Elemental characterization of bones dating back to 17-18th century by ICP-MS and CV-AAS

(1)

1

UNIVERSITÀ DI PISA

Dipartimento di Chimica e Chimica Industriale

CORSO DI LAUREA MAGISTRALE IN

CHIMICA

Curriculum: analitico

Classe: LM-54 Scienze chimiche

Elemental characterization of bones dating back to

17-18th century by ICP-MS and CV-AAS

Relatore:

Dott.ssa Ilaria Degano

Controrelatore:

Prof.ssa Stefania Giannarelli

Candidato:

Paolo D’Imporzano

(2)

(3)

3

CHAPTER 1 STATE OF ART

9

1.1 FRANCISCAN FRIARY SAN FRANCESCO A FOLLONI IN MONTELLA 9

1.2 THE FRANCISCAN FRIARY OF SVENDBORG 12

1.3 CHARACTERISTICS OF BONES 14

1.3.1 BONE TISSUE HISTOLOGY 15

1.3.1.1 Organic fraction 16

1.3.1.2 Inorganic fraction 17

1.3.1.3 Compact tissue 18

1.3.1.4 Spongy tissue 19

1.3.2 BONE REMODELLING 19

1.3.2.1 Surface remodeling of bones 20

1.3.2.2 Internal bone remodeling 20

1.3.3 ELEMENTAL DISTRIBUITION IN SKELETON 21

1.3.3.1 Correlation between diet and bone element concentration 21

1.3.3.2 Incorporation of strontium and barium in the bone 22

1.3.3.3 Sr/Ca and Ba/Ca ratio 23

1.3.3.4 Mercury 25

1.4 DIAGENESIS 26

1.5 ANALYTICAL TECHNIQUES FOR ELEMENTAL ANALYSIS IN BONES 28

CHAPTER 2 EXPERIMENTAL

30

2.1 SAMPLES 30

2.1.1 Iannelli chapel samples 30

2.1.2 Montella friary sidewalk samples 33

2.1.3 Hardenberg chapel samples 34

2.2 CHEMICALS AND INSTRUMENTS 35

2.2.1 Chemicals and materials 35

2.2.2 ICP-MS 36

2.2.2.1 Preparation of calibration solution 37

2.2.3 FIMS 38

2.2.3.1 Preparation of calibration solution 38

2.3 SAMPLE TREATMENT 39

2.3.1 Sampling 39

(4)

4

2.4 DATA ANALYSIS 42

2.4.1 ICP-MS quantitative analysis 42

2.4.2 analysis of reference materials 44

CHAPTER 3 RESULTS AND DISCUSSION

.45

3.1 IANNELLI’S CHAPEL 45

3.2 MONTELLA INDIVIDUALS 48

3.3 HARDENBERG’S CHAPEL 53

3.4 STATISTICAL DATA ANALYSIS 55

3.4.1 Iannelli group 55

3.4.2 Montella sidewalk individuals 60

3.4.3 Hardenberg group 62

3.4.4 comparison among data sets 63

3.4.5 Mercury analysis… 71

CONCLUSION

74

(5)

(6)

6

RESUME

Inductively coupled plasma mass spectrometry ( ICP-MS) is an analytical technique that is able of detecting a wide number of elements with atomic mass ranging from 7 to 250 (from Li to U) at concentration levels as low as one part per trillion to 100 milligrams per liter, around 8 orders of magnitude of concentration units. These characteristics, achieved by ionizing the sample with inductively coupled plasma connected with a mass spectrometer, make the ICP-MS the ideal tool for elemental and trace-elemental analysis.

In this thesis, ICP-MS was used to carry out elemental analyses on bones dating back to XVII-XVIII century, in order to characterize the diet and life style of several people in their last years of life. This thesis is part of a project, carried out by the University of Southern Denmark, focused at understanding the nutritional habits of ancient populations in Europe. The measurements and data interpretation were performed at the University of Southern Denmark, Department of Physics, Chemistry and Pharmacy (Odense), under the supervision of dr.scient. Kaare Lund Rasmussen.

The bone samples were collected from 3 different sites: 14 come from a private chapel in the Franciscan friary of Svendborg and 81 come from the Franciscan friary in Montella (Italy). The Italian samples were collected from 2 different places of the friary: 35 from the tombs in the Iannelli’s family private chapel and the others were from burial in a sidewalk inside the friary

All the samples date back to the 1600-1700 ca., and the ones taken from the chapels were collected from individuals belonging to noble families.

For each bone sample, 21 elements were quantified with ICP-MS, and their Hg content was analyzed by CV-AAS, a dedicated instrument for Hg analysis.

In order to achieve information about life style, the data analysis was focused on specific elements that are known diet markers such Ba, Sr, Mg, Zn and Ca, and on some elements that can be environmental pollution markers as Fe, Mn, Pb. With the help of statistical treatment of the data, especially with the use of PCA (principal components analysis), the samples were divided in 3 main groups, which corresponded to the burial sites. The data analysis shows also that the samples collected from the chapels had an elemental composition suggesting that the people buried there followed a rich protein diet, which is consistent with their social status.

