Abstract
This paper presents a combination of elemental and isotopic spatial distribution imaging with near-infrared hyperspectral imaging (NIR-HSI) to evaluate the diagenetic status of skeletal remains. The aim is to assess how areas with biogenic n(87Sr)/n(86Sr) isotope-amount ratios may be identified in bone material, an important recorder complementary to teeth. Elemental (C, P, Ca, Sr) and isotopic (n(87Sr)/n(86Sr)) imaging were accomplished via laser ablation (LA) coupled in a split stream to a quadrupole inductively coupled plasma mass spectrometer (ICP-QMS) and a multicollector inductively coupled plasma mass spectrometer (MC ICP-MS) (abbreviation for the combined method LASS ICP-QMS/MC ICP-MS). Biogenic areas on the bone cross section, which remained unaltered by diagenetic processes, were localized using chemical indicators (I(C)/I(Ca) and I(C) × 10/I(P) intensity ratios) and NIR-HSI at a wavelength of 1410 nm to identify preserved collagen. The n(87Sr)/n(86Sr) isotope signature analyzed in these areas was in agreement with the biogenic bulk signal revealed by solubility profiling used as an independent method for validation. Elevated C intensities in the outer rim of the bone, caused by either precipitated secondary minerals or adsorbed humic materials, could be identified as indication for diagenetic alteration. These areas also show a different n(87Sr)/n(86Sr) isotopic composition. Therefore, the combination of NIR-HSI and LASS ICP-QMS/MC ICP-MS allows for the determination of preserved biogenic n(87Sr)/n(86Sr) isotope-amount ratios, if the original biogenic material has not been entirely replaced by diagenetic material.
Similar content being viewed by others
Abbreviations
- ICP-QMS:
-
Inductively coupled plasma quadrupole mass spectrometer
- LA:
-
Laser ablation
- LASS ICP-QMS/MC ICP-MS:
-
Laser ablation coupled via a split stream to a quadrupole inductively coupled plasma mass spectrometer and a multicollector inductively coupled plasma mass spectrometer
- MC ICP-MS:
-
Multicollector inductively coupled plasma mass spectrometer
- NIR:
-
Near-infrared
- NIR-HSI:
-
Near-infrared hyperspectral imaging
- PCA:
-
Principal component analysis
- ROI:
-
Region of interest
References
Bentley RA. Strontium isotopes from the earth to the archaeological skeleton: a review. J Archaeol Method Theory. 2006;13(3):135–87.
Slovak NM, Paytan A. Applications of Sr isotopes in archaeology. In: Baskaran M, editor. Handbook of environmental isotope geochemistry, vol. I. Berlin, Heidelberg: Springer; 2012. p. 743–68. https://doi.org/10.1007/978-3-642-10637-8_35.
Szostek K, Mądrzyk K, Cienkosz-Stepańczak B. Strontium isotopes as an indicator of human migration—easy questions, difficult answers. Anthropol Rev. 2015;78(2):133–56. https://doi.org/10.1515/anre-2015-0010.
Sehrawat JS, Kaur J. Role of stable isotope analyses in reconstructing past life-histories and the provenancing human skeletal remains: a review. Anthropol Rev. 2017;80(3):243–58. https://doi.org/10.1515/anre-2017-0017.
Prohaska T, Latkoczy C, Schultheis G, Teschler-Nicola M, Stingeder G. Investigation of Sr isotope ratios in prehistoric human bones and teeth using laser ablation ICP-MS and ICP-MS after Rb/Sr separation. J Anal At Spectrom. 2002;17(8):887–91. https://doi.org/10.1039/b203314c.
Prohaska T, Teschler-Nicola M, Galler G, Přichystal A, Stingeder G, Jelenc M, et al. Non-destructive determination of 87Sr/86Sr isotope ratios in early upper Paleolithic human teeth from the Mladeč caves—preliminary results. In: Teschler-Nicola M, editor. Early modern humans at the Moravian Gate. Vienna: Springer-Verlag; 2006. p. 505–14. https://doi.org/10.1007/978-3-211-49294-9_18.
