Journal of Archaeological Science (1995) 22, 115-l 19

Geochemical Criteria for Thermal Alteration of Bone

R. E. Taylor
Radiocarbon Laboratory, Department of Anthropology, Institute of Geophysics and Planetary Physics,
University of California, Riverside, CA 92521-0418, U.S.A.

P. E. Hare
Geophysical Laboratory, Carnegie Institution of Washington, 5251 Broad Branch Road, N. W., Washington,
DC20015-1305, U.S.A.

T. D. Whitk
Department of Anthropology, University of California, Berkeley, CA 94720, U.S. A.

(Received 7 December 1993, revised manuscript accepted 31 March 1994)

In bones not influenced by other diagenetic effects, heating events can be reflected in changes in the relative
concentrations of constituent amino acids that alter the collagen-like pattern in bone to a non-collagen-like pattern with
an increase in the relative NH, levels. The ratio of glycine (Gly) to glutamic acid (Glu) can be used as an index of a
collagen-like or non-collagen-like pattern. For a group of eight bone samples from a southwestern United States Pueblo
II period Anasazi site dated to about AD 1100, amino acid composition, as characterized by Gly/Glu values, along with
NH, data, clearly indicated which bone fragments had been subjected to one or more heating events. The use of
Gly/Glu and NH, values for the purpose of inferring heating events may not be accurate for bones which have
experienced other significant diagenetic effects not related to heating.

Keywords: BONE, AMINO ACIDS, GEOCHEMISTRY, BIOGEOCHEMISTRY, HEATING, FIRE, THERMAL

EFFECTS, GLYCINE, GLUTAMIC ACID, AMMONIA, COLLAGEN. DIAGENESIS.

Introduction unambiguous indication that a bone had been sub-

jected to some type of a heating event, since heating
ver the last two decades, various types of bio-

0 geochemical analysis of bone obtained from

archaeological or palaeoanthropological
texts have been employed in efforts to make chrono-
con-
has been postulated as one factor responsible for
anomalous D/L ratios in fossil bone (Bada, 1985).

logical, palaeoenvironmental, and palaeodietary
inferences (Price, 1989). Amino acid biogeochemistry
has been employed in attempts to distinguish diagenetic
from temporal or phylogenetic alteratious (Hare, 1980;
Hare, Hoering & King, 1980). For example, significant
improvements in the potential use of amino acid race-
mization to provide chronometric placement for fossil
bone resulted from the recognition of the importance of
the chemical state of amino acids on measured D/L
ratios (Hare, 1969; Bada 1985; Taylor et al., 1989).
This study derives, in part, from an iuterest by one of
us (TDW) in ascertaining if heating rather than some
other biogeochemical effect could be objectively docu-
mented for human bone suspected of having been
roasted in the course of being prepared as a food item
(White, 1992). Also, one of us (RET) wished to deter-
mine if any biogeochemical signal could provide an
Previous Approaches
Various approaches to quantifying the physical and
chemical processes involved in heat-altered bone have
been previously reported. Attempts to validate cate-
gories based on coloration have been widely employed.
For example, using thermogravimetry, differential
thermal analysis, X-ray diffraction (XRD) and scan-
ning electron microscopy (SEM), Bonucci & Graziani
(1975) examined various physical effects that could be
correlated with bone colour (“white”, “ochraceous/
black”, “black-grayish”, and “white-grayish”) on
fossil, subfossil (4th/5th centuries BC) and known
temperature heat-treated modern bone. They con-
cluded that changes in bone colour could indeed be
used to deduce approximate ( f 50°C) temperatures to
which a bone had been exposed in the range from
200-650°C.