(7)

7

INTRODUCTION

Archaeological excavations aim at retrieving information about past societies. In particular, since several artifacts are human remains, direct insights about life in the past are provided by studying the skeletal remains, first from the archaeological context, then from an anthropological point of view and thirdly by chemical analyses. Chemical analyses of bones are particularly useful because through the knowledge of their chemical composition the chemist can achieve information about diet, life style and diseases. Studying bones allows the reconstruction of people’s life because skeleton plays a main role in human body. Bones not only provide mechanical function by protecting internal organs and giving structure support, but are also important for biological functions as blood production (haematopoiesis), mineral storage, fat storage, acid-base balance and detoxification processes (bones store toxic heavy metals). These metabolic pathways rule the distribution of minerals in bone tissues. Thus, chemical analyses can provide information about nutrition, exposition to pollution agents and diseases [1-5].

The information obtained from elemental analysis are valid only within the limits in which the elements concentration do not suffer any change during the burial time and is thus representative of ante-mortem mineral content. This condition is not always fulfilled, depending on burial ground conditions as pH, temperature and water. These phenomena in particular can lead to ion exchange between bones and the ground. All the processes that can change the original bones chemical composition are called diagenesis, which is the main problem regarding chemical analysis of archeological bones [6,7].

The techniques described for bones elemental analysis in the literature are atomic absorption spectroscopy (AAS) [8,9], atomic emission spectroscopy with inductively coupled plasma (ICP-AES) [10,11], mass spectrometry with inductively coupled plasma [1,12-15] and instrumental neutron activation analysis (INAA) [16-19]. INAA with its high sensitivity, wide dynamic range, low detection limits and its non destructive operation mode is be the best option for the analysis of archeological remains, but its high costs prevent its widespread use. The other three techniques are the most used, even if destructive. In this work ICP-MS was chosen due to its good balance between costs, operating time, sensitivity and detection limits. Cold vapor atomic absorption spectroscopy (CV-AAS) was also used to measure the concentration of Hg in samples.

The aim of this thesis is to analyze the bones of 59 people, all died in 1600-1700, originating from different countries and social classes, in order to assess how life style affects bones elemental composition. Moreover, we evaluated the possibility to apply statistical analysis to our data set to separate different groups of individuals according to their bone elemental composition. This work was carried out in the context of an anthropological and archaeological research project aimed at studying the eating habits of ancient peoples (paleonutrition). From a chemical point of view, paleonutrition studies are accomplished by quantifying those elements that can be used as markers for the determination of the food type of diet followed by the individual when he was alive. In particular, high levels of strontium, magnesium and barium are index of a predominantly

(8)

8 vegetarian diet, while zinc and copper indicate a mainly protein-based diet [43,48]. Hg analysis was also carried out to check if the people were treated for leprosy or syphilis, because since medieval age these diseases were cured with Hg based compounds [20].

(9)

9

CHAPTER 1 STATE OF THE ART

1.1 FRANCISCAN FRIARY SAN FRANCESCO A FOLLONI IN

MONTELLA

« Fu questo Venerabile Monisterio edificato nell'anno del Signore 1222 al tempo di Onorio terzo sommo

pontefice, e propriamente nell'anno settimo del suo pontificato: nell'anno 12 dell'imperio di Federico Secondo detto Barbarossa e nell'anno quarto decimo della Religione de' Minori. Fin dalla sua origene il Signore

dimostròssi impegnatissimo a proteggerlo; anzi con un stupendo miracolo volle, e dié motivo alla sua fondazione »

(Platea Venerabilis Conventus Sancti Francisci, Sebastiano Guerruccio, 1740-41)

San Francesco a Folloni friary is located on the edge of the city Montella up on the slope of the Monti Picentini, a mountain range in Southern Italy. Montella is located in Avellino province, which is part of the Campania region, which includes the city of Naples, Vesuvius and Pompei and Sorrento-peninsula with the famous Amalfi Coast. San Francesco a Folloni friary owes its name to the place where it was founded, Folloni woods, by San Francesco himself, during his trip to San Michele sul Gargano sanctuary in January 1221-2. The story tells us that Francesco and his friars could not find any hospitable people who would host them, so the Saint decided to spend the night under an oak in the Folloni forest. In spite of heavy snowfall at night, it turned out that the next morning the wood under which Francesco had spent the night was untouched by the snow. The news quickly spread by word of mouth and locals demanded to establish a monastery. Thus San Francesco d’Assisi left two brothers by the tree, and they built a monastery dedicated to the SS Annunziata. The oak is reportedly hidden in the foundations of the monastery.

