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AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques
Nuclear Instruments and Methods in Physics Research B xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques C. Solis a,⇑, E. Solis-Meza a, M.E. Morales a, M. Rodriguez-Ceja a, M.A. Martínez-Carrillo b, D. Garcia-Calderon a, A. Huerta a, E. Chávez a a b
Instituto de Física Universidad Nacional Autónoma de México, Avenida Universidad 3000, Circuito de la Investigación Científica S/N Ciudad Universitaria, 04510, Mexico Facultad de Ciencias Universidad Nacional Autónoma de México, Avenida Universidad 3000, Circuito de la Investigación Científica S/N Ciudad Universitaria, 04510, Mexico
a r t i c l e
i n f o
Article history: Received 5 August 2016 Received in revised form 31 December 2016 Accepted 28 February 2017 Available online xxxx Keywords: Radiocarbon Bomb-pulse dating Carbon-14 Forensics
a b s t r a c t AMS-14C analysis of modern teeth has become important for forensic studies. 14C content in human teeth reflects the 14C atmospheric concentration during its formation and allows the calculation of the actual year of birth. Through AMS, it is possible to measure the 14C concentrations in a tissue with high precision. However, there is a debate about which should be the best fraction for teeth carbon dating: collagen or enamel. This work focuses on the results obtained from enamel and collagen extracted from Mexican people in order to compare them. Collagen from dental pieces donated from people older than 60-yearsold have been included to understand the turnover process and usefulness of collagen to determine the date of birth. Our results indicate that when a single dental piece is available, enamel method allows the determination of the tooth formation date. Dating collagen of the same tooth helps to discriminate if the formation date belongs to the left or the right side of the peak bomb, but also corroborates, the ages obtained through enamel analysis. Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction In Mexico, research in any of the forensic disciplines is extremely scarce. For this reason, forensic experts have frequently resorted to scientific knowledge generated abroad. However, this situation is changing and there is an increasing interest to apply the scientific methods available in Mexico to the study of materials found in criminal scenes, with the aim of having multidisciplinary tools to complement the expert studies and so contribute to the procurement and administration of justice in the prevention of offenses. Analysis of 14C content in human tissues such as teeth and bones has proved to be a useful way to establish the time elapsed since individual’s birth and death. In forensic cases, the usefulness of modern tissues 14C dating comes from the fact that 14C is incorporated into tissues through metabolism. This exchange with the atmosphere stops when the organism dies, so 14C contents within the tissue, reflects 14C level that existed in the atmosphere [1,2]. Cosmic ray interactions with atmospheric 14N produces 14C, which oxidizes to CO2 and distributes throughout the planet. Before 1950, the 14C content in the atmosphere was more or less ⇑ Corresponding author. E-mail address: [email protected] (C. Solis).
stable [3]. During the so-called nuclear era, atmospheric 14C content increased abruptly due to atmospheric nuclear weapon testing during the 50s and early 60s. From the 1963 peak, after the nuclear test banning, 14CO2 levels have slowly decreased mainly due to exchange with carbon in the oceans and the biosphere [4]. These changes in atmospheric 14CO2 levels are reflected in organisms born after 1950, who assimilate 14C through food chain. Their tissues, which assimilate at different rates, reflect the atmospheric 14 CO2 levels, at the time of formation, being then possible to determine their formation date through 14C content. Forensic application is based on the 14C/12C ratio (normalized to a 13C value) which reflects the atmospheric isotopic ratio at the time when these tissues were formed. This method is called bomb/pulse dating [5]. Teeth are formed by organic and inorganic fractions. Inorganic teeth enamel contains approximately 2–3% of carbon while organic fraction, mainly collagen, has 30% of carbon. Because of their hardness and strength, teeth are frequently the unique useful remaining body parts to determine the date when a person was born. This can be an important step to establish the identity of victims that are classified as unknown because a lack of any other information. Forensic studies have been focused on determine, through the analysis of 14C in different dental pieces, the year-of-birth of an individual [6–10]. Analysis with Accelerator Mass Spectrometry
http://dx.doi.org/10.1016/j.nimb.2017.02.085 0168-583X/Ó 2017 Elsevier B.V. All rights reserved.
Please cite this article in press as: C. Solis et al., AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques, Nucl. Instr. Meth. B (2017), http://dx.doi.org/10.1016/j.nimb.2017.02.085
2
C. Solis et al. / Nuclear Instruments and Methods in Physics Research B xxx (2017) xxx–xxx
(AMS) is the ideal technique for teeth because of its high precision and the possibility to measure this radionuclide in milligram or less-sized samples [11]. With the AMS facility (LEMA) installed in 2013 at the Physics Institute of the UNAM, it is now possible to perform high precision analyses of radionuclides [12]. Each dental piece has different development periods: crown dentine forms after the enamel and root dentine forms after crown dentine. The time span of the crown and root formation process is well known for each tooth, by what is a good candidate for determine the date of birth of a person [13,14]. How close is the 14C contents in teeth related to that of the atmosphere, depends on the tooth type, if the formation was completed before or after the peak bomb, among other factors. However, there is a debate about which should be the best fraction to be used for dating: collagen or enamel. Several studies show that, after being formed, enamel is not replaced and 14C content is close to the atmospheric level at the time of crown formation [2,7–10]. Dentine collagen has also been used in a number of studies, since it accounts for a greater amount of carbon than tooth enamel. It also helps in distinguishing the side of the peak bomb where the sample belongs. Nonetheless, collagen’s use to determine the year-of-birth seems to be controversial. Some authors base the use of collagen in the fact that racemization experiments have shown that there is no turnover [2,7,15]. However, some others have observed that collagen’s carbon continues to recycle even when the tooth is already complete [9,16]. Wang et al. declare that collagen could capture 14C even until the individual’s death. The aim of this work is to compare the dates coming from enamel and collagen analysis. With this purpose in mind, collagen or enamel was extracted from a set of dental pieces from individuals of known ages, in order to calculate the year of complete development. For enamel and collagen extraction from a single piece, third molars were chosen because their high carbon yield. A sample set of collagen from dental pieces donated from people older than 60-years-old whose teeth were extracted between 2014 and 2015, have been included to understand the turnover process and usefulness of collagen to determine the date of birth. 2. Material and methods All dental pieces were supplied by a dentist and were extracted between 2014 and 2016. They belonged to Mexicans living in Mexico City and San Luis Potosi, two urban cities. Four teeth were chosen for enamel and collagen extraction, nine for collagen and seven for enamel. Each tooth was cleaned with distilled water in an ultrasonic bath and dried at 70 C for 72 h. Four third molars were chosen for collagen and enamel extraction. Each molar was cut away lengthwise; one-half for enamel and the other half for collagen extraction. Collagen from crown and root was extracted from one tooth half. The other half was cut at the cervical line in order to
Fig. 1. Diagram showing the teeth fractions used: a) Whole tooth b) One half was used for collagen extraction; c) the crown of the other half was cut at the cervical line in order to extract the enamel.
