AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 000:000–000 (2009)
Technical Note: Applicability of Tooth Cementum
Annulation to an Archaeological Population
Mirjana Roksandic,1* Dejana Vlak,2 Michael A. Schillaci,3 and Diana Voicu4
1
Department of Anthropology, University of Winnipeg, Winnipeg, Canada
2
Department of Anthropology, University of Toronto, Toronto, Ontario, Canada
3
Department of Social Sciences, University of Toronto Scarborough, Scarborough, Canada
4
Albion Library, Etobicoke, Ontario, Canada
KEY WORDS age determination; osteology; dental anthropology; histology; taphonomy; tooth
cementum annulations
ABSTRACT The use of tooth cementum annulations
for age determination has been deemed promising,
exhibiting high correlations with chronological age.
Despite its apparent potential, to date, the tooth
cementum annulations method has been used rarely for
estimating ages in archaeological populations. Here we
examine the readability of cementum annulations
and the consistency of age estimates using a sample
of 116 adults from the Iron Gates Gorge Mesolithic/Neo-
Accurate age determination from skeletal and dental
remains is an important goal for biological anthropolo-
gists. Sequential changes during growth and develop-
ment facilitate estimation of biological age in nonadults.
Once growth is over, however, assessing age at death
becomes more problematic as the degenerative process of
aging is variable and influenced by lifestyle and the
environment.
Since the publication of several articles critical of our
ability to accurately assess age in adult skeletons (e.g.,
Bocquet-Appel and Masset, 1982; Jackes, 1992; Wood
et al., 1992), more attention has been paid to improving
the already existing methods of age determination and
developing new ones. One of the techniques deemed
promising utilizes tooth cementum annulations (TCA).
Based on the hypothesis that the annual formation of
one dark and one light incremental line reflects a natu-
ral metabolic rhythm of seasonal changes in diet and
hormonal cycles (Laws, 1952; Grue and Jensen, 1979;
Kay et al., 1984; Geiger, 1993), the TCA method involves
counting lines visible on the accelular cementum band of
thin root crossections. Histological age of the individual
studies is obtained by adding the number of observed
lines to the age at which the tooth erupted (Wittwer-
Backofen et al., 2004).
Originally developed for nonhuman mammalian spe-
cies (e.g., Spinage, 1973; Grue and Jensen, 1979), the
TCA technique was first applied to humans by Stott et
al. (1982). Technical improvements (e.g., Naylor et al.,
1985; Kvaal et al., 1996; Maat et al., 2006) which have
yielded high correlations with chronological age in analy-
ses of known-age samples (e.g., Jankauskas et al., 2001;
Kagerer and Grupe, 2001; Wittwer-Backofen et al., 2004)
led to the suggestion that TCA might be superior to
other methods of age estimation. Despite seemingly
exceptional results, several scholars (Lipsinic et al.,
1986; Lucas and Loh, 1986; Miller et al., 1988; Renz and
C 2009
V WILEY-LISS, INC.
lithic series. Our examination of the method pointed to
several sources of error that call into question the use of
this method for estimating the chronological ages of
archaeologically derived dental samples. The poor per-
formance of the method in our analysis might
be explained by taphonomic influences, including the
effect of chemical and biological agents on dental
microstructures. Am J Phys Anthropol 000:000–000,
2009. V 2009 Wiley-Liss, Inc.
C
Radlanski, 2006) have drawn attention to the method’s
weaknesses.
Given the reported high correlations with chronologi-
cal age, it is surprising that the application of the TCA
method of age determination to past populations has not
been more widely adopted (Großkopf, 1990; Cipriano-
Bechtle et al., 1996; Geusa et al., 1999; Wittwer-Backo-
fen et al., 2008). With the exception of two studies that
have reported problems with the TCA technique applied
to past populations (Geusa et al., 1999; Wittwer-Backo-
fen et al., 2008), all previous studies have assumed the
method’s superior performance for age determination in
adults.
We analyzed the applicability of the TCA method for
estimating age in an archaeological population. Because
we could not establish the correlation between histologi-
cal and chronological age for archaeological specimens,
we concentrated on evaluating inter- and intraobserver
error, and consistency of results obtained by reading
several segments of the same tooth. The influence of
previous experience with the TCA method was also
examined.