Just two years later another miracle happened, also involving an heavy snowfall: after some days of snowing, the friars in the monastery had nothing left to eat. Even this time no one brought them food from the city because it was impossible to walk through the deep snow. Suddenly, there was a knock at the abbey gate, but when the monks opened there was no one outside. Instead there was a bag filled with fragrant bread. The monks were looking for their savior’s footprints in the snow, but there was none. The bread was in a sack with the French lily. At that moment Francesco d’Assisi was at Louis VIII’s court, and the legend says that the Saint had entrusted bread to angels for his brothers, asked to the French King by Francesco for charity. The canvas of the bag was kept for three centuries as a tablecloth altar. In the sixteenth century it was cut in pieces and distributed as a relic in many churches and monasteries. After

(10)

10 the friary was suppressed under the Napoleonic government, the leftover relic was moved to the Chiesa Madre di Montella. In 1828, after the reopening of the friary, the bishop of Nusco decided to divide the relic between Montella Mother Church and that of St. Francesco at Folloni. In 1998, Brother Agnello Stoia managed to find the last vestiges of the bag, and the relic is now preserved in a reliquary made on purpose and placed in the Cappella del Crocifisso, on the right of the altar of the church. The original monastery was built at the turn of the XIII century, and the first written document that proves the existence of the monastery is dated January 5, 1322. The present monastery was built in the mid-1700s, because the old buildings collapsed during the great earthquake in 1732. A church was built in Baroque-Roccocò style, rotated by 90° with respect to the previous East-West oriented church [21-23]. In 2009 four bachelor students from the Duke University went to Montella where they cooperated with the monastery's prior to establish an animated reconstruction of the history of the monastery and its buildings. The reconstruction tells “the story of the early Franciscan architecture as an additive process propped (or even driven) on donations from private citizens who demanded burial in the church or in monastery walkway”. The monastery has been gradually expanded as the need arose and as new benefactors joined on the field, and the entire monastery complex has been developed over a period of several hundred years [24,25].

In this project we measured the elemental bone composition of individuals buried in a private chapel, who belong to Iannelli’s family, and in the inner sidewalk of the monastery.

In Figure 1.1.1

(11)

11

(12)

12

1.2 THE FRANCISCAN FRIARY OF SVENDBORG

The Franciscan friary of Svendborg, Figure 1.2.1, dedicated to St Catherine, was founded in 1236 as one of the earliest friars in Denmark. In 1247 the building was destroyed when the town was burned to the ground by King Abel of Denmark. A new friary was built in 1288 as the result of a gift of land for a chapel for the Franciscans from Herr Astrad Frakke, Herr Niels Bille, Herr Niels Beger and several other local nobles. In 1268 Lady Gro Gunnarsdatter Vint, the extremely wealthy widow of Esbjørn Vognsen, gave away her considerable fortune to abbeys, priories and friaries throughout Denmark when she joined the Poor Clares in Roskilde. The "brothers' chapel at Svendborg" is specifically mentioned in the list of her beneficiaries. In 1332 Minister Laurens Jonsen gave to the monastery the ownership of Sankt Jorgensgarden as long as lepers received wine, bread, oil and wax for religious services from Franciscans. The gothic church was built in 1361 and dedicated to St. Catherine. In 1388 a chapel in the church was founded by Niels Finsen, his son Fin Nielsen, Fin Aagesen Ulfeld, Niels Bicker, Niels Bild and their wives. In 1389 the city was destroyed again by Hanseatic Fleet, and then the friary was rebuilt again. During the 15th century it expanded considerably, and the buildings were extended several times. The main friary precinct of this period consisted of a rectangular enclosure containing a church, dormitory, refectory, and servants' quarters, as well as a cloister surrounding a central garden. The buildings were constructed in red bricks, the most common building material in Denmark at the time. The friary had a close connection with St. George's Chapel and Hospital just outside Svendborg, the last remaining medieval leper hospital in Denmark. Queen Chaterine’s household accounts show her favor towards the Franciscans and her considerateness was probably characteristic of the alms-giving of the upper classes. In 1500 Queen Christina, who was at that point in direct control of Svendborg, gave the whole of Bysen Street to the Franciscans to use as accommodation for the town's poor and sick in their care. In 1505 a great sum was paid out to a skipper from Rudkobing for conveying 6000 bricks from Langeland to Svendborg.

During the early phase of the reformation, the mendicant orders were hardly hit, starting with the expulsion of the gray friars in 1528, and after 4 years only 7 of the 26 Franciscan friaries were left. In 1530 king Frederik I transferred the church and friary to the mayors and corporations so that a hospital could be set up in the buildings. The friary church became a parish church for the people of Svendborg. The East wing was demolished shortly after the Reformation. The West wing of the priory was converted into a Latin school that operated until 1740. The buildings where the Latin School were located were torn down in 1875. In 1586 the North wing of the friary was turned into a hospital founded by Lady Helvig Hardenberg. The old hospital was torn down in 1870. In the Renaissance period, at the end of 16th century, the Hardenberg chapel was built to the North of the choir and since then the church seems not to have undergone great changes. At the time it was still used for burials, including in the floor

(13)

13 and in the Hardenberg chapel. By 1828 the former friary church fell into serious disrepair and the town council decided to demolish it. The buildings were blown up, the ground leveled and the site in the town centre redeveloped with houses and shops: some buildings used parts of the old friary walls as foundations. The churchyard containing thousands of common and noble graves was cleared and the most recently buried bodies transferred to a cemetery a few streets away. Today a train station is located where the monastery was built.