extract the enamel from the crown (Fig. 1). Enamel was obtained by hydrolysis [7]. Every crown fraction was treated with 7 mL of 10 M NaOH for one week at 50 C. The sample was placed in a sonic bath at 50 C, NaOH was replaced every day before the sonic bath. After that week the samples were washed at least three times in 0.25 M HCl followed by several rinses with deionized water until pH = 7. Dentine and other tissues were removed by blunt dissection. Collagen was extracted with a Longin modified method with ultrafiltration. The corresponding half was pulverized by the mortar and pestle. Samples were demineralized with HCl (0.5 M) at 5 C temperature over 24 h, then, rinsed 3 times. A further acid wash was carried out with HCl (0.2 M), at 76 C overnight to obtain the gelatin. The supernatant was ultra-filtered and centrifuged and the fraction higher than 30KD (soluble collagen) was lyophilized [17]. Collagen samples containing 1 mg of C were converted to graphite in Automated Graphitization Equipment (AGE III Ion Plus), using a Peltier cooler ( 5 C) to retain the water produced in the reaction. Samples were processed for enamel extraction using a Carbonate Handling System (CHS Ion Plus), each sample reacted with orthophosphoric acid to obtain CO2 which was graphitized in the AGE III. AMS 14C analyses of graphite pressed in Al cathodes were performed in the HVEE LEMA-AMS system. Oxalic acid standard (NIST SRM4990C Oxalic Acid II) was employed for normalization and blanks (Phthallic acid (C8H6O4) and marble (IAEA-C1) with no 14C, were also processed and subtracted to correct for background. Graphite obtained from Phthalic acid gives a F14C close to 0.0030 ± 0.0001. Obtained 14C/12C ratios were converted to 14C ages using a computer code developed at LEMA [11]. Calibrated ages were obtained with CALIBomb computer code, and the curve IntCal 13 and NHZ2 [18–20]. The 14C results are expressed as a fraction modern carbon (F14C ± 2r) [21]. In routine operation the normal precision obtained at LEMA for 1 mg of modern carbon is around 0.3%.
Table 1 Fraction modern carbon values in collagen and crown enamel from 4 third molars, calibrated date of formation ranges. Calculations were performed using CALIBomb. LEMA ID 273 273 223 223 269 269 399 399 +
Collagen Enamel Collagen Enamel Collagen Enamel Collagen Enamel
Year of birth
AFY+
F14C
Calibrated date of formation range (2r)
CFY++
1936 1936 1944 1944 1995 1995 1999 1999
1958 ± 4 1950 ± 2 1966 ± 4 1958 ± 2 2014 ± 4 2009 ± 2 2021 ± 4 2013 ± 2
1.107 ± 0.004 0.975 ± 0.003 1.438 ± 0.007 1.177 ± 0.005 1.044 ± 0.005 1.030 ± 0.006 1.042 ± 0.004 1.046 ± 0.004
1957.8–1958.2 1934–1954 1973.0–1974.9 1958.4–1959.1 2009.5 2009.4–2009.5 2009.5 2009.5
1958.0 ± 0.2 1944 ± 10 1973.9 ± 0.9 1958.8 ± 0.4 2009.5 2009.43 ± 0.03 2009.5 2009.5
AFY-CFY 0.5 6 8.4 0.8 0.4
AFY: Actual formation year. CFY: Calculated formation year.