Horstwood MSA, Evans JA, Montgomery J. Determination of Sr isotopes in calcium phosphates using laser ablation inductively coupled plasma mass spectrometry and their application to archaeological tooth enamel. Geochim Cosmochim Acta. 2008;72(23):5659–74. https://doi.org/10.1016/j.gca.2008.08.016.
Simonetti A, Buzon MR, Creaser RA. In-situ elemental and Sr isotope investigation of human tooth enamel by laser ablation-(MC)-ICP-MS: successes and pitfalls. Archaeometry. 2008;50(2):371–85. https://doi.org/10.1111/j.1475-4754.2007.00351.x.
Copeland SR, Sponheimer M, Lee-Thorp JA, le Roux PJ, de Ruiter DJ, Richards MP. Strontium isotope ratios in fossil teeth from South Africa: assessing laser ablation MC-ICP-MS analysis and the extent of diagenesis. J Archaeol Sci. 2010;37(7):1437–46. https://doi.org/10.1016/j.jas.2010.01.003.
Montgomery J, Evans JA, Horstwood MSA. Evidence for long-term averaging of strontium in bovine enamel using TIMS and LA-MC-ICP-MS strontium isotope intra-molar profiles. Environ Archaeol. 2010;15(1):32–42. https://doi.org/10.1179/146141010x12640787648694.
Le Roux PJ, Lee-Thorp JA, Copeland SR, Sponheimer M, de Ruiter DJ. Strontium isotope analysis of curved tooth enamel surfaces by laser-ablation multi-collector ICP-MS. Palaeogeogr Palaeoclimatol Palaeoecol. 2014;416:142–9. https://doi.org/10.1016/j.palaeo.2014.09.007.
Lewis J, Coath CD, Pike AWG. An improved protocol for 87Sr/86Sr by laser ablation multi-collector inductively coupled plasma mass spectrometry using oxide reduction and a customised plasma interface. Chem Geol. 2014;390:173–81. https://doi.org/10.1016/j.chemgeo.2014.10.021.
Capo RC, Stewart BW, Chadwick OA. Strontium isotopes as tracers of ecosystem processes: theory and methods. Geoderma. 1998;82:197–225.
Coplen TB. Guidelines and recommended terms for expression of stable-isotope-ratio and gas-ratio measurement results. Rapid Commun Mass Spectrom. 2011;25(17):2538–60. https://doi.org/10.1002/rcm.5129.
Blum JD, Taliaferro EH, Weisse MT, HR T. Changes in Sr/Ca, Ba/Ca and 87Sr/86Sr ratios between two forest ecosystems in the northeastern USA. Biogeochemistry. 2000;49:87–101.
Sealy JC, van der Merwe NJ, Sillen A, Kruger FJ, Krueger HW. 87Sr86Sr as a dietary indicator in modern and archaeological bone. J Archaeol Sci. 1991;18(3):399–416. https://doi.org/10.1016/0305-4403(91)90074-Y.
Grupe G, Price TD, Schröter P, Söllner F, Johnson CM, Beard BL. Mobility of Bell Beaker people revealed by strontium isotope ratios of tooth and bone: a study of southern Bavarian skeletal remains. Appl Geochem. 1997;12:517–25.
Lee-Thorp J, Sponheimer M. Three case studies used to reassess the reliability of fossil bone and enamel isotope signals for paleodietary studies. J Anthropol Archaeol. 2003;22(3):208–16. https://doi.org/10.1016/s0278-4165(03)00035-7.
Schweissing MM, Grupe G. Stable strontium isotopes in human teeth and bone: a key to migration events of the late Roman period in Bavaria. J Archaeol Sci. 2003;30(11):1373–83. https://doi.org/10.1016/S0305-4403(03)00025-6.
Tütken T, Vennemann TW, Pfretzschner H-U. Nd and Sr isotope compositions in modern and fossil bones—proxies for vertebrate provenance and taphonomy. Geochim Cosmochim Acta. 2011;75(20):5951–70. https://doi.org/10.1016/j.gca.2011.07.024.
Ortega LA, Guede I, Zuluaga MC, Alonso-Olazabal A, Murelaga X, Niso J, et al. Strontium isotopes of human remains from the San Martín de Dulantzi graveyard (Alegría-Dulantzi, Álava) and population mobility in the Early Middle Ages. Quat Int. 2013;303:54–63. https://doi.org/10.1016/j.quaint.2013.02.008.