0305~4403/95/010115+05 $08.00/O 0 1995 Academic Press Limited

116 R. E. Taylor, P. E. Hare and T. D. White
Based on macro- and microscopic analysis, XRD,
and SEM studies on known-temperature heat-
treated modem bone (and teeth), Shipman, Foster &
Schoeninger (1984) argued that colour alone is an
imprecise criterion. This is, in part, because burnt
bones may change colour in a post-depositional envi-
ronment depending on trace elements in the containing
sediment and, as noted by Susini, Baud & Tochon-
Danguy (1988), differential soil moisture content. Also,
since meat insulates bones during heating, preparation
of meat with fire may not, in some cases, have raised
bone temperature to a sufficient level to be detected.
Brain & Sillen (1988) examined carbon/nitrogen (C/N)
ratios on a series of known temperature heat-treated
modem bones and fossil bones whose surface was
obscured by heavy CaCO, and MnO, encrustations.
They concluded that C/N criteria on fossil bone can
be used to distinguish black discoloration due to
heat treatment from that due to the incorporation of
MnO,.
Based on an inspection of a selection of archaeologi-
cal site reports on tile at the Eastern Archaeological
Information Center, a repository of archaeological
field cultural resource management reports filed for a
three-county region of interior southern California,
colour criteria alone were employed to identify
“burned bone”. The use of criteria based on analytical
data-such as that reported in the previous
paragraph-was absent in all of the site reports exam-
ined. No attempt was made to distinguish between
food processing-related (e.g. roasting, cooking or heat-
ing) and discard-related heating, such as damage by
direct exposure to a heat source that would occur as
the result of a cremation.
In other sources consulted, gross macroscopic sur-
face characteristics, such as colour, cracking and warp-
ing, and degree of calcination, are typically employed
as the criteria to characterize the degree of exposure to
a heat source or lack thereof. For example, typical
categories employed include “carbonized”, “calcined”,
“smoked” and “unburned” (Baby, 1954; Buikstra &
Goldstein, 1973). The most widely-employed criteria
used for burning (carbonization/“charring”, calci-
nation, discoloration) are, in most cases, confined to
relatively small regions of most bones. We suggest that
the incidence of thermally-altered bone is probably
under-reported in general archaeological literature and
non-calcined burned specimens are probably most
commonly labeled as “weathered”.
Materials and Methods
For our studies, we employed bone samples obtained
from a Pueblo II period Anasazi site dated at about
AD 1100 in Mancos Canyon (5MTUMR-2346),
Colorado (White, 1992). The site is situated as a low
mound on the first terrace above the present floodplain
of the Mancos River, approximately 10 km south of
Table 1. G!,JGIII rind NH, wlrre~ of urrburned and burned bone from
Mancos SMTUMR-2346
Act. Thermally-erected GlylGlu
number character* ratio NH, (“W
Tibia1 fragment
1@t‘k3 unburned 4.23 441
1044b burned 1.26 37.11
Occipital fragment
I762a unburned 4.38 4.83
I762b burned 1.32 14.32
*Evaluation based on visual inspection by TDW.
the southern boundary of Mesa Verde National Park.
Modem site cover is primarily sagebrush, grass and
cactus, with juniper and pinyon pine on adjacent rocky
slopes. The soil at the site is well-drained, a mixture
of locally derived sandstone-based colluvium, water-
deposited gravels and sandy loam, and yellow loess-
like soil of more uniform grain size. The bone samples
were derived from pueblo floor and fill deposits buried
at least 1 m below the modem ground surface
(Nordby, 1974). As summarized by Nickens (198 l), the
region has a cold, mid-latitude, semi-arid climate, with
200-500 mm of annual precipitation that is distributed
seasonally, and with a mean annual temperature of
about 10°C on the tops of adjacent mesas. Tempera-
ture in Mancos Canyon is much warmer than on the
mesa tops in summer months. While the annual pre-
cipitation fluctuates greatly, the annual figure averages
about 200 mm. The in situ bone assemblage would
have been subject to seasonal temperature and
moisture variations. However, the bones would have
been buffered from these seasonal changes by the
over-burden. Unfortunately, subsurface data on
temperature and moisture are not available.
Previous unpublished studies of fossil bone biochem-
istry suggested to one of us (PEH) that bones exposed
to open fire heating were characterized by a significant
increase in the relative amount of ammonia (NH,) as
compared to normal diagenetic reactions in the pres-
ence of water or water vapour. In addition, collagen in
heated bone is altered and leached preferentially so
that the amino acid pattern in heated bone tends to
lose its collagen-like amino acid pattern.
Two sets of bone fragments were prepared by one of
us (TDW) to test this hypothesis. A tibia1 fragment
(1044) and an occipital fragment (1762) from
5MTUMR-2346 were selected as controls. Both speci-
mens had adjacent segments of unburned and burned
bone identified by TDW on the basis of characteristic
surface discolouration. Two chips, one burned and one
unburned, were sampled from each specimen at lo-
cations separated by 1 cm (Table 1). The burned and
unburned samples in both cases were identified to the
collaborators (RET and PEH). A second set of eight
bone samples was obtained from six other cranial and
postcranial fragments (Table 2). On the basis of visual
 Geochemical Criteria for Thermal Alteration of Bone 117