Grant sponsors: Max Planck Institute for Demographic Research,
Connaught New Staff Matching Grant, University of Toronto.
*Correspondence to: Mirjana Roksandic, Department of Anthro-
pology, University of Winnipeg, Winnipeg, Canada, MB, R3B 2E9.
E-mail: m.roksandic@uwinnipeg.ca
Received 2 January 2009; accepted 9 June 2009
DOI 10.1002/ajpa.21136
Published online in Wiley InterScience
(www.interscience.wiley.com).
2 M. ROKSANDIC ET AL.
MATERIALS AND METHODS
Our sample consisted of 116 adults from the Iron Gates
Gorge skeletal collection (Serbia), representing both
Mesolithic (Padina, Lepenski Vir, Vlasac, and Hajducka
Vodenica) and Early Neolithic (Velesnica) sites on the
right bank of the Danube dating from 8500 BC to 5500
BC calibrated (Bonsall et al., 2008). Of the 263 adults in
the collection, only 116 had at least one of the monoradi-
cal teeth preserved. One tooth was extracted from each
individual. Each tooth was measured, photographed,
described, and processed at the TCA Laboratory at Max
Planck Institute for Demographic Research (MPIDR) in
Rostock, Germany. Description included archaeological
provenience, tooth type, level of occlusal wear, hyperce-
mentosis, and dental pathology (paradotosis, caries). The
crowns and the upper third of the root were removed
using a microtome. The remaining two thirds of the
tooth root were embedded in a two component epoxy
resin and dried in a vacuum chamber. Three sequential
70–80 lm undecalcified crossections were prepared for
each tooth from the middle third of the root using a
Leica SP 1600 microtome fitted with a diamond coated
blade following the protocols of the MPIDR TCA lab
(Wittwer-Backofen et al., 2004). Unstained sections were
mounted on slides for further analysis. Visual scanning
was done at the laboratory of Paleoethnobotany at
the University of Toronto Mississauga using a Nikon1
LABOPHOT2-POL microscope at 4003 magnification.
Segments that showed readable lines were captured as
JPEG images (1,600/1,200 pixels) with a Nikon DS Cam-
era Control Unit DS-L1 and DS Camera Head DS-5M,
DS Cooled Camera Head DS-5Mc fitted on the micro-
scope. Readings were done on selected segments in
Adobe1 Photoshop1 CS2.
Three observers were involved in the evaluation pro-
cess: one undergraduate student (DiV) with limited lab
experience in biological anthropology (Observer 1), a
graduate student (DeV) with substantial experience with
both forensic and archaeological populations, though no
previous experience with the TCA method (Observer 3),
and the primary author (MR) with previous experience
using the TCA method (Observer 2). Initially, three
segments were selected and evaluated by all three
authors together, allowing the senior author to instruct
the student observers. Subsequent readings were per-
formed independently. To calculate intraobserver error,
segments were scanned and read twice at 2-month inter-
vals after the first reading.
Statistical analyses included an evaluation of interob-
server error for three sets of readings, intraobserver
error for each author, and differences in reading between
different segments of the same tooth. Inter- and intraob-
server error was tested using a normal approximation of
the Wilcoxon sign-rank test of equal medians (Sokal and
Rohlf, 1995). For the tests of intraobserver error, we
looked at the differences between original counts (T1)
and counts taken during two additional data collection
sessions (T2 and T3) by each of the three observers inde-
pendently. To evaluate differences between different seg-
ments of the same tooth we used original counts (T1) for
all three observers, reporting minimum and maximum
difference in line counts for nine teeth with multiple
sections. The odds of teeth being excluded from the anal-
ysis based on the presence of hypercementosis, periodon-
tal disease, dental caries, or occlusal wear were assessed
American Journal of Physical Anthropology
using a bootstrapped odds ratio (OR). Statistical signifi-
cance (i.e., the probability that OR 5 1) was estimated
using a Fisher’s Exact test.