The samples analyzed in this work were collected in the Hardenberg chapel, which was a small almost squared structure (5.6 x 6 m) built of bricks on a granite plinth. The entrance was through the above mentioned door from the choir of the church. The only illumination seems to have been from a elliptically-headed window whit an inner rabbet in the north gabble.

The chapel itself had almost disappeared but a burial crypt was discovered inside the chapel, its inner measurements were 4 x 2.6 m, the walls were constructed of brick one layer thick and built up against the sides of the pit dug for the crypt. The floor was paved with unglazed tiles. Both chapel and crypt were used for burials, and the name of the chapel suggests that it was erected by Helvig Hardenberg (1540-99) a widow who owned part of the friary in 1586 [26,27].

Figure 1.2.1 Illustration of Svensborg Friary just prior to demolition in 1828. Watercolor by C.F. Thorin 1828, now at the National Museum of Denmark

(14)

14

1.3 CHARACTERISTICS OF BONES

Bone, like other connective tissue, consists of cells, fibers and ground substance, but unlike the others, its extracellular components are calcified, making it an hard and unyielding substance suited for its supportive and protective functions.

Bones provide for the internal support of the body and for the attachment of the muscles and tendons essential for locomotion. They protect the vital organs of the cranial and abdominal cavities, and it encloses the blood-forming elements of the bone marrow. Skeleton also plays an important metabolic role as a mobilizable store of calcium, which can be drawn on as needed in the homeostatic regulation.

Bone possesses a remarkable combination of physical proprieties: high tensile and compressive strength, elasticity and moreover it is a relatively lightweight material. Bone structure ensures great strength with economy of material and minimal weight. Skeleton, despite its hardness and strength, is a living organ ant it is constantly renewed and reconstructed during lifetime. The outside part of the bone consists of a layer of connective tissue called the periosteum. Under this part, the outer shell of bone is constituted by a tissue called compact bone that surrounds the inner part called cancellous bone (spongy bone) which contains red bone marrow. The interior part of the long bone is the medullary cavity and it is filled with yellow marrow in adults, and red marrow in young children, Figure 1.3.1

(15)

15

1.3.1 BONE TISSUE HISTOLOGY

Upon a macroscopic inspection it is possible to see two different kind of bone tissues,

compact (substantia compacta) and spongy or cancellous bone (substantia spongiosa).

The latter consists of a three dimensional lattice of branching bony spicules, or trabeculae, delimiting a labyrinthine system of interspaces that are occupied by bone

marrow. The compact bone appears as a solid continuous mass in which spaces can be

seen only with the aid of a microscope. The two forms of bone grade into one other without a sharp boundary Figure 1.3.1.1.

To a microscopic level, bone tissue, it is composed by a large amount of extra-cellular matrix that surrounds and divides all the cells in the bone. In living bones the matrix consist in 15% water, 30% organic matter and 55% in crystallized mineral salt.

(16)

16

1.3.1.1

Organic fraction

The organic phase of the bone is formed by collagen, for the 98% , and it is responsible of bone elasticity, tensile and torsion strength. The remaining 2% it is composed by different kind of cells, the most important are osteoprogenitor, osteoblasts, osteocytes and osteoclasts Figure 1.3.1.1.1.

Osteoprogenitors: like other connective tissues, develops originally from embryonic

mesenchymal cells that have a very board range of development potentialities. Along the pathway of their differentiation into bone-forming cells, a population of them become units that have more limited potential. These are able to proliferate only in chondroblasts and osteoblasts. Osteoprogenitors cells persist throughout postnatal life and are found near all of the free surfaces of bones. Their nuclei are pale-staining and oval or elongated and their scant cytoplasm is acidophil or faintly basophilic. Osteoprogenitors cells are most active during the growth of bones but are reactivated in adult life in order to repair bone fractures and other forms of injury.

Osteoblasts: Osteoblasts are the bone forming cells present in developing and mature

bones. During active deposition of new matrix, they are arranged as an epithelioid layer of cuboidal or columnar cells on the bone surface, the nucleus is usually located at the end of the cell, farthest away from the bone surface. The cytoplasm is intensely basophilic and a prominent Golgi complex appears as a paler staining area between the nucleus and the cell base. With the periodic-acid-Shiff reaction, a number of small cytoplasmic vacuoles are found contain pink-staining material believed to represent precursor of bone matrix. Although osteoblasts are polarized toward the underlying bone, the release of their products is apparently not confined to the basal pole because some cells among them gradually become enveloped by their own secretion and are transformed in osteocytes, imprisoned in lacunae within the newly formed bone matrix.