Visual GlylGlu Analytical

Context* characterizationt ratio NH, (U/(I) characterization

El unburned 3.63 2.40 unburned

B8a burned 2.20 20.49 burned
B8b burned 2.52 23.11 burned
B8c burned 3.08 16.84 burned
B9 unburned 3.96 3.42 unburned
FA3 burned I.51 12.91 burned
FS19 unburned 4.33 3.53 unburned
FS30 unburned 4.18 3.15 unburned

*Site SMTUMR - 2346 context designations: B=“burial”; FA=“floor artefact”; FS=“floor sample”.
tEvaluation based on visual characterization by TDW.

inspection of the areas from which the samples were

taken, each bone fragment was characterized by TDW
as to whether it had been subjected to heating. The
identification of “burned” or “unburned” on these
small fragments was made by TDW using macroscopic
criteria of discoloration, cracking, exfoliation, and
opacity; only one specimen had evidence of carboniz-
ation, and no fragment was large enough to attribute it
to a specific skeletal element beyond “postcranial” or
“cranial vault”. The collaborators (RET and PEH) 0.050 -
8
were not informed which samples in the second set 0
were deemed by TDW to have been thermally-altered
until after the analytical data measurements had been
obtained and reported to TDW.
0.010:
Amino acid composition and NH, values were ob-
tained on each bone fragment using ion-exchange 0.005 -
liquid chromatography with post-column derivatiz-
ation using OPA (o-phthaldialdehyde), and fluores-
cence detection (Benson & Hare, 1975). The system l
used was optimized to detect very low levels of amino
acids but did not detect hydroxyproline. Bone samples 0
0~00kidiT~ 310 &I 510 /
were dissolved in 6 N HCl and hydrolyzed at 150°C for Gly/Glu
20 min. After evaporation of the HCl, samples were
dissolved in dilute HCl (pH=2), centrifuged, filtered, Figure I. Plot of relationship of amino acid nitrogen content (N,%)
and ratio of glycine and glutamic acid (GlylGlu) in total hydrolysate
and injected on an ion-exchange column. Amino acid of bones from Unit 8 at CA-LAn-43, the Encino Village site (taken
and ammonia concentrations were determined by from Taylor et al., 1989).
comparison to standard amino acid mixtures.
Table 1 summarizes data obtained from the amino
acid composition and NH, values on the first set of Gly/Glu values below 3 indicate bone samples in which
bone samples where unburned and burned portions of the amino acid nitrogen content has dropped below
the same bone were measured. There was a significant 0.1%. This typically is characterized as a “non-
increase in the relative NH, values in both of the collagen-like profile”. Using the data from Table 1, we
burned bone samples examined. In addition, there was have plotted in Figure 2 the NH, and GlylGlu values
a significant alteration in the amino acid profile. The of the two non-burned (open square [1044a] and open
most characteristic change is reflected in the ratio circle [1762a]) and two burned segments (solid square
between glycine (Gly) and glutamic acid (Glu). The [1044b] and solid circle [1762b]) of the two bone
Gly/Glu values can be used as one means of expressing samples. Collagen and unheated modem bone typically
the degree to which a “collagen-like profile” is or is not exhibit Gly/Glu values above 3.5 and NH, values of
present in a bone. less than 5%.
Figure 1 illustrates the relationship between the Table 2 summarizes results obtained on eight bone
amino acid nitrogen content (N,%) and ratio of glycine samples supplied by TDW. After visual inspection of
and glutamic acid in a series of late Holocene bone the portions of the bone from which the samples were
samples (Taylor et al., 1989). With two exceptions, obtained, TDW characterized whether, based on his
118 R. E. Taylor, P. E. Hare and T. D. White
Gly/Glu
Figure 2. Plot of GlylGlu and NH, values for burned and unburned
bones from SMTUMR-2346, Mancos Canyon, Colorado,
from Tables I and 2. Squares designate tibia1 fragments
circles indicate occipital fragments (I 762). Solid symbols represent
known burned samples; open symbols represent known
samples (Table I). Crosses indicate bone samples on which GlylGlu
and NH, values were obtained to identify thermally-altered
(Table 2).
visual criteria, a sample had been subjected to heating:
these characterizations are shown in Table 2. The
