by ICP-MS was not hindered by weathering effects on the
carcasses. ICP-MS of tissue digests may be useful to a pathologist in the identiﬁcation of
suspected gunshot wounds when decomposition has precluded conventional means of

wound identiﬁcation.

4.2 FUTURE WORK

Now that it is established that GSR components can be detected in decomposing
tissue through all stages of decomposition, there are a great many possibilities for future
projects. In general, more studies should be conducted to expand the sample population
and thus provide some information as to the statistical signiﬁcance of the ﬁndings
previously presented. Also, it is necessary to perform the same study over a longer
sampling time frame as the latest time points in the studies discussed in this work still
exhibited detectable levels of GSR components. It is thought that GSR remains as long as
skin is present with the remains, and so leaving some remains with gunshot wounds

outside and sampling until the tissue is gone could test this theory.

The usefulness of ICP-MS to determine the ﬁring range could also be investigated
by ﬁring shots of known distances and quantifying the GSR components in the tissue in
order to see how their concentration varies with ﬁring distance. This study could be

conducted preliminarily with fresh gunshot wounds and then again with decomposed

68

 

 

wounds to see if the results differ with decomposed tissue. Additionally, a sampling study
should be conducted on fresh gunshot wounds to see how much variability exists around
a wound and ﬁom wound to wound. This information would be useful in order to
determine an optimal sampling scheme ﬁom the wounds and could be used to help
provide statistical information about the technique itself and about the precision of the

ﬁring distance determinations method.

Additionally, it would be useful to perform similar studies using pigs that had
been shot then buried. When bodies are recovered after burial, it is often the case that the
tissue has decomposed to a point that makes a visual and histological assessment of
gunshot wounds extremely difﬁcult. The skin of the victim may have a great deal of dirt
embedded in it, which could affect the background concentrations of the metals. The
samples could still be microwave digested and analyzed for the characteristic GSR

components by ICP-MS.

A simple study could demonstrate the ability of ICP-MS digested tissue to
discriminate entrance wounds from exit wounds in decomposed tissue. In such a study, a
euthanized pig could be shot so as to incur through and through wounds and tissue could
be sampled over time from both the entrance and exit wounds to determine if the
concentration of GSR components varies from entrance to exit, and, if the variation

exists, the extent to which it is exhibited.

Other studies could investigate different brands of ammunition. It is possible that
different types and brands of ammunition would deposit different amounts of GSR with
different elemental distribution ratios. The concentrations of Pb, Sb, and Ba could be

measured in the tissue surrounding the wound along with other trace elements to see if a

69

statistical difference exists between different brands of jacketed ammunition along with
unjacketed and semi-jacketed types of ammunition. It would also be beneﬁcial to see if
the trace elemental proﬁle of various types of ammunition changes with decomposition,

for example, if the concentration of zinc decreases over time.

Finally, further work should be done to optimize the collection of blowﬂy larvae
from gunshot wounds for ICP-MS analysis. Larvae could be collected from different
wounds from various depths inside the wound tract in order to see if the sampling depth

affects the number of days after death that blowﬂy larvae might be useful for GSR

component detection and subsequent gunshot wound identiﬁcation.

The research presented in this thesis has shown the ability to use ICP-MS to
chemically identify GSR components in decomposing porcine tissue over many stages of
decomposition and for at least sixty days following death. The similarities between
porcine and human tissue make it likely that the techniques outlined in Chapter 2 could
be applied to gunshot wounds in decomposing human remains. It was also shown that
analyzing blowﬂy larvae in suspected gunshot wounds for the presence of GSR
components using ICP-MS could also be useful to identify gunshot wounds while the

larvae are still in early developmental stages.