Osteocytes: These are the principal cells of mature bone, which reside in lacunae

within the calcified matrix. Their cell body is flatten conforming to the shape of the lenticular cavity that they occupie, but there are numerous slender cell processes that extend for some distance in canaliculi that radiate from the lacuna in the surrounding matrix. This canaliculi make sure that osteocytes are not isolated in their lacunae but are in communication with one another, and with cells in surface via a series of junctions of low electrical resistance, permitting flow of ions and possibly of small molecules from cell to cell. Nuclear and cytoplasmic characteristics of osteocytes are similar to the osteoblasts, except that the Golgi area is less conspicuous and the surrounding cytoplasm exhibits less affinity for basic dyes.

Osteoclasts: Throughout life, bone undergoes a continuous process of internal

remodeling that involves removal of bone matrix at multiples sites, followed by its replacement by newly deposited bone. In this process, the agents of bone resorption are osteoclasts, huge cells containing as many as 50 nuclei. These cells are located in shallow concavities, called Howships lacunae, produced by erosive action of the osteoclast on the underlying bone. Osteoclasts are long lived, but they are not

(17)

17 continuously active. A response to an unusual metabolic demand for mobilization of calcium from bone does not imply a generation of new osteoclasts but only the activation of quiescent members of these cells. When the demand for calcium has been met, osteoclasts revert again to a resting phase.

Figure 1.3.1.1. 1 Bone cells

1.3.1.2 Inorganic fraction

The inorganic phase of the bone matrix gives power and mechanical resistance to the bones. It is composed by many mineral salts, the most abundant is calcium phosphate [Ca3(PO4)2], that reacts with calcium hydroxide [Ca(OH)2] to give hydroxyapatite

crystals [Ca10(PO4)6(OH)2]. In mature bone hydroxyapatite is in the form of slender

rod-like crystals about 40 nm in length and 1.5-3 nm in thickness. The crystals are not randomly distributed but disposed at regular intervals of 60-70 nm along the length of the collagen fibers. The mineral bone hydroxyapatite is not pure but contains many impurity, as citrate ions C6H5O73- and the ion carbonate CO32- , the last one can be

situated on the crystal surface or it can replace PO42- in the crystal structure.

Substitution of fluoride ion F- for OH- in the apatite crystal is common, and the amount of fluoride in bones depends mainly on the concentrations of this element in drinking water. Mg and Na, which are normal constituents of the body fluids, are also present in bone hydroxyapatite, which serves as a storage depot for these elements. The isotopes 45Ca and 32P in the hydroxyapatite can situate for the stable 40Ca and 31P in the crystal. Cations, such as Pb2+, Sr2+ and 226Ra2+, may also substitute for Ca2+. In the fission of uranium and plutonium during the detonation of nuclear weapons, a large number of radioactive elements are released. When these elements reach the body, some of them are incorporated in bones. During growth, the amount of organic material per unit volume of bone remains relatively constant, but the amount of water

(18)

18 decreases and the proportion of bone mineral increases attaining, in adults, a maximum of about 65% of the fat-free dry weight. If bone is exposed to a weak acid or a chelating agent, the inorganic salt are removed. Compact tissue and spongy tissue, the two tissue that constitutes the bone, are about the 80% and 20% of the skeletal mass.

1.3.1.3 Compact tissue

The compact tissue, also called cortical or dense, is situated on the surface of the bone. It is formed by a large number of structural units called osteons (Figure 1.3.1.3.1). These units are made up of concentric lamellae arranged around a central channel, also called Haversian canal. These straws are nothing more than circular plates of mineralized extracellular matrix surrounding a small network of blood vessels, lymphatics vessels and nerves. Between one lamellae and the other there are small spaces that are called lacunae, in which osteocytes are contained . From these spaces canaliculi spread in all directions, canaliculi are small channels containing extracellular fluids, that connect the lacunae between them and with the central channel, creating a small system of grooves through the bone, which allows the oxygen and nutrients to reach osteocytes .

(19)

19

1.3.1.4 Spongy tissue

The spongy tissue, also called trabecular, shows difference from compact tissue mainly for the lacks of osteons. This tissue is always situated in the bone inner part, and it is surrounded by cortical tissue. Trabecula bone is constituted by lamellae, that are arranged in an irregular manner in narrow columns Figure 1.3.1.4.1. Among these there are wide areas visible to the naked eye, which are filled with red or yellow bone marrow. The distinction between compact and spongy tissue can be discussed on the basis of their porosity. In fact, while in the compact tissue porosity varies between 5% to 20% (mainly due to the Haversian canal, and to a lesser extent of lamellae and canaliculi), in the trabecular one the porosity goes from 40% even up to 95% (the space between the trabeculae typically ranges from 100 to 500 µm).