RESULTS
From 116 processed teeth, we selected 40 that had
at least one readable segment of any of the three cros-
sections. On the remaining 76 teeth we could not identify
any sections that exhibited continuously visible lines
from the cemento/dentine junction to the edge of the
root. Each of these 76 teeth contained segments of cros-
sections that were visibly destroyed by diagenesis, seg-
ments with faint to invisible lines, or segments where
lines were interspersed with pits and either vertical and/
or horizontal breaks. In several cases saw marks parallel
to TCAs (n 5 7), and/or breakage of cementum adjacent
to the embedding medium (n 5 6), were observed in seg-
ments where the lines were visible. Out of the remaining
40 teeth for which photographs of selected segments
were obtained, 23 teeth (represented by 40 segments)
were judged by the first author to exhibit lines that were
clear enough to allow readings. These were read by all
three readers at three different times (Table 1). Given
the conservative selection process resulting in a high
number of teeth rejected prior to analysis, we expected
reasonably consistent results.
The odds of teeth being excluded from the analysis
based on the presence of hypercementosis (OR 5 2.901,
P 5 0.096), periodontal disease (OR 5 0.787, P 5 0.834),
dental caries (OR 5 0.365, P 5 0.908), or level of occlu-
sal wear (OR 5 0.460, P 5 0.194) were not significant
statistically. These odds ratio tests suggest that the pres-
ence of dental pathology and attrition have not biased
the study sample. The results of our intraobserver error
tests revealed one significant comparison out of a total of
six comparisons (Table 2). Mean differences between the
readings ranged from 1.333 to 4.8 years, while maximum
difference between readings ranged from 6 to 17 years.
One out of nine of our tests of interobserver error was
significant (Table 3). The results of these observer error
tests indicate that observer error likely has not substan-
tially affected the results of the study.
More than one readable segment was available in only
9 out of the 23 teeth. Comparison of the variation in the
number of lines counted between different segments for
these nine teeth, given in the Table 4, revealed a range
of age estimates of up to 24 years. This level of uncer-
tainty in age estimates is similar to that obtained using
standard macroscopic methods of age determination.
DISCUSSION
In our study a large number of teeth were discarded
(93 teeth, 80.17%) because cementum annulations within
crossections presented clear evidence of advanced diage-
netic process, no visible lines (see Fig. 1), wavy lines
interspersed with ‘‘pits’’ (see Fig. 2), numerous impur-
ities represented by bifurcating lines (see Fig. 3), par-
tially obscured lines (see Fig. 4). Other studies on
archaeological samples have also reported problems with
unreadable segments (Cipriano-Bechtle et al., 1996;
Wittwer-Backofen et al., 2008). The determination of
readability of a tooth segment is necessarily subjective.
Such subjectivity was evident in recent research by
Wittwer-Backofen et al. (2008) where the senior author
rejected 14.1% of the teeth, while the second observer in
 TOOTH CEMENTUM ANNULATION 3
TABLE 1. Reported readings for the 23 scanned and read teeth TABLE 2. Results for the tests of intraobserver error
represented by 40 segments
T1 vs. T2 T1 vs. T3
Observer 1 Observer 2 Observer 3
Observer 1
Slide T1 T2 T3 T1 T2 T3 T1 T2 T3 N 40 40
HV13 12 8 8 8 11 10 13 9 8 Mean difference 1.25 1.325
HV15 42 43 48 32 45 37 37 36 40 Max difference 16 19
LV2 38 38 38 34 38 32 29 30 34 Wilcoxon (Z5) 0.936 0.421
LV4 31 31 35 31 44 28 30 30 33 P-value 0.349 0.674
LV8 34 34 45 42 48 43 47 47 47 Observer 2
LV8 24 27 24 32 33 31 29 26 27 N 40 40
LV8 36 35 38 45 41 40 42 45 40 Mean difference 4.800 4.275
LV8 43 42 43 51 39 42 45 46 45 Max difference 13 13
LV39 40 46 42 60 54 49 41 42 44 Wilcoxon (Z5) 1.442 1.045
LV39 41 44 43 65 59 58 52 52 52 P-value 0.149 0.296
LV39 57 61 57 64 60 56 57 56 55 Observer 3
LV62 43 34 31 38 40 37 32 35 35 N 40 40
LV69 83 67 70 70 78 69 82 89 79 Mean difference 2.550 3.325
LV74 63 58 54 58 60 50 70 58 64 Max difference 12 17
LV83A 45 47 41 55 47 52 42 47 40 Wilcoxon (Z5) 2.324 1.444
LV83A 48 48 48 50 41 38 47 46 43 P-value 0.020 0.149
LV88 49 52 52 48 53 49 53 56 58
A significance level of a 5 0.05 was used for all tests.