70

APPENDIX 1

Weather data for the fall collection period:31

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2007 Temperature (°F) Rainfall (in)
Day Date Min Max Average Today
Wed 5-Sep 62.4 88.4 75.4 --
Thu 6-Sep 67.0 83.6 75.3 --
Fri 7-Sep 68.2 79.5 73 .9 0.61
Sat 8-Sep 59.5 78.5 69.0 --
Sun 9-Sep 60.3 80.3 70.3 --
Mon 10-Sep 53.3 66.7 60.0 0.46
Tues ll-Sep 52.9 67.2 60.0 0.07
Wed 12-Sep 45 .9 60.9 53 .4 --
Thu l3-Sep 42.8 73 .1 58.0 --
Fri 14-Sep 48.8 63.3 56.1 0.02
Sat lS-Sep 41.1 58.8 49.9 --
Sun 16-Sep 35.3 65.8 50.6 --
Mon l7-Sep 43 .0 72.3 57.6 --
Tues 18-Sep 54.4 82.1 68.2 --
Wed l9-Sep 58.0 82.5 70.3 --
Thu 20-Sep 56.8 80.6 68.7 --
Fri 21-Sep 57.7 85.5 71.6 --
Sat 22-Sep 54.2 74.2 64.2 0.05
Sun 23-Sep 42.5 77.3 59.9 --
Mon 24-Sep 52.6 88.6 70.6 --
Tues 25-Sep 66.3 81.5 73.9 0.59
Wed 26-Sep 57.3 70.8 64.0 0.13
Thu 27-Sep 50.4 70.1 60.3 --
Fri 28-Sep 47.9 70.1 59.0 --
Sat 29-Sep 40.7 73 .3 57.0 --
Sun 30-Sep 50.3 77.0 63 .6 --
Mon l-Oct 55.8 69.7 62.7 0.93
Tues 2-Oct 5 5.1 73 .4 64.2 --
Wed 3-Oct 47.0 72.3 59.7 0.04
Thu 4-Oct 42.0 79.4 60.7 --
Fri S-Oct 60.8 86.6 73.7 --
Sat 6-Oct 64.4 85.2 74.8 --
Sun 7-Oct 63 .6 87.1 75 .4 --
Mon 8-Oct 66.2 86.4 76.3 --
Tues 9-Oct 51.5 73.1 62.3 0.07
Wed lO-Oct 43.3 53.0 48.2 0. I 2
Thu ll-Oct 43.7 51.6 47.6 0.01
Fri 12-Oct 39.9 50.8 45.3 --

 

71

 

APPENDIX 2

Weather data for the winter collection periodz3'

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2008 Temperature (F) Rainfall (in.)
Day Date Min Max Avergge TodaL
Fri 11-Jan 33.6 44.4 39.0 0.03
Sat 12-Jan 31.4 38.3 34.9 --
Sun 13-Jan 31.1 40.1 35.6 0.05
Mon 14-Jan 26.3 32.3 29.3 --
Tues 15-Jan 24.7 29.5 27.1 0.01
Wed 16-Jan 14.7 33.0 23.8 --
Thu l7-Jan 20.0 34.7 27.3 0.02
Fri 18-Jan 16.0 25.7 20.8 --
Sat 19-Jan 2.7 20.3 1 1.5 --
Sun 20-Jan 1.7 12.4 7.1 --
Mon 21-Jan 4.2 23.1 13.6 --
Tues 22-Jan 16.0 28.4 22.2 --
Wed 23-Jan 11.0 17.9 14.5 --
Thu 24-Jan 5.4 19.6 12.5 --
Fri 25-Jan 2.0 18.9 10.4 --
Sat 26—Jan 13 .9 24.5 19.2 --
Sun 27-Jan 14.8 28.9 21.9 --
Mon 28-Jan 14.0 44.5 29.2 0.18
Tues 29-Jan 27.8 46.6 37.2 0.40
Wed 30-Jan 6.7 27.9 17.3 0.01
Thu 31-Jan 8.6 24.2 16.4 --
Fri l-Feb 20.0 28.7 24.3 --
Sat 2-Feb 24.4 28.8 26.6 --
Sun 3-Feb 27.3 30.4 28.9 0.06
Mon 4-Feb 27.7 40.5 34.1 0.20
Tues 5-Feb 31.7 45.7 38.7 0.03
Wed 6-Feb 24.0 32.2 28.1 --
Thu 7-Feb 18.3 28.9 23 .6 --
Fri 8-Feb 24.7 32.0 28.4 --
Sat 9-Feb 26 34.6 30.3 0.04
Sun lO-Feb -4.3 26.7 1 1.2 --
Mon 11-Feb -5.4 1 1.7 3.2 --
Tues 12-Feb 0.3 14.9 7.6 --
Wed 13-Feb 0.3 23.5 1 1.9 0.01
Thu 14-Feb 13.4 33.2 23.3 0.01
Fri 15-Feb 13.1 28.8 20.9 --
Sat 16-Feb 8.5 28.4 18.5 --
Sun 17-Feb 20.4 46.3 33.3 0.74
Mon 18-Feb 13.] 33.8 23.5 --
Tues 19-Feb 7.8 18.9 13.3 --
Wed 20-Feb 4.4 18.8 1 1.6 --