Figure 1.3.1.4.1 Head of femur section

1.3.2 BONE REMODELING

Such as skin, bone tissue is formed before birth and then continues to renew for the entire life of the individual. The replacement of old tissues with the new ones takes the name of bone remodeling, and involves two mechanisms closely joined: resorption, that occurs when minerals and collagen fibers are removed by osteoclasts, and the deposition, that consists in the addition of minerals and collagen by osteoblasts. This process is very important because allows the bone to be renewed in order to adapt to the external stress and furthermore allows to remove any damaged parts replacing them with the new tissue. In addition, the remodeling plays an important role in mineral homeostasis, by activating hormone-type controls, and releasing calcium.

(20)

20

1.3.2.1

Surface remodeling of bones

Although growing bones are continuously changing their internal organization, they retain approximately the same external form the whole life. In fact the shape of the bone is maintained during growth by a continual remodeling or sculpturing of its surface, which involves bone deposition under some areas of the peristeum and bone absorption in other areas. It means that peristeum plays opposite roles in neighboring regions of the same bone when the remodeling of it is in progress. Subperiosteal bone deposition is occurring in the cylindrical shaft while subperiosteal bone resorption is taking place in the conical region. Similarly bone is formed at the endosteal surface of the conical region and adsorbed on the inner part of the cylinder. As a result of these processes, the midportion of the shaft is expanding radially and its marrow cavity is being enlarged. As the bone is elongating by growth at the epiphyseal plate, the diverging walls of the conical region of the shaft are straightened by bone deposition on its outer surface. In this way the lengthening of cylindrical portion of the shaft is guaranteed while general shape of the bone is maintained.

1.3.2.2

Internal bone reorganization

The conversion from the primary lattice of trabeculae, produced by intramembranous ossification, into compact bone is attributable to thickening of the trabeculae and the progressive encroachment of bone on the perivascular spaces. As this process advances, bone is deposited in ill-defined layers containing randomly orientated collagen fibers, but due to the fact that these layers are disposed more or less concentrically around the vascular channel, they bear a superficial resemblance to harversian system. They are sometimes called primitive harversian system, but they should be clearly distinguished from the more precisely ordered lamellar system comprising the definitive harversian system in adult bone. The latter are first formed only during the internal reorganization of primary compact bone that is referred to as secondary bone formation. In this process, cavities appear in the primary compact bone as a result of local osteoclast activity. Such absorption cavities are enlarged by continuing osteoclastic activity to form long cylindrical wich are invaded in blood vessels growing out from the embryonic bone marrow. When the cavities reach considerable length, bone absorption ceases and the osteoclast are replaced by osteoblast that begin to deposit concentric lamellae of bone on the wall of the cylindrical cavity until it is filled on to the form a typical harversian system. Internal bone resorption and reconstruction don’t end with the replacement of the primary by secondary bone, but continue actively throughout life. The formation time of an haversian system in the adult is 4-5 weeks, but different values are obtained in young growing bone and in pathological states. The newly deposited lamellar bone continues to calcify over a considerable period of time after osteon is completed [27].

(21)

21

1.3.3 ELEMENTAL DISTRIBUITION IN SKELETON

The inorganic composition of bones changes according to the type of bone and the elemental distribution may also changes within the same bone. An example of the latter statement can be seen in Figure 1.3.3.1, where are reported the results of a study in the literature [28], which shows how the concentrations of various elements vary along a humerus. In particular it is shown how the values are approximately constant in the central part of the bone (epiphysis), as was expected, since this is the portion less affected by the process of remodeling.

Figure 1.3.3.1 Elemental distribuition in long bone

1.3.3.1

Correlation between diet and bone element concentration

Carbon, Nitrogen, Hydrogen and Oxigen constitute the building blocks of all human organic molecules, and are found in large quantities in the human body [29]. Other elements, called trace elements, constitute less than 0.1% of body mass and can be divided into four groups: essential, potentially essential, non-essential and toxic trace elements. Fifteen trace elements are classified as essential, including iron, zinc, manganese and selenium. To the toxic trace elements belong lead, mercury and cadmium, while Sr and Ba are not essential trace elements.

Every element found in the human body can be traced to a specific class of food in which this element is particularly concentrated. Therefore, considering the

(22)

22 concentration levels of these elements it is possible to go back to the diet followed by the individual. The main trace elements used as food marker are: strontium, magnesium and barium, as indicators of a mostly vegetarian diet, and zinc and copper, as indicators of a type of protein diet .

Strontium is one of the most studied within paleonutritional analysis. It is a non-essential element, it means that does not take part in metabolic processes that could vary or rule its concentration. Strontium is contained entirely within the bones, as calcium substitute because of their similar chemical behavior. In addition, strontium is scarcely subject to diagenetic phenomena [30]. It is particularly concentrated in leafy green vegetables such as spinach, chard, arugula, cauliflower and cabbage, which absorb it from the soil through the roots, but it can also be found in large quantity in small fish and shellfish, both marine and fresh water [31-33] .