LV126 34 36 35 39 39 34 30 34 32
P5A 37 38 38 32 30 44 37 38 34
P5A 37 38 38 31 30 30 34 31 33
TABLE 3. Results for the tests of interobserver error
P12 20 20 20 22 16 23 27 29 29
P12 28 32 29 26 25 25 22 23 26 T1 T2 T3
P12 20 20 21 19 18 22 23 26 23
P18 36 35 40 33 34 33 44 42 40 DiV-MR Z 5 0.841 Z 5 1.791 Z 5 0.094
P18 41 50 45 40 46 40 37 39 37 P 5 0.400 P 5 0.073 P 5 0.925
P24 39 31 29 33 40 40 39 42 38 DiV-DeV Z 5 0.451 Z 5 2.079 Z 5 1.132
VEL2B 37 34 42 29 35 36 40 43 36 P 5 0.652 P 5 0.038 P 5 0.258
VEL2G 32 31 32 28 27 29 30 34 33 MR-DeV Z 5 1.017 Z 5 0.606 Z 5 0.957
VL26 39 39 31 36 36 33 34 37 30 P 5 0.309 P 5 0.544 P 5 0.339
VL26 34 31 30 30 31 33 31 33 35
A significance level of a 5 0.05 was used for all tests.
VL26 41 41 41 40 48 36 37 39 54
VL32 30 29 32 33 27 29 28 32 28
VL45 88 81 83 77 81 76 78 82 77
VL45 64 64 62 76 78 72 82 83 73 TABLE 4. Minimum and maximum difference in line counts
VL45 86 72 67 75 87 88 86 86 83 associated with observations of different tooth sections
VL47 27 26 27 23 29 30 34 36 27 Burial no. Observer 1 Observer 2 Observer 3
VL55 42 28 31 35 38 37 37 40 36
VL55 35 34 34 33 31 27 37 32 32 Lepenski Vir 8 2–19 (4) 3–19 (4) 3–18 (4)
VL55 29 31 29 28 34 34 37 35 33 Lepenski Vir 39 1–17 (3) 1–5 (3) 5–16 (3)
VL55 35 34 34 31 35 31 38 38 32 Lepenski Vir 83A 3 (2) 5 (2) 5 (2)
Padina 5A 0 (2) 1 (2) 3 (2)
Repeat slides indicate more than one segment read per tooth. Padina 12 0–8 (3) 3–7 (3) 1–5 (3)
HV, Hajducka Vodenica; LV, Lepenski Vir; P, Padina; VEL, Padina 18 5 (2) 7 (2) 7 (2)
Velesnica; VL, Vlasac. Vlasac 26 5 (2) 4–10 (3) 3–6 (3)
Vlasac 45 2–24 (3) 1–2 (3) 4–8 (3)
Vlasac 55 0–13 (4) 2–7 (4) 0–1 (4)
the study rejected 22.3% of the specimens as unreadable.
In our study, the decision to discard teeth was made sub- Number of sections is given in parenthesis.
jectively by the senior author on the basis of clarity and
continuity of observable annulations in each crossection. the formation of a mold of an original histological struc-
We suggest that in our archaeological sample poor mi- ture (Retallack, 1984; Henderson, 1987; Francillon-Veil-
croscopic preservation might be one of the contributing liellot et al., 1990; Garland, 1993). Thus, despite the mac-
factors leading to a large number of discarded teeth. It roscopic morphology being maintained, there can be a
has been established that good macroscopic preservation total or partial substitution of the components at a micro-
of skeletal tissue does not necessarily mean good micro- scopic level (Lambert et al., 1979; Francillon-Veilliellot
scopic preservation as well (Schultz, 1997; Pfeiffer, 2000; et al., 1990; Gilber, 1997; Gill-King, 1997).