++
Please cite this article in press as: C. Solis et al., AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques, Nucl. Instr. Meth. B (2017), http://dx.doi.org/10.1016/j.nimb.2017.02.085
3
C. Solis et al. / Nuclear Instruments and Methods in Physics Research B xxx (2017) xxx–xxx Table 2 Enamel and root complete development ages for each tooth. Taken from Schour [13]. Upper teeth
Central incisor Lateral incisor Canine First premolar Second premolar First molar Second molar Third Molar
Lower teeth
Enamel
Root
Enamel
Root
4–5 4–5 6–7 5–6 6–7 2.5–3 7–8 12–16
10 11 13–15 12–13 12–14 9–10 14–16 18–25
4–5 4–5 6–7 5–6 6–7 2.5–3 7–8 12–16
9 10 12–14 12–13 13–14 9–10 14–15 18–25
3. Results Table 1 shows the comparison of AMS-14C results obtained with enamel and collagen extracted from third molars. Two pieces correspond to the left and two pieces correspond to the right side of the peak bomb. Levels of 14C are shown as fraction of modern carbon (F14C). The actual formation year (AFY) was calculated from year-of-birth (YOB) plus the tooth formation time (TF) from Schour et al. [13] shown in Table 2. Calibrated years were obtained from F14C by means of CALIBomb computer code using the data set corresponding to the Northern Atmospheric 14C Levels (Zone 2) from 1950 to 2009 AD. The 14C fixation in teeth was assumed to occur at the end of the tooth development. From the intervals obtained by calibration of the fraction of modern carbon, only the dates close to YOB are shown in Table 1. The last column indicates the difference between actual enamel or dentine formation year and the mean value of the calculated range. Results show that in general collagen and enamel content reproduce the elapsed time for the complete development of each dental pieces. In samples with a formation time before AD 1950 precision is lower because the slight change in atmospheric 14C levels, this may cause greater errors than in samples after AD 1950 where atmospheric 14C dramatically changed due to all ground level nuclear bomb tests. This is the case for enamel from sample 273 in Table 1, formed before 1950 which may explain the uncertainty. Between enamel and col-
lagen from third molars, there is an expected difference of 6– 9 years for complete development periods. This difference is confirmed in Table 1. Dating collagen of the same tooth helps to discriminate if the formation date belongs to the upward or downward side of the peak bomb, but also corroborates, the ages obtained through enamel analysis. For those teeth whose formation year falls after 2009, the F14C values cannot be distinguished, since calibration curve used by the CALIBomb computer code has a data set until 2009. Table 3 shows the AMS-14C results obtained for collagen extracted from dentine from the whole tooth. Replicates of same samples shows reproducible values. The obtained results allow to predict the formation year within 3.8 years. It is worthy to notice that all examined teeth were extracted between 2014 and 2016. In people older than 60 years old, extraction occurred many years after teeth completed their formation. Extrapolation of the calibration curve predicts a F14C value of 0.99 for 2014 and 2015. If there was a replacement in the collagen of the third molars as it has been suggested before [9], a F14C value close to 0.99 would be expected for these dental pieces. However, this is not observed, and even
Fig. 2. Comparison of calculated formation time for collagen (values shown in Table 3) and enamel (values from Tables 1 and 3) with actual formation time. A a one to one correspondence line representing an ideal correspondence between actual formation time and measured formation time is also shown.
Table 3 Fraction modern carbon values in root collagen from 12 individual teeth and enamel from 7 individuals. Calibrated date of formation ranges (SM: second molar, FP: first premolar, FM: first molar, TM: third molar, C: central, CI: central incisor, C: canine, U: upper, L: lower). All teeth were extracted between 2014 and 2015. Calculations were performed using CALIBomb. LEMA ID
Year of birth
Tooth type
AFY+
F14C
Calibrated formation date range (2r)
CFY++
AFY-CFY
272.1.1 219.1.1 219.1.2 274.1.2 218.1.1 218.1.2 268.1.1 220.1.1 220.1.2 221.1.1 275.1.2 271.1.1
Collagen 1939 1942 1942 1943 1944 1944 1944 1949 1949 1952 1965 1974
SM-L SM-U SM-U SM-U FP-L FP-L SM-U FM-L FM-L FM-L SM-U TM-U
1954 ± 1 1957 ± 1 1957 ± 1 1958 + 1 1957 ± 1 1957 ± 1 1959 ± 1 1959 ± 1 1959 ± 1 1962 ± 1 1980 ± 1 1996 ± 4
1.124 ± 0.004 1.108 ± 0.005 1.119 ± 0.007 1.126 ± 0.003 1.002 ± 0.005 0.999 ± 0.005 1.086 ± 0.005 0.993 ± 0.006 0.997 ± 0.007 1.020 ± 0.006 1.288 ± 0.004 1.205 ± 0.005
1957.9–1958.6 1957.7–1958.3 1957.8–1958.6 1958.0–1958.6 1954.6–1956.0 1955.0–1956.0 1957.5–1958.1 1953.0–1956.0 1953.0–1956.0 1955.5–1956.4 1979.1–1980.8 1983.9–1987.7
1958.2 ± 0.3 1958.0 ± 0.3 1958.2 ± 0.4 1958.3 ± 0.3 1955.3 ± 0.7 1955.5 ± 0.5 1957.8 ± 0.3 1954.5 ± 1.5 1954.5 ± 1.5 1955.9 ± 0.4 1980.0 ± 0.9 1985.8 ± 1.9
4.7 1.0 1.2 0.3 1.2 1.0 1.2 4.0 4.0 5.6 0 9.7
637.2.1 552.2.2 556.2.2 595.2.1 596.2.1 373.2.1 374.2.1
Enamel 1964 1991 1991 1993 1993 2002 2002
CI-L TM TM C-U C-U FP-U FP-U
1968.5 ± 0.5 2005 ± 2 2005 ± 2 1999.5 ± 0.5 1999.5 ± 0.5 2007.5 ± 0.5 2007.5 ± 0.5
1.575 ± 0.007 1.089 ± 0.004 1.079 ± 0.004 1.098 ± 0.004 1.095 ± 0.004 1.049 ± 0.003 1.054 ± 0.003
1967.7–1969.9 2000.9–2004.7 2001.0–2005.0 1997.1–2001.1 1998.1–2001.9 2007.9–2009.5 2006.9–2009.5
1968.8 ± 1.1 2002.8 ± 1.9 2003.0 ± 2.0 1999.1 ± 2.0 2000.0 ± 1.9 2008.7 ± 0.8 2008.2 ± 1.3
0.3 2.2 2.0 0.4 0.5 1.2 0.7
+AFY: Actual formation year. ++ CFY: calculated formation year.