 

72

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Thu 21-Feb -0.9 21.2 10.1 --
Fri 22-Feb 10.3 29.0 19.7 0.01
Sat 23-Feb 3.3 30.5 16.9 --
Sun 24-Feb 10.1 34.6 22.3 --
Mon 25-Feb 23.6 31.1 27.4 --
Tues 26-Feb 13.7 30.8 22.3 --
Wed 27-Feb 8.0 21.1 14.5 --
Thu 28-Feb 10.2 24.1 17.1 --
Fri 29-Feb 15.8 34.2 25.0 0.13
Sat l-Mar 15.8 31.7 23.7 0.01
Sun 2-Mar 23 .0 41.9 32.5 --
Mon 3-Mar 28.5 54.0 41.3 0.09
Tues 4-Mar 20.6 29.0 24.8 --
Wed 5-Mar 18.8 36.6 27.7 0.06
Thu 6-Mar 23.4 35.1 29.2 --
Fri 7-Mar 18.8 25.8 22.3 --
Sat 8-Mar 1 1.2 25.5 18.4 --
Sun 9-Mar 11.7 31.4 21.5 --
Mon lO-Mar 19.3 35.3 27.3 --

 

 

 

 

 

73

 

REFERENCES

1. Chase, K. Firearms: a Global History to 1 700; Cambridge University Press, New
York, New York, 2003.

2. FBI Crime in the United States report.
http://www.fbi.gLov/ucr/ciusr2006/data/table_20.htrnl. (March 2008).

3. Di Maio VJM. Gunshot Wounds: Practical Aspects of Firearms, Ballistics, and
Forensic Techniques. CRC Press, Boca Raton, Florida, 1999.

4. Basu S. Formation of Gunshot Residues. J Forensic Sci, 27 (1982) 72-91.

5. Romolo FS and Margot P. Identification of gunshot residue: a critical review.
Forensic Sci Int, 119 (2001) 195-211.

6. Steinberg M, Leist Y, Goldschmidt P, and Tassa M. Spectrophotometric
determination of nitrites in gunpowder residue on shooters’ hands. J Forensic Sci, 29
(1984) 464-470.

7. Harrison HC and Gilroy R. Firearms discharge residues. J Forensic Sci, 9 (1964)
119-132.

8. Ruch RR, Buchanan JD, Guinn VP, Bellanca SC, and Pinker RH. Neutron activation
analysis in scientiﬁc crime detection. J Forensic Sci, 9 (1964) 119-132.

9. Krishnan SS. Rapid detection of ﬁrearms discharge residues by atomic absorption
and neutron activation analysis. J Forensic Sci, 16 (1971) 144-151.

10. Koons RD, Havekost DG, Peters CA. Analysis of gunshot primer residue collection
swabs using ﬂameless absorption spectrophotometry: a re-examination of extraction
and instrument procedures. J Forensic Sci, 32 (1987) 846-865.