Nonato do Rosario Marinho et al., 2006). Chemistry (e.g., We suggest that cementum, like bone, is subject to
ground water ionic exchange and mineral deposition) and/ these same diagenic processes. Recent research analyz-
or postmortem biological activity (e.g., bacteria and fungi) ing animal teeth from an archaeological context by Stutz
can alter microscopic tissue, yielding cloudy, incomplete, (2002: p 1343) has demonstrated that ‘‘chemical diage-
or structurally altered histological images (Pfeiffer, 2000). netic processes of collagen leaching and apatite recrys-
While water and microorganisms decompose the organic tallization can create bands that mimic true biogenetic
components of bone (Henderson, 1987; Heuck, 1993), the growth increments in archaeological cementum.’’
mineral portion undergoes intense chemical action and Our inability to get consistent results represents a
degradation by microorganisms, which facilitates the major problem in terms of applicability of the method to
impregnation by minerals such as CaCO3, Fe2O3, and archaeological populations. Similar to the findings by
SiO2, leached from the soil. In many cases this results in Renz and Radlinski (2006), we recognized problems in

American Journal of Physical Anthropology

4 M. ROKSANDIC ET AL.
Fig. 1. A segment with no clearly visible lines and substan-
tial diagenic? destruction. The few faint lines can not be fol-
lowed throughout the segment.
Fig. 2. Wavy lines (a) interspersed with ‘‘pits’’ (b). The por-
tion closer to the dentine is not showing any clear and consist-
ent lines (c); the upper portion of the segment shows lines (a)
interspersed with pits (b) and vertical breaks (d).
visually identifying a number of TCA lines: the first and
the last, faint, bifurcating, inconsistent, and other prob-
lematic lines. Because previous experience does not seem
to play a significant role, we suggest that the authors
American Journal of Physical Anthropology
Fig. 3. A segment showing a number of bifurcating lines of
unclear origin (indicated by letters a, b, c, and d). These lines
appear as bands of varying width that obscure the flow of the
annulations.
Fig. 4. The portion of the segment closer to the dentine (a)
shows clear regular lines, the top part (b) is much more difficult
to read as lines are not clear and are at times overlapping.
who claim high performance and high reliability of the
method should provide a much more thorough descrip-
tion of the expert knowledge guiding their decision to
count or discard problematic or ambiguous lines.
Our strongest reservation regarding the method stems
from readings obtained from teeth where more than one
segment was available. A range of age estimates of up to
24 years is comparable to ranges obtained by other
methods, and while certainly useful where no other
observations are possible, the method does not warrant
destruction of archaeological material where other
means of age assessment are available.
CONCLUSION
The results of our application of the TCA method to an
archaeologically derived dental sample pointed to several
 TOOTH CEMENTUM ANNULATION
sources of error which might be related to taphonomic
changes at the microscopic level. What is troubling is
the fact that even when individual segments produced
consistent results between observers, differences
between readable segments of one tooth were substan-
tial. When all ages obtained through the reading of mul-
tiple crossections were taken into consideration, the
range of age estimates was similar to that which can be
obtained by standard macroscopic osteological methods.
The destruction of archaeological specimens for TCA age
determination, therefore, may not be warranted until
the biology and diagenic processes influencing cementum
annulations are fully understood. We suggest that future
research examine the effects of taphonomy on dental his-
tological structures. Such research is needed before the
TCA method can be applied to—especially rare and old—
archaeological samples.
ACKNOWLEDGMENTS
The authors thank Prof. G. Crawford at the University
of Toronto in Mississauga for use of his laboratory for
processing the samples and the Faculty of Philosophy at
the University of Belgrade (Serbia) for access to the skel-
etal material used in our analysis. We also wish to thank
the anonymous reviewers for their thoughtful and con-
structive comments.
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