Please cite this article in press as: C. Solis et al., AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques, Nucl. Instr. Meth. B (2017), http://dx.doi.org/10.1016/j.nimb.2017.02.085
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C. Solis et al. / Nuclear Instruments and Methods in Physics Research B xxx (2017) xxx–xxx
third molars from people older than 60 years-old, had F14C values of 1.4, contradicting the occurrence of a replacement of the collagen. Our results indicate more likely, that collagen (even from third molars) predicts the formation year of a dental piece and in consequence the date-of-birth of an individual. Fig. 2 shows calculated formation year for collagen and enamel compared with a one to one correspondence line that represents an ideal correlation between actual and measured formation year. In general, a good correlation between expected and measured values was obtained. Collagen results show a slightly higher scattering around the one to one line, relative to enamel results. Enamel from sample 273, corresponding to an individual born in 1936 showed the lowest F14C value and the highest scattering. As explained before, more scattered values are due to the slightly changes in pre 1950 atmospheric 14C levels. 4. Conclusion In this work dentine collagen and enamel were extracted from a set of dental pieces. Analyses were carried out to explore if it is more convenient to use collagen or enamel 14C contents to determine the formation year of teeth. Samples included root collagen and enamel from teeth with different development periods. Results obtained show that 14C content in enamel and collagen allow to determine the year of complete development. When both fractions of a same tooth are available, the difference between collagen and enamel development times, and therefore of the 14C fixation in each tissue, can be used for a precise determination of the tooth formation date and therefore the year-of-birth of an individual. By dating collagen of the same tooth, which has a longer formation time than enamel, serves to discriminate if the formation date belongs to the upward or downward side of the peak bomb. Collagen values also corroborate the ages obtained through enamel fraction. Data obtained from enamel analysis is more precise for dating due to its shorter formation time, relative to collagen that takes longer for a complete development. To overcome this problem, collagen samples could be taken from the dentine outer layers, the most recently formed. Analyses performed on collagen and enamel from all teeth (even from third molars) indicate in an accurate way that 14C levels reflect the formation year and in consequence the date-of-birth of an individual, contradicting the occurrence of a replacement of the collagen. Our results shown confirms previous observations that establish that measuring 14C content in tissues with two different formation time from same person as dental collagen and enamel helps to determinate the actual formation year of those, eliminating the inconvenience of two possible formation ranges in the right or left side of the peak bomb.
Acknowledgements The authors thank Graciela Solis for providing the dental pieces, Ivet Gil for the discussion of results and Sergio Martínez for technical support. This research was partially funded by grants DGAPA-UNAM IG100216 and CONACyT 264245, 205317, 271802 and 232718. References [1] R.M. Kalin, K. Burns, A.J.T. Jull, Studies of residence time of 14C in human bones-An application of AMS to forensic-science, Abstr. Pap. Am. Chem. Soc. 209 (1995) 35. [2] G.W. Hodgins, Measuring atomic bomb-derived 14C levels in human remains to determine Year of Birth and/or Year of Death. US Department of Justice. (2009) S 98. [3] M. Martini, M. Piacentini (Eds.), Physics Methods in Archaeometry, vol. 154, IOS Press, 2004. [4] M.J.S. Falso, B.A. Buchholz, Bomb pulse biology, Nucl. Instrum. Methods Phys. Res., Sect. B 294 (2013) 666–670. [5] U. Zoppi, Z. Skopec, J. Skopec, G. Jones, D. Fink, Q. Hua, A. Ellipsis & Williams, Forensic applications of 14 C bomb-pulse dating, Nucl. Instrum. Methods Phys. Res., Sect. B 223 (2004) 770–775. [6] P. Bulman, D. McLeod-Henning, Applying carbon-14 dating to recent human remains, NIJ J. 269 (2012) 8–10. [7] G.T. Cook, E. Dunbar, S.M. Black, S. Xu, A preliminary assessment of age at death determination using the nuclear weapons testing 14C activity of dentine and enamel, Radiocarbon 48 (3) (2006). [8] G.T. Cook, L.A.N. Ainscough, E. Dunbar, Radiocarbon analysis of modern skeletal remains to determine year of birth and death: a case study, Radiocarbon 57 (3) (2015) 327–336. [9] N. Wang, C.D. Shen, P. Ding, W.X. Yi, W.D. Sun, K.X. Liu, L.P. Ellipsis & Zhou, Improved application of bomb carbon in teeth for forensic investigation, Radiocarbon 52 (2) (2010) 706–716. [10] K.L. Spalding, B.A. Buchholz, L.E. Bergman, H. Druid, J. Frisén, Forensics: age written in teeth by nuclear tests, Nature 437 (7057) (2005) 333–334. [11] A.J.T. Jull, A.E. Scott, Encyclopedia of Quaternary Science (2007) 2911. [12] C. Solís, E. Chávez-Lomelí, M.E. Ortiz, A. Huerta, E. Andrade, E. Barrios, A new AMS facility in Mexico, Nucl. Instrum. Methods Phys. Res., Sect. B 331 (2014) 233–237. [13] I. Schour, M. Massler, Studies in tooth development: the growth pattern of human teeth, J. Am. Dent. Assoc. 27 (12) (1940) 1918–1931. [14] C.M. Nolla, The Development of Permanent Teeth, University of Michigan, 1952. [15] P.M. Helfman, J.L. Bada, Aspartic Acid Racemisation in Dentine as a Measure of Ageing, 1976. [16] B.A. Buchholz, K.L. Spalding, Year of birth determination using radiocarbon dating of dental enamel, Surf. Interface Anal. 42 (5) (2010) 398–401. [17] I. Hajdas, A. Michczynski, G. Bonani, L. Wacker, H. Furrer, Dating bones near the limit of the radiocarbon dating method: study case mammoth from Niederweningen, ZH Switzerland, Radiocarbon 51 (2) (2009) 675. [18] P.J. Reimer, R. Reimer, CALIBomb radiocarbon calibration, 2004, Interactive program available on-line at:. [19] I. Levin, The tropospheric (super 14) CO (sub 2) level in mid-latitudes of the Northern Hemisphere (1959–2003), Radiocarbon 46 (3) (2004) 1261–1272. [20] Q. Hua, M. Barbetti, A.Z. Rakowski, Atmospheric radiocarbon for the period 1950–2010, Radiocarbon 55 (4) (2013) 2059–2072. [21] P.J. Reimer, T.A. Brown, R.W. Reimer, Discussion: reporting and calibration of post-bomb 14C data, Radiocarbon 46 (3) (2004) 1299–1304.