Hoogewerff J, Papesch W, Kralik M, Berner M, Vroon P, Miesbauer H, et al. The last domicile of the iceman from Hauslabjoch: a geochemical approach using Sr, C and O isotopes and trace element signatures. J Archaeol Sci. 2001;28(9):983–9. https://doi.org/10.1006/jasc.2001.0659.
Wilson L, Pollard M. Here today, gone tomorrow? Integrated experimentation and geochemical modeling in studies of archaeological diagenetic change. Acc Chem Res. 2002;35(8):644–51.
Nelson B, Deniro MJ, Schoeninger MJ, De Paolo DJ. Effects of diagenesis on strontium, carbon, nitrogen and oxygen concentration and isotopic composition of bone. Geochim Cosmochim Acta. 1986;50:1941.
Kohn MJ, Schoeninger MJ, Barker WW. Altered states: effects of diagenesis on fossil tooth chemistry. Geochim Cosmochim Acta. 1999;63(18):2737–47.
Nielsen-Marsh CM, Hedges REM. Patterns of diagenesis in bone I: the effects of site environments. J Archaeol Sci. 2000;27(12):1139–50. https://doi.org/10.1006/jasc.1999.0537.
Hoppe KA, Koch PL, Furutani TT. Assessing the preservation of biogenic strontium in fossil bones and tooth enamel. Int J Osteoarchaeol. 2003;13(1–2):20–8. https://doi.org/10.1002/oa.663.
Kyle JH. Effect of post-burial contamination on the concentrations of major and minor elements in human bones and teeth—the implications for palaeodietary research. J Archaeol Sci. 1986;13(5):403–16. https://doi.org/10.1016/0305-4403(86)90011-7.
Driessens FCM, Verbeeck RK. Biominerals. Boca Raton: CRC Press; 1990.
Lambert JB, Xue L, Buikstra JE. Physical removal of contaminative inorganic material from buried human bone. J Archaeol Sci. 1989;16(4):427–36. https://doi.org/10.1016/0305-4403(89)90017-4.
Price TD, Blitz J, Burton J, Ezzo JA. Diagenesis in prehistoric bone: problems and solutions. J Archaeol Sci. 1992;19(5):513–29.
Sillen A. Biogenic and diagenetic Sr/Ca in Plio-Pleistocene fossils of the Omo Shungura Formation. Paleobiology. 1986;12(3):311–23.
Reynard B, Balter V. Trace elements and their isotopes in bones and teeth: diet, environments, diagenesis, and dating of archeological and paleontological samples. Palaeogeogr Palaeoclimatol Palaeoecol. 2014;416:4–16. https://doi.org/10.1016/j.palaeo.2014.07.038.
Trueman CN, Palmer MR, Field J, Privat K, Ludgate N, Chavagnac V, et al. Comparing rates of recrystallisation and the potential for preservation of biomolecules from the distribution of trace elements in fossil bones. Comptes Rendus Palevol. 2008;7(2):145–58. https://doi.org/10.1016/j.crpv.2008.02.006.
Koenig AE, Rogers RR, Trueman CN. Visualizing fossilization using laser ablation–inductively coupled plasma–mass spectrometry maps of trace elements in Late Cretaceous bones. Geology. 2009;37(6):511–4. https://doi.org/10.1130/G25551A.1.
Fernandes R, Hüls M, Nadeau M-J, Grootes PM, Garbe-Schönberg CD, Hollund HI, et al. Assessing screening criteria for the radiocarbon dating of bone mineral. Nucl Inst Methods Phys Res B. 2013;294(Supplement C):226–32. https://doi.org/10.1016/j.nimb.2012.03.032.
Benson A, Kinsley L, Willmes M, Defleur A, Kokkonen H, Mussi M, et al. Laser ablation depth profiling of U-series and Sr isotopes in human fossils. J Archaeol Sci. 2013;40(7):2991–3000. https://doi.org/10.1016/j.jas.2013.02.028.
Willmes M, Kinsley L, Moncel MH, Armstrong RA, Aubert M, Eggins S, et al. Improvement of laser ablation in situ micro-analysis to identify diagenetic alteration and measure strontium isotope ratios in fossil human teeth. J Archaeol Sci. 2016;70:102–16. https://doi.org/10.1016/j.jas.2016.04.017.