Zinc is an important indicator of protein intake. In this case we are dealing with an essential element, that takes part in many metabolic processes, and whose changes are carefully controlled by the body. This translates to less variations in its concentration in bone when an individual changes his diet. It is found mainly in legumes, red meat, milk, dairy products and also in molluscs and crustaceans of marine origin [34].

Copper, as well as zinc, is an element related to high protein intake and mainly due to the consumption of offal and shellfish. Magnesium is linked to the assumption of vegetables, legumes and cereals consumed without milling [35]. Are also particularly rich in Magnesium the wheat germ flakes and oatmeal, corn, beans, lentils and some types of crustaceans [36]. Unfortunately, both of these elements, Cu and Mg, are often subject to diagenesis [37]. Barium, which has similar chemical properties to Strontium, is considered a good indicator for vegetarian diet [38], and most of all it allows to discriminate marine food, which has high levels of strontium but is poor in barium [39] .

Other elements that are commonly considered are aluminum, iron, manganese and potassium which suffer strongly phenomena of diagenesis, and therefore are most useful for assessing any changes post mortem. Lead analysis is also interesting, because this element is mainly assimilate by the body due to the pollution of food and drink caused by the use of cooking utensils and storage containers. This allows to obtain useful information on the methods of preparation and storage of food [40] .

1.3.3.2

Incorporation of strontium and barium in the bone

Strontium fate in biological systems has been extensively studied, especially in the 1950s and 1960s, when there was concern about the biological effects of the radioactive and carcinogenic 90Sr isotope, emitted in the atmosphere by the numerous atomic test which was performed after World War II [41]. Concern with 90Sr occurred because Strontium behaves like Calcium in the food chain, it easily absorbed by plants, animals and people along the same paths as Calcium [42]. Barium it is absorbed by living organisms as well [43,44]. This process occurs due to the effective ionc radius of Ca2+, Ba2+ and Sr2+, that is roughly equal: in fact in hydroxyapatite Ca2+

(23)

23 has a coordination number of 8 and the effective ionic radius is 1.12 Ǻ. Sr2+ and Ba2+, have similar characteristics with coordination number 8, and an effective ionic radius of 1.26 and 1.42 Ǻ [45]. This explain how strontium and barium, as other elements of the second group of the periodic table, (that can form bivalent cations) can be substituted in hydroxiapatit structure during the remodeling of bones [46].

Ca, Sr, and Ba are largely present in various minerals and salts dissolved in the sea. The main minerals that contains these elements are dolomite, celestit, strontianite and barite. The natural abundance of Calcium, however, is much greater than Strontium and Barium [47]. The soil may have elements leach into groundwater or absorbed by plants and thereby become part of food chain. People assume Strontium and Barium through diet by drinking ground water, eating plants that have taken these elements from the soil or eating animal that has assumed this elements from the soil or plants [43,48]. One example of a very simple food chain could be: grass-cow-man. However, Strontium and Barium decrease from one trophic level to a higher trophic one because the absorption of Calcium, rather than Sr or Ba, is favored. In fact strontium and barium, due of their larger ionic radius than calcium, find more problem to pass through the cell membrane, thus the intestine can detain these elements and isolate them favoring the absorption of calcium.This may explain why plants contain more Sr and Ba than herbivores that contain more of these elements than carnivores that eat them. Omnivors, as man, who eat both plants and meats, lie somewhere in between herbivores and carnivores, and this will be reflected in the bones Sr and Ba levels [43].

1.3.3.3

Sr/Ca and Ba/Ca ratio

Because Sr and Ba concentration is higher in plants than meat, paleonutritional studies have attempted to determine if past people have been eating mostly one of them by studing Ca, Sr, and Ba concentrations in bones. However, it is a rough simplification to assume that the various diet components are reflected proportionately in the skeleton. Especially for people whose nutrition usually consists in several different dishes, the picture is complex. If diet contains a lot of calcium, it will hinder the inclusion of strontium and barium, although the diet may be rich on these elements [49]. If diet, for example, consists of two components, meats and beans, the mineral-rich beans mask meat, so long as meat constitute less than 60% of the diet Figure 1.3.3.3.1.