Please cite this article in press as: C. Solis et al., AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques, Nucl. Instr. Meth. B (2017), http://dx.doi.org/10.1016/j.nimb.2017.02.085
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques C. Solis a,⇑, E. Solis-Meza a, M.E. Morales a, M. Rodriguez-Ceja a, M.A. Martínez-Carrillo b, D. Garcia-Calderon a, A. Huerta a, E. Chávez a a b
Instituto de Física Universidad Nacional Autónoma de México, Avenida Universidad 3000, Circuito de la Investigación Científica S/N Ciudad Universitaria, 04510, Mexico Facultad de Ciencias Universidad Nacional Autónoma de México, Avenida Universidad 3000, Circuito de la Investigación Científica S/N Ciudad Universitaria, 04510, Mexico
a r t i c l e
i n f o
Article history: Received 5 August 2016 Received in revised form 31 December 2016 Accepted 28 February 2017 Available online xxxx Keywords: Radiocarbon Bomb-pulse dating Carbon-14 Forensics
a b s t r a c t AMS-14C analysis of modern teeth has become important for forensic studies. 14C content in human teeth reflects the 14C atmospheric concentration during its formation and allows the calculation of the actual year of birth. Through AMS, it is possible to measure the 14C concentrations in a tissue with high precision. However, there is a debate about which should be the best fraction for teeth carbon dating: collagen or enamel. This work focuses on the results obtained from enamel and collagen extracted from Mexican people in order to compare them. Collagen from dental pieces donated from people older than 60-yearsold have been included to understand the turnover process and usefulness of collagen to determine the date of birth. Our results indicate that when a single dental piece is available, enamel method allows the determination of the tooth formation date. Dating collagen of the same tooth helps to discriminate if the formation date belongs to the left or the right side of the peak bomb, but also corroborates, the ages obtained through enamel analysis. Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction In Mexico, research in any of the forensic disciplines is extremely scarce. For this reason, forensic experts have frequently resorted to scientific knowledge generated abroad. However, this situation is changing and there is an increasing interest to apply the scientific methods available in Mexico to the study of materials found in criminal scenes, with the aim of having multidisciplinary tools to complement the expert studies and so contribute to the procurement and administration of justice in the prevention of offenses. Analysis of 14C content in human tissues such as teeth and bones has proved to be a useful way to establish the time elapsed since individual’s birth and death. In forensic cases, the usefulness of modern tissues 14C dating comes from the fact that 14C is incorporated into tissues through metabolism. This exchange with the atmosphere stops when the organism dies, so 14C contents within the tissue, reflects 14C level that existed in the atmosphere [1,2]. Cosmic ray interactions with atmospheric 14N produces 14C, which oxidizes to CO2 and distributes throughout the planet. Before 1950, the 14C content in the atmosphere was more or less ⇑ Corresponding author. E-mail address: [email protected] (C. Solis).
stable [3]. During the so-called nuclear era, atmospheric 14C content increased abruptly due to atmospheric nuclear weapon testing during the 50s and early 60s. From the 1963 peak, after the nuclear test banning, 14CO2 levels have slowly decreased mainly due to exchange with carbon in the oceans and the biosphere [4]. These changes in atmospheric 14CO2 levels are reflected in organisms born after 1950, who assimilate 14C through food chain. Their tissues, which assimilate at different rates, reflect the atmospheric 14 CO2 levels, at the time of formation, being then possible to determine their formation date through 14C content. Forensic application is based on the 14C/12C ratio (normalized to a 13C value) which reflects the atmospheric isotopic ratio at the time when these tissues were formed. This method is called bomb/pulse dating [5]. Teeth are formed by organic and inorganic fractions. Inorganic teeth enamel contains approximately 2–3% of carbon while organic fraction, mainly collagen, has 30% of carbon. Because of their hardness and strength, teeth are frequently the unique useful remaining body parts to determine the date when a person was born. This can be an important step to establish the identity of victims that are classified as unknown because a lack of any other information. Forensic studies have been focused on determine, through the analysis of 14C in different dental pieces, the year-of-birth of an individual [6–10]. Analysis with Accelerator Mass Spectrometry
http://dx.doi.org/10.1016/j.nimb.2017.02.085 0168-583X/Ó 2017 Elsevier B.V. All rights reserved.
Please cite this article in press as: C. Solis et al., AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques, Nucl. Instr. Meth. B (2017), http://dx.doi.org/10.1016/j.nimb.2017.02.085
2
C. Solis et al. / Nuclear Instruments and Methods in Physics Research B xxx (2017) xxx–xxx
(AMS) is the ideal technique for teeth because of its high precision and the possibility to measure this radionuclide in milligram or less-sized samples [11]. With the AMS facility (LEMA) installed in 2013 at the Physics Institute of the UNAM, it is now possible to perform high precision analyses of radionuclides [12]. Each dental piece has different development periods: crown dentine forms after the enamel and root dentine forms after crown dentine. The time span of the crown and root formation process is well known for each tooth, by what is a good candidate for determine the date of birth of a person [13,14]. How close is the 14C contents in teeth related to that of the atmosphere, depends on the tooth type, if the formation was completed before or after the peak bomb, among other factors. However, there is a debate about which should be the best fraction to be used for dating: collagen or enamel. Several studies show that, after being formed, enamel is not replaced and 14C content is close to the atmospheric level at the time of crown formation [2,7–10]. Dentine collagen has also been used in a number of studies, since it accounts for a greater amount of carbon than tooth enamel. It also helps in distinguishing the side of the peak bomb where the sample belongs. Nonetheless, collagen’s use to determine the year-of-birth seems to be controversial. Some authors base the use of collagen in the fact that racemization experiments have shown that there is no turnover [2,7,15]. However, some others have observed that collagen’s carbon continues to recycle even when the tooth is already complete [9,16]. Wang et al. declare that collagen could capture 14C even until the individual’s death. The aim of this work is to compare the dates coming from enamel and collagen analysis. With this purpose in mind, collagen or enamel was extracted from a set of dental pieces from individuals of known ages, in order to calculate the year of complete development. For enamel and collagen extraction from a single piece, third molars were chosen because their high carbon yield. A sample set of collagen from dental pieces donated from people older than 60-years-old whose teeth were extracted between 2014 and 2015, have been included to understand the turnover process and usefulness of collagen to determine the date of birth. 2. Material and methods All dental pieces were supplied by a dentist and were extracted between 2014 and 2016. They belonged to Mexicans living in Mexico City and San Luis Potosi, two urban cities. Four teeth were chosen for enamel and collagen extraction, nine for collagen and seven for enamel. Each tooth was cleaned with distilled water in an ultrasonic bath and dried at 70 C for 72 h. Four third molars were chosen for collagen and enamel extraction. Each molar was cut away lengthwise; one-half for enamel and the other half for collagen extraction. Collagen from crown and root was extracted from one tooth half. The other half was cut at the cervical line in order to
Fig. 1. Diagram showing the teeth fractions used: a) Whole tooth b) One half was used for collagen extraction; c) the crown of the other half was cut at the cervical line in order to extract the enamel.