Shemesh A. Crystallinity and diagenesis of sedimentary apatites. Geochim Cosmochim Acta. 1990;54(9):2433–8.
Weiner S, Bar-Yosef O. States of preservation of bones from prehistoric sites in the Near East: a survey. J Archaeol Sci. 1990;17(2):187–96. https://doi.org/10.1016/0305-4403(90)90058-D.
Greene EF, Tauch S, Webb E, Amarasiriwardena D. Application of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) for the identification of potential diagenesis and crystallinity changes in teeth. Microchem J. 2004;76(1):141–9. https://doi.org/10.1016/j.microc.2003.11.006.
Fernández-Jalvo Y, Andrews P, Pesquero D, Smith C, Marín-Monfort D, Sánchez B, et al. Early bone diagenesis in temperate environments. Palaeogeogr Palaeoclimatol Palaeoecol. 2010;288(1–4):62–81. https://doi.org/10.1016/j.palaeo.2009.12.016.
Patonai Z, Maasz G, Avar P, Schmidt J, Lorand T, Bajnoczky I, et al. Novel dating method to distinguish between forensic and archeological human skeletal remains by bone mineralization indexes. Int J Legal Med. 2012;127(2):529–33. https://doi.org/10.1007/s00414-012-0785-4.
Gutierrez MA. Bone diagenesis and taphonomic history of the Paso Otero 1 Bone Bed, Pampas of Argentina. J Archaeol Sci. 2001;28(12):1277–90. https://doi.org/10.1006/jasc.2000.0648.
Boskey AL, Moore DJ, Amling M, Canalis E, Delany AM. Infrared analysis of the mineral and matrix in bones of osteonectin-null mice and their wildtype controls. J Bone Miner Res. 2009;18(6):1005–11. https://doi.org/10.1359/jbmr.2003.18.6.1005.
Longato S, Woss C, Hatzer-Grubwieser P, Bauer C, Parson W, Unterberger SH, et al. Post-mortem interval estimation of human skeletal remains by micro-computed tomography, mid-infrared microscopic imaging and energy dispersive X-ray mapping. Anal Methods. 2015;7(7):2917–27. https://doi.org/10.1039/c4ay02943g.
Woess C, Unterberger SH, Roider C, Ritsch-Marte M, Pemberger N, Cemper-Kiesslich J, et al. Assessing various infrared (IR) microscopic imaging techniques for post-mortem interval evaluation of human skeletal remains. PLoS One. 2017;12(3):e0174552. https://doi.org/10.1371/journal.pone.0174552.
Stathopoulou ET, Psycharis V, Chryssikos GD, Gionis V, Theodorou G. Bone diagenesis: new data from infrared spectroscopy and X-ray diffraction. Palaeogeogr Palaeoclimatol Palaeoecol. 2008;266(3–4):168–74. https://doi.org/10.1016/j.palaeo.2008.03.022.
Thomas D, McGoverin C, Chinsamy A, Manley M. Near infrared analysis of fossil bone from the Western Cape of South Africa. J Near Infrared Spectrosc. 2011;19(3):151–9.
Linderholm J, Fernández Pierna J, Vincke D, Dardenne P, Baeten V. Identification of fragmented bones and their state of preservation by using near infrared hyperspectral image analysis. J Near Infrared Spectrosc. 2013;21(6):459. https://doi.org/10.1255/jnirs.1082.
Vincke D, Miller R, Stassart E, Otte M, Dardenne P, Collins M, et al. Analysis of collagen preservation in bones recovered in archaeological contexts using NIR hyperspectral imaging. Talanta. 2014;125:181–8. https://doi.org/10.1016/j.talanta.2014.02.044.
Koehler FW, Lee IE, Kidder LH, Lewis EN. Near infrared spectroscopy: the practical chemical imaging solution. Spectrosc Eur. 2002;14(3).
Doneus M, Verhoeven G, Atzberger C, Wess M, Ruš M. New ways to extract archaeological information from hyperspectral pixels. J Archaeol Sci. 2014;52:84–96. https://doi.org/10.1016/j.jas.2014.08.023.