Gly/Glu and NH, values for the eight samples pro-
vided by TDW and, for each bone, the analytical
characterization of the bone made by RET and PEH
based on the “collagen-like/non-collagen like” GlylGlu
values and the amount of NH, exhibited in each
sample are also shown in Table 2. When PEH and
RET made their “analytical characterization” of each
sample, they had not been informed of the “visual
characterization” by TDW.
Results and Discussion
For comparison with the data in Table 1, we have
plotted the results summarized in Table 2 on Figure 2.
From Table 2, we note that, in all cases, the visual
characterizations by TDW and the analytical charac-
terizations by RET/PEH based on the Gly/Glu values
and NH, criteria are in agreement. Samples exhibiting
the collagen-like/relatively low NH, characteristics of
unburned bone can be seen to be tightly clustered in
Figure 2, while the non-collagen-like, relatively higher
NH, type bone samples exhibit greater variability in
both of these parameters. This variability may reflect
variation in the length of exposure to and/or higher
effective temperature experienced by the different bone
samples. Variation in the amount of available moisture
during the period(s) of exposure to heating may also be
a factor, since water is a critical element in protein
diagenesis (Hare, 1980).
In our view, it is not currently possible to distinguish
heating events from other biogeochemical effects in
bones subjected to long-term diagenetic processes
using amino acid compositional criteria. In the suite of
samples examined in this study, the inference that all
bones would retain their collagen composition unless
they had been heated could be reasonably assumed in
light of the known environmental conditions, age of
the site, and spatial proximity of the skeletal fragments
in the deposit (White, 1992). This assumption is most
likely to be violated in older bones. As a possible
approach to distinguishing long-term diagenetic from
specific heating effects, we might note that other reac-
tions such as racemization are affected by heating and
the relative in situ water content of the bone. In cases
where the heat has driven off the in situ water in the
bone, racemization reactions essentially stop. In heat-
ing experiments in the presence of water, the racemiz-
ation reactions are accelerated. In addition, other
reactions such as decarboxylation and deamination
taken
(1044);
appear to be independent of water content. Laboratory
studies are currently underway to correlate these vari-
unburned ous reactions in bone with its thermal history to enable
researchers to separate out effects due to heating from
bone those due to other diagenetic factors.
Conclusions
In cases where long-term diagenetic effects have not
altered a bone, it appears that GlylGlu and NH,
criteria can provide a valuable objective basis on which
to infer whether a bone sample has been subjected to
one or more heating events. Using the criteria we have
examined, there are not suflicient data to characterize
definitively the severity of the heating events with
respect to either duration or maximum temperature
and to distinguish heating effects from other longer-
term diagenetic effects such as those which would be
exhibited in some Pleistocene age bones.
Acknowledgements
The studies reported in this paper were supported, in
part, by the National Science Foundation, BNS-
9119958 (Anthropology [Archaeometry] Program) and
the Gabrielle 0. Vierra Memorial Fund to RET.
Additional support by the University of California/
Lawrence Livermore National Laboratory (UC/
LLNL), the Intermural Research Fund and the College
of Humanities and Social Sciences, University of
California, Riverside is gratefully acknowledged. An
earlier version of this paper was presented at the 28th
International Symposium on Archaeometry, 24 March
1992, held at the University of California, Los Angeles.
We appreciate the comments of Richard Burky and
Philip Wilke on a draft of this paper. This is contribu-
tion 93113 of the Institute of Geophysics and Planetary
Physics, University of California, Riverside.
 Geochemical Criteria for Thermal Alteration of Bone 119

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