extract the enamel from the crown (Fig. 1). Enamel was obtained by hydrolysis [7]. Every crown fraction was treated with 7 mL of 10 M NaOH for one week at 50 C. The sample was placed in a sonic bath at 50 C, NaOH was replaced every day before the sonic bath. After that week the samples were washed at least three times in 0.25 M HCl followed by several rinses with deionized water until pH = 7. Dentine and other tissues were removed by blunt dissection. Collagen was extracted with a Longin modified method with ultrafiltration. The corresponding half was pulverized by the mortar and pestle. Samples were demineralized with HCl (0.5 M) at 5 C temperature over 24 h, then, rinsed 3 times. A further acid wash was carried out with HCl (0.2 M), at 76 C overnight to obtain the gelatin. The supernatant was ultra-filtered and centrifuged and the fraction higher than 30KD (soluble collagen) was lyophilized [17]. Collagen samples containing 1 mg of C were converted to graphite in Automated Graphitization Equipment (AGE III Ion Plus), using a Peltier cooler ( 5 C) to retain the water produced in the reaction. Samples were processed for enamel extraction using a Carbonate Handling System (CHS Ion Plus), each sample reacted with orthophosphoric acid to obtain CO2 which was graphitized in the AGE III. AMS 14C analyses of graphite pressed in Al cathodes were performed in the HVEE LEMA-AMS system. Oxalic acid standard (NIST SRM4990C Oxalic Acid II) was employed for normalization and blanks (Phthallic acid (C8H6O4) and marble (IAEA-C1) with no 14C, were also processed and subtracted to correct for background. Graphite obtained from Phthalic acid gives a F14C close to 0.0030 ± 0.0001. Obtained 14C/12C ratios were converted to 14C ages using a computer code developed at LEMA [11]. Calibrated ages were obtained with CALIBomb computer code, and the curve IntCal 13 and NHZ2 [18–20]. The 14C results are expressed as a fraction modern carbon (F14C ± 2r) [21]. In routine operation the normal precision obtained at LEMA for 1 mg of modern carbon is around 0.3%.
Table 1 Fraction modern carbon values in collagen and crown enamel from 4 third molars, calibrated date of formation ranges. Calculations were performed using CALIBomb. LEMA ID 273 273 223 223 269 269 399 399 +
Collagen Enamel Collagen Enamel Collagen Enamel Collagen Enamel
Year of birth
AFY+
F14C
Calibrated date of formation range (2r)
CFY++
1936 1936 1944 1944 1995 1995 1999 1999
1958 ± 4 1950 ± 2 1966 ± 4 1958 ± 2 2014 ± 4 2009 ± 2 2021 ± 4 2013 ± 2
1.107 ± 0.004 0.975 ± 0.003 1.438 ± 0.007 1.177 ± 0.005 1.044 ± 0.005 1.030 ± 0.006 1.042 ± 0.004 1.046 ± 0.004
1957.8–1958.2 1934–1954 1973.0–1974.9 1958.4–1959.1 2009.5 2009.4–2009.5 2009.5 2009.5
1958.0 ± 0.2 1944 ± 10 1973.9 ± 0.9 1958.8 ± 0.4 2009.5 2009.43 ± 0.03 2009.5 2009.5
AFY-CFY 0.5 6 8.4 0.8 0.4
AFY: Actual formation year. CFY: Calculated formation year.