ElMasry G, Sun D-W. CHAPTER 1 - principles of hyperspectral imaging technology. In: Sun D-W, editor. Hyperspectral imaging for food quality analysis and control. San Diego: Academic; 2010. p. 3–43. https://doi.org/10.1016/B978-0-12-374753-2.10001-2.
Firtha F. Argus hyperspectral acquisition software. 2010.
Prohaska T, Irrgeher J, Zitek A. Simultaneous multi-element and isotope ratio imaging of fish otoliths by laser ablation split stream ICP-MS/MC ICP-MS. J Anal At Spectrom. 2016;31(8):1612–21. https://doi.org/10.1039/c6ja00087h.
Brand WA, Coplen TB, Vogl J, Rosner M, Prohaska T. Assessment of international reference materials for isotope-ratio analysis (IUPAC technical report). Pure Appl Chem. 2014;86(3):425–67. https://doi.org/10.1515/pac-2013-1023.
Galler P, Limbeck A, Boulyga SF, Stingeder G, Hirata T, Prohaska T. Development of an on-line flow injection Sr/ matrix separation method for accurate, high-throughput determination of Sr isotope ratios by multiple collector-inductively coupled plasma-mass spectrometry. Anal Chem. 2007;79:5023–9.
Galler P, Limbeck A, Uveges M, Prohaska T. Automation and miniaturization of an on-line flow injection Sr/matrix separation method for accurate, high throughput determination of Sr isotope ratios by MC-ICP-MS. J Anal At Spectrom. 2008;23(10):1388. https://doi.org/10.1039/b803964j.
Romaniello SJ, Field MP, Smith HB, Gordon GW, Kim MH, Anbar AD. Fully automated chromatographic purification of Sr and Ca for isotopic analysis. J Anal At Spectrom. 2015;30(9):1906–12. https://doi.org/10.1039/c5ja00205b.
Woodhead J, Swearer S, Hergt J, Maas R. In situ Sr-isotope analysis of carbonates by LA-MC-ICP-MS: interference corrections, high spatial resolution and an example from otolith studies. J Anal At Spectrom. 2005;20(1):22. https://doi.org/10.1039/b412730g.
Irrgeher J, Galler P, Prohask T. 87Sr/86Sr isotope ratio measurements by laser ablation multicollector inductively coupled plasma mass spectrometry: reconsidering matrix interferences in bioapatites and biogenic carbonates. Spectrochim Acta B. 2016;125:31–42. https://doi.org/10.1016/j.sab.2016.09.008.
Draxler J, Zitek A, Meischel M, Stranzl-Tschegg SE, Mingler B, Martinelli E, et al. Regionalized quantitative LA-ICP-MS imaging of the biodegradation of magnesium alloys in bone tissue. J Anal At Spectrom. 2015. https://doi.org/10.1039/C5JA00354G10.1039/c5ja00354g.
Zitek A, Aleon J, Prohaska T. CHAPTER 9 chemical imaging. In: Prohaska T, Irrgeher J, Zitek A, Jakubowski N, editors. Sector field mass spectrometry for elemental and isotopic analysis: The Royal Society of Chemistry; 2015. p. 152–82. https://doi.org/10.1039/9781849735407-00152.
Schultheiss G. Analysis of isotope ratios in anthropological and archaeological samples by high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). Vienna: University of Natural Resources and Life Sciences; 2003.
Theiner S. The use of strontium isotope ratio measurements by MC-ICP-MS for fundamental studies on diagenesis and for the reconstruction of animal migration at the Celtic excavation site Roseldorf Diplomarbeit. Vienna: University of Vienna; 2011.
Swoboda S, Brunner M, Boulyga SF, Galler P, Horacek M, Prohaska T. Identification of Marchfeld asparagus using Sr isotope ratio measurements by MC-ICP-MS. Anal Bioanal Chem. 2008;390(2):487–94. https://doi.org/10.1007/s00216-007-1582-7.