11. Wolten GM, Nesbitt RS, Calloway AR, Loper GL, and Jones PF. Particle Analysis
for the Detection of Gunshot Residue. 1: Scanning Electron Microscopy/Energy
Dispersive X-Ray Characterization of Hand Deposits ﬁom Firing. J Forensic Sci, 24
(1979) 409-422.

12. DeGaetano D, Siegel JA, and Klomparens KL. A Comparison of Three Techniques
for Sampling and Analysis of GSR by SEM/EDX Analysis. J Forensic Sci, 37 (1992)
281-300.

74

l3. Torre C, Mattutino G, Vasino V, and Robino C. A Source ofNon-GSR Particles
Containing Lead, Barium, and Antimony. J Forensic Sci, 47 (2002) 494-504.

14. Koons RD. Analysis of Gunshot Primer Residue Collection Swabs by Inductively
Coupled Plasma- Mass Spectrometry. J Forensic Sci, 43 (1998) 748-754.

15. Santos A, Magalhaes T, Vieira DN, Almeida AA, and Sousa AV. Firing Distance
Estimation Through the Analysis of the Gunshot Residue Deposit Pattern Around the

Bullet Entrance Hole by Inductively Coupled Plasma-Mass Spectrometry: An
Experimental Study. Am J Forensic Med Pathol, 28 (2007) 24-30.

16. Klintean W, Durongkadech P, Minarni T, Ruangyuttikam W, Tohno S, Vichairat K,
Azuma C, Sribanditmongkol P, Yoshiyuki T. Differences in the Element Contents
Between Gunshot Entry Wounds with F uIl-jacketed Bullet and Lead Bullet. Biol
Trace Elem Res 120 (2007) 74-81.

17. Schaeffer L. The Persistence of Gunshot Residue in Decomposing Tissue. MS Thesis,
Michigan State University, 2007.

18. Ohshima T. Forensic wound examination. Forensic Sci Int, 1 13 (2000) 153-164.

19. Firearms Tutorial.
http://librarv.med.utahedu/WebPath/TUTORIAL/GUNS/GUNINJ.htm1. (March

2008)

20. Collins KA and Lantz PE. Interpretation of fatal, multiple, and exiting gunshot
wounds by trauma specialists. J Forensic Sci, 39 (1994) 94-99.

21. Catts EP. Problems in estimating the PM] in death investigations. J Agr Ento, 82
(1992) 1-62.

22. Nuorteva P and Hasanen E. Transfer of mercury ﬁom ﬁshes to in sarcosaprophagous
ﬂies. Ann Zool Penn, 9 (1972) 23-27.

23. Beyer JC, Enos WP, and Stajic M. Drug identification through the analysis of
maggots. J Forensic Sci, 25 (1980) 411-412.

24. Roeterdink EM, Dadour IR, and Watling RJ. Extraction of gunshot residues from the
larvae of the forensically important blowﬂy Calliphora dubia (Macquart) (Diptera:
Calliphoridae). Int J Legal Med, 118 (2004) 63-70.

25. Gunshot Residue Examinations. http://www.ﬁrearm_sid.com/A distanceExams.htm.
(April 2008).

75

26. Practical Pathology of Gunshot Wounds.
http://f1ndarticles.com/D/articles/mi qa3725/is 200609/ai n16717329/pg 5. (April
2008).

27. Haag M and Wolberg G. Scientiﬁc Examination and Comparison of Skin Simulants
for Distance Determinations. AF TE J. 32 (2000) 136-142.

28. F legler SL, Heckman JW, and Klomparens KL. Scanning and Transmission Electron
Microscopy: An Introduction. Oxford University Press, New York, 1993.

29. Lamble KJ and Hill SJ. Microwave digestion procedures for environmental matrices.
Analyst, 123 (1998) 103R-133R.

30. Harris DC. Quantitative Chemical Analysis. W.H. Freeman and Company, New
York, 2003.

3 1. Enviroweather.
http://www.enviroweather.m§u.edu/run.asp?stn=msu&mod=w sum&vr=&mol=&da
1=&m02=&da2=&ds=. (April 2008).

76

 

   

11111111111111111111111111