++
Please cite this article in press as: C. Solis et al., AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques, Nucl. Instr. Meth. B (2017), http://dx.doi.org/10.1016/j.nimb.2017.02.085
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C. Solis et al. / Nuclear Instruments and Methods in Physics Research B xxx (2017) xxx–xxx Table 2 Enamel and root complete development ages for each tooth. Taken from Schour [13]. Upper teeth
Central incisor Lateral incisor Canine First premolar Second premolar First molar Second molar Third Molar
Lower teeth
Enamel
Root
Enamel
Root
4–5 4–5 6–7 5–6 6–7 2.5–3 7–8 12–16
10 11 13–15 12–13 12–14 9–10 14–16 18–25
4–5 4–5 6–7 5–6 6–7 2.5–3 7–8 12–16
9 10 12–14 12–13 13–14 9–10 14–15 18–25
3. Results Table 1 shows the comparison of AMS-14C results obtained with enamel and collagen extracted from third molars. Two pieces correspond to the left and two pieces correspond to the right side of the peak bomb. Levels of 14C are shown as fraction of modern carbon (F14C). The actual formation year (AFY) was calculated from year-of-birth (YOB) plus the tooth formation time (TF) from Schour et al. [13] shown in Table 2. Calibrated years were obtained from F14C by means of CALIBomb computer code using the data set corresponding to the Northern Atmospheric 14C Levels (Zone 2) from 1950 to 2009 AD. The 14C fixation in teeth was assumed to occur at the end of the tooth development. From the intervals obtained by calibration of the fraction of modern carbon, only the dates close to YOB are shown in Table 1. The last column indicates the difference between actual enamel or dentine formation year and the mean value of the calculated range. Results show that in general collagen and enamel content reproduce the elapsed time for the complete development of each dental pieces. In samples with a formation time before AD 1950 precision is lower because the slight change in atmospheric 14C levels, this may cause greater errors than in samples after AD 1950 where atmospheric 14C dramatically changed due to all ground level nuclear bomb tests. This is the case for enamel from sample 273 in Table 1, formed before 1950 which may explain the uncertainty. Between enamel and col-
lagen from third molars, there is an expected difference of 6– 9 years for complete development periods. This difference is confirmed in Table 1. Dating collagen of the same tooth helps to discriminate if the formation date belongs to the upward or downward side of the peak bomb, but also corroborates, the ages obtained through enamel analysis. For those teeth whose formation year falls after 2009, the F14C values cannot be distinguished, since calibration curve used by the CALIBomb computer code has a data set until 2009. Table 3 shows the AMS-14C results obtained for collagen extracted from dentine from the whole tooth. Replicates of same samples shows reproducible values. The obtained results allow to predict the formation year within 3.8 years. It is worthy to notice that all examined teeth were extracted between 2014 and 2016. In people older than 60 years old, extraction occurred many years after teeth completed their formation. Extrapolation of the calibration curve predicts a F14C value of 0.99 for 2014 and 2015. If there was a replacement in the collagen of the third molars as it has been suggested before [9], a F14C value close to 0.99 would be expected for these dental pieces. However, this is not observed, and even
Fig. 2. Comparison of calculated formation time for collagen (values shown in Table 3) and enamel (values from Tables 1 and 3) with actual formation time. A a one to one correspondence line representing an ideal correspondence between actual formation time and measured formation time is also shown.
Table 3 Fraction modern carbon values in root collagen from 12 individual teeth and enamel from 7 individuals. Calibrated date of formation ranges (SM: second molar, FP: first premolar, FM: first molar, TM: third molar, C: central, CI: central incisor, C: canine, U: upper, L: lower). All teeth were extracted between 2014 and 2015. Calculations were performed using CALIBomb. LEMA ID
Year of birth
Tooth type
AFY+
F14C
Calibrated formation date range (2r)
CFY++
AFY-CFY
272.1.1 219.1.1 219.1.2 274.1.2 218.1.1 218.1.2 268.1.1 220.1.1 220.1.2 221.1.1 275.1.2 271.1.1
Collagen 1939 1942 1942 1943 1944 1944 1944 1949 1949 1952 1965 1974
SM-L SM-U SM-U SM-U FP-L FP-L SM-U FM-L FM-L FM-L SM-U TM-U
1954 ± 1 1957 ± 1 1957 ± 1 1958 + 1 1957 ± 1 1957 ± 1 1959 ± 1 1959 ± 1 1959 ± 1 1962 ± 1 1980 ± 1 1996 ± 4
1.124 ± 0.004 1.108 ± 0.005 1.119 ± 0.007 1.126 ± 0.003 1.002 ± 0.005 0.999 ± 0.005 1.086 ± 0.005 0.993 ± 0.006 0.997 ± 0.007 1.020 ± 0.006 1.288 ± 0.004 1.205 ± 0.005
1957.9–1958.6 1957.7–1958.3 1957.8–1958.6 1958.0–1958.6 1954.6–1956.0 1955.0–1956.0 1957.5–1958.1 1953.0–1956.0 1953.0–1956.0 1955.5–1956.4 1979.1–1980.8 1983.9–1987.7
1958.2 ± 0.3 1958.0 ± 0.3 1958.2 ± 0.4 1958.3 ± 0.3 1955.3 ± 0.7 1955.5 ± 0.5 1957.8 ± 0.3 1954.5 ± 1.5 1954.5 ± 1.5 1955.9 ± 0.4 1980.0 ± 0.9 1985.8 ± 1.9
4.7 1.0 1.2 0.3 1.2 1.0 1.2 4.0 4.0 5.6 0 9.7
637.2.1 552.2.2 556.2.2 595.2.1 596.2.1 373.2.1 374.2.1
Enamel 1964 1991 1991 1993 1993 2002 2002
CI-L TM TM C-U C-U FP-U FP-U
1968.5 ± 0.5 2005 ± 2 2005 ± 2 1999.5 ± 0.5 1999.5 ± 0.5 2007.5 ± 0.5 2007.5 ± 0.5
1.575 ± 0.007 1.089 ± 0.004 1.079 ± 0.004 1.098 ± 0.004 1.095 ± 0.004 1.049 ± 0.003 1.054 ± 0.003
1967.7–1969.9 2000.9–2004.7 2001.0–2005.0 1997.1–2001.1 1998.1–2001.9 2007.9–2009.5 2006.9–2009.5
1968.8 ± 1.1 2002.8 ± 1.9 2003.0 ± 2.0 1999.1 ± 2.0 2000.0 ± 1.9 2008.7 ± 0.8 2008.2 ± 1.3
0.3 2.2 2.0 0.4 0.5 1.2 0.7
+AFY: Actual formation year. ++ CFY: calculated formation year.