Irrgeher J, Teschler-Nicola M, Leutgeb K, Weiß C, Kern D, Prohaska T. Migration and mobility in the latest Neolithic of the Traisen Valley, Lower Austria: Sr isotope analysis. In: Kaiser E, Burger J, Schier W, editors. Population dynamics in prehistory and early history. New approaches by using stable isotopes and genetics, vol. 5. Berlin, Boston: De Gruyter; 2012. p. 213–26. https://doi.org/10.1515/9783110266306.
Retzmann A, Zimmermann T, Pröfrock D, Prohaska T, Irrgeher J. A fully automated simultaneous single-stage separation of Sr, Pb, and Nd using DGA resin for the isotopic analysis of marine sediments. Anal Bioanal Chem. 2017;409(23):5463–80. https://doi.org/10.1007/s00216-017-0468-6.
Irrgeher J, Prohaska T, Sturgeon RE, Mester Z, Yang L. Determination of strontium isotope amount ratios in biological tissues using MC-ICPMS. Anal Methods. 2013;5(7):1687. https://doi.org/10.1039/c3ay00028a.
Horsky M, Irrgeher J, Prohaska T. Evaluation strategies and uncertainty calculation of isotope amount ratios measured by MC ICP-MS on the example of Sr. Anal Bioanal Chem. 2016;408(2):351–67. https://doi.org/10.1007/s00216-015-9003-9.
Burton JH, Price TD, Middleton WD. Correlation of bone Ba/Ca and Sr/Ca due to biological purification of calcium. J Archaeol Sci. 1999;26(6):609–16. https://doi.org/10.1006/jasc.1998.0378.
Osborne BG. Near-infrared spectroscopy in food analysis. In: Encyclopedia of analytical chemistry. Major reference works: Wiley; 2006. p. 1–14. https://doi.org/10.1002/9780470027318.a1018.
Wopenka B, Pasteris JD. A mineralogical perspective on the apatite in bone. Mater Sci Eng C. 2005;25(2):131–43. https://doi.org/10.1016/j.msec.2005.01.008.
White EM, Hannus LA. Chemical weathering of bone in archaeological soils. Am Antiq. 1983;48(2):316–22.
Tzaphlidou M, Zaichick V. Calcium, phosphorus, calcium-phosphorus ratio in rib bone of healthy humans. Biol Trace Elem Res. 2003;93(1):63–74. https://doi.org/10.1385/BTER:93:1-3:63.
Keenan SW. From bone to fossil: a review of the diagenesis of bioapatite. Am Mineral. 2016;101(9):1943–51. https://doi.org/10.2138/am-2016-5737.
Acknowledgments
The authors would like to acknowledge Maria Teschler-Nicola as former director of the Department of Anthropology at the Museum of Natural History, Vienna, who permitted the use and selected the human femur samples of this study in 2015. The authors would like to acknowledge Barbara Hinterstoisser from the University of Natural Resources and Life Sciences (Vienna, Austria) for enabling access to the NIR-HSI instrument. Very warm thanks to Ferenc Firtha from Szent Istvan University (Budapest, Hungary), who provided us with his expertise in NIR-HSI measurements/setup, who placed his software tools (Cubrowser, Argus) at our disposal and taught us how to use it. Furthermore, the authors would like to acknowledge the two anonymous reviewers for their positive and constructive feedback, which helped to improve this manuscript. Finally, we would like to thank Melanie Diesner and Tine Opper (VIRIS Laboratory) for their support in the lab.
Funding
This project was supported by the COMET-K1 competence center FFoQSI. The COMET-K1 competence center FFoQSI is funded by the Austrian ministries BMVIT, BMDW, and the Austrian provinces Niederoesterreich, Upper Austria, and Vienna within the scope of COMET - Competence Centers for Excellent Technologies. The program COMET is handled by the Austrian Research Promotion Agency FFG. We acknowledge the ERASMUS+ program for financial support.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Published in the topical collection Elemental and Molecular Imaging by LA-ICP-MS with guest editor Beatriz Fernández García.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(PDF 572 kb)
Rights and permissions
About this article
Cite this article
Retzmann, A., Blanz, M., Zitek, A. et al. A combined chemical imaging approach using (MC) LA-ICP-MS and NIR-HSI to evaluate the diagenetic status of bone material for Sr isotope analysis. Anal Bioanal Chem 411, 565–580 (2019). https://doi.org/10.1007/s00216-018-1489-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-018-1489-5