Please cite this article in press as: C. Solis et al., AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques, Nucl. Instr. Meth. B (2017), http://dx.doi.org/10.1016/j.nimb.2017.02.085
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C. Solis et al. / Nuclear Instruments and Methods in Physics Research B xxx (2017) xxx–xxx
third molars from people older than 60 years-old, had F14C values of 1.4, contradicting the occurrence of a replacement of the collagen. Our results indicate more likely, that collagen (even from third molars) predicts the formation year of a dental piece and in consequence the date-of-birth of an individual. Fig. 2 shows calculated formation year for collagen and enamel compared with a one to one correspondence line that represents an ideal correlation between actual and measured formation year. In general, a good correlation between expected and measured values was obtained. Collagen results show a slightly higher scattering around the one to one line, relative to enamel results. Enamel from sample 273, corresponding to an individual born in 1936 showed the lowest F14C value and the highest scattering. As explained before, more scattered values are due to the slightly changes in pre 1950 atmospheric 14C levels. 4. Conclusion In this work dentine collagen and enamel were extracted from a set of dental pieces. Analyses were carried out to explore if it is more convenient to use collagen or enamel 14C contents to determine the formation year of teeth. Samples included root collagen and enamel from teeth with different development periods. Results obtained show that 14C content in enamel and collagen allow to determine the year of complete development. When both fractions of a same tooth are available, the difference between collagen and enamel development times, and therefore of the 14C fixation in each tissue, can be used for a precise determination of the tooth formation date and therefore the year-of-birth of an individual. By dating collagen of the same tooth, which has a longer formation time than enamel, serves to discriminate if the formation date belongs to the upward or downward side of the peak bomb. Collagen values also corroborate the ages obtained through enamel fraction. Data obtained from enamel analysis is more precise for dating due to its shorter formation time, relative to collagen that takes longer for a complete development. To overcome this problem, collagen samples could be taken from the dentine outer layers, the most recently formed. Analyses performed on collagen and enamel from all teeth (even from third molars) indicate in an accurate way that 14C levels reflect the formation year and in consequence the date-of-birth of an individual, contradicting the occurrence of a replacement of the collagen. Our results shown confirms previous observations that establish that measuring 14C content in tissues with two different formation time from same person as dental collagen and enamel helps to determinate the actual formation year of those, eliminating the inconvenience of two possible formation ranges in the right or left side of the peak bomb.
Acknowledgements The authors thank Graciela Solis for providing the dental pieces, Ivet Gil for the discussion of results and Sergio Martínez for technical support. This research was partially funded by grants DGAPA-UNAM IG100216 and CONACyT 264245, 205317, 271802 and 232718. References [1] R.M. Kalin, K. Burns, A.J.T. Jull, Studies of residence time of 14C in human bones-An application of AMS to forensic-science, Abstr. Pap. Am. Chem. Soc. 209 (1995) 35. [2] G.W. Hodgins, Measuring atomic bomb-derived 14C levels in human remains to determine Year of Birth and/or Year of Death. US Department of Justice. (2009) S 98. [3] M. Martini, M. Piacentini (Eds.), Physics Methods in Archaeometry, vol. 154, IOS Press, 2004. [4] M.J.S. Falso, B.A. Buchholz, Bomb pulse biology, Nucl. Instrum. Methods Phys. Res., Sect. B 294 (2013) 666–670. [5] U. Zoppi, Z. Skopec, J. Skopec, G. Jones, D. Fink, Q. Hua, A. Ellipsis & Williams, Forensic applications of 14 C bomb-pulse dating, Nucl. Instrum. Methods Phys. Res., Sect. B 223 (2004) 770–775. [6] P. Bulman, D. McLeod-Henning, Applying carbon-14 dating to recent human remains, NIJ J. 269 (2012) 8–10. [7] G.T. Cook, E. Dunbar, S.M. Black, S. Xu, A preliminary assessment of age at death determination using the nuclear weapons testing 14C activity of dentine and enamel, Radiocarbon 48 (3) (2006). [8] G.T. Cook, L.A.N. Ainscough, E. Dunbar, Radiocarbon analysis of modern skeletal remains to determine year of birth and death: a case study, Radiocarbon 57 (3) (2015) 327–336. [9] N. Wang, C.D. Shen, P. Ding, W.X. Yi, W.D. Sun, K.X. Liu, L.P. Ellipsis & Zhou, Improved application of bomb carbon in teeth for forensic investigation, Radiocarbon 52 (2) (2010) 706–716. [10] K.L. Spalding, B.A. Buchholz, L.E. Bergman, H. Druid, J. Frisén, Forensics: age written in teeth by nuclear tests, Nature 437 (7057) (2005) 333–334. [11] A.J.T. Jull, A.E. Scott, Encyclopedia of Quaternary Science (2007) 2911. [12] C. Solís, E. Chávez-Lomelí, M.E. Ortiz, A. Huerta, E. Andrade, E. Barrios, A new AMS facility in Mexico, Nucl. Instrum. Methods Phys. Res., Sect. B 331 (2014) 233–237. [13] I. Schour, M. Massler, Studies in tooth development: the growth pattern of human teeth, J. Am. Dent. Assoc. 27 (12) (1940) 1918–1931. [14] C.M. Nolla, The Development of Permanent Teeth, University of Michigan, 1952. [15] P.M. Helfman, J.L. Bada, Aspartic Acid Racemisation in Dentine as a Measure of Ageing, 1976. [16] B.A. Buchholz, K.L. Spalding, Year of birth determination using radiocarbon dating of dental enamel, Surf. Interface Anal. 42 (5) (2010) 398–401. [17] I. Hajdas, A. Michczynski, G. Bonani, L. Wacker, H. Furrer, Dating bones near the limit of the radiocarbon dating method: study case mammoth from Niederweningen, ZH Switzerland, Radiocarbon 51 (2) (2009) 675. [18] P.J. Reimer, R. Reimer, CALIBomb radiocarbon calibration, 2004, Interactive program available on-line at:
Please cite this article in press as: C. Solis et al., AMS-14C analysis of modern teeth: A comparison between two sample preparation techniques, Nucl. Instr. Meth. B (2017), http://dx.doi.org/10.1016/j.nimb.2017.02.085