(24)

24

Figure 1.3.3.3.1 log (Sr / Ca) for a diet of meat and potatoes (Kød og kartofler) or meat and bean (Kød og bønner)

Therefore, Sr/Ca and Ba/Ca ratio is not suitable to know the precise diet follows by the analyzed individuals, but can give general diet information useful to compare different groups of individuals. In fact Sr/Ca and Ba/Ca ratios are able to distinguish different populations on specified local elemental conditions that existed in the area where they lived. In a comparison of the southern German populations from early medieval age there was a clear difference on the Sr/Ca ratio of people from the mountainous regions and people from low-lying areas [50,51]. Sr/Ca ratio was lower for the population in upland, indicating a Calcium rich diet containing animal feed items as milk, probably because the mountain life allowed cattle and pasture economy. People from low-lying areas had higher Sr/Ca ratios, indicating a greater proportion of plant food items in the diet, probably because arable cultivation has played a major role on the fertile soil in lower-lying areas. Sr/Ca and Ba/Ca ratios are also used to detect difference within the same population, leading to know the affiliation to a particular social status. Often difference in social status, in early medieval populations, are reflected in bones. In a study of skeletons from the southern German town of Weingarten, individuals belonging to high social class are distinguish by calcium-rich diet (lower Sr/Ca and Ba/Ca ratios) [52]. Reverse, the majority of individuals belonged to lower social rank, are recognizable by mineral diet (higher Sr/Ca or Ba/Ca ratio), probably more based on vegetable products Figure 1.3.3.3.2. Another study carried on in Wenigumstadt, Southern Germany, based on Sr/Ca ratio shows correlations between nutritional habits and social status affiliation within the same population [53].

(25)

25

Figure 1.3.3.3.2 Sr/Ca- and Ba / Ca ratios in skeletons from Wenigumstadt. The individuals belong to different familys

1.3.3.4

Mercury

Mercury was used in Europe in many fields since medieval times, this allow this element to be present in the human body for several reasons since then.

Victims of leprosy and syphilis have pronounced expression on the skin, and it is known that diseases visible on the skin were treated with mercury-containing compounds, at least since late medieval times.

It is well known that mercury sulphide was used throughout medieval Europe as a color pigment for the bright red ink used extensively in illuminated manuscript.

Mercury can be accumulated in human bones, but the mechanism is not yet clear. Presumably the Hg2+ ion substitutes for Ca2+ in the bone carbonate or hydroxyapatite [54] although it is also possible that other species of mercury, e.g. Hg(CH3)2 could be

present in the organic parts of the bone. It is considered a general fact that the concentration in bones is lower than in many other tissues of the human body. This is supported by Garcia et al. who found concentrations of Hg in human bone remains be lower than 0.05 µg /g, whereas they found levels of 0.25 and 0.14 µg/g in kidney and liver, respectively [55]. Very few studies have reported Hg levels in exhumed human bones. Studies measured the Hg concentrations in Japanese individuals from two different burial sites and from two different time periods (6–7th century and 12–17th century). They found concentrations ranging from 175 to 1700 µg/g in a few cases. They also measured the concentration of Hg in soil from the graves. In most cases the Hg concentration in the soil was below the detection limit. When this was not the case,

(26)

26 there was no correlation between soil and bone concentrations [56]. Other studies investigate on Danish monks and normal people, focused on three different groups of people potentially exposed to mercury. The first two groups were victims of leprosy and syphilis and the third group investigated consists of individuals, presumably friars or monks, who lived at the friary or monastery and that can have access to the scriptorium, where mercury based ink were used, or they may have access to the medicine in order to heal sick people [57]

1.4 DIAGENESIS

In order to produce meaningful results, it is necessary that the concentration of the elements within the bone is the same ante-mortem. This unfortunately is not always true due to post mortem changes in the chemical composition that go under the name of diagenetic effects [58]. Diagenesis is "a set of physical, chemical and biological

changes that acts on all the archaeological buried finds; these processes alter the chemical or structural original object” [59]. From this it is clear that this

phenomenon constitutes the biggest problem related to the elemental analysis of archaeological samples. Indeed, no element is immune to this type of alteration, although diagenesis does not affect all elements in the same way.

The causes related to this phenomenon are numerous. It is mostly related to soil conditions, such as pH and temperature (at pH less than 4 Hydroxyapatite is very soluble, there is dissolution and recrystallization of the inorganic part of the bone) [60], which favor the dissemination and exchange of certain elements. Moreover, the presence of metal artifacts or burial gifts is a significant source of pollution, and therefore the bones found in the vicinity of certain metallic objects are not suitable for paleonutritional analysis. No analyses of archaeological remains are possible without an assessment of the presence of diagenesis. Determination of the Ca/P ratio provides to understand if pollution occurs. In fact calcium is one of the elements more prone to diagenesis, and if the value of Ca/P ratio is contained around 2,16 [61,62], it means that hydroxyapatite is good conserved. The value 2.16 is the Ca/P ratio present in modern body hydroxyapatite . Another method widely used for bone analysis, is to compare the data obtained from human bone with those obtained from the skeletons of carnivorous and herbivorous animals present within the same burial site. This method, called "correction with the site”, allows to notice any interference in the performance of elementary profiles. If the levels of the elements do not coincide with the theoretical profiles (eg strontium in human bone should be at an intermediate level between herbivorous animals and those carnivores, reflecting the omnivorous character of man) then the information obtained may have been obscured by the diagenetic effects [63-64]. Others ways to understand if pollution occurs are the comparison of the bones remain data with data obtained from modern bones analysis (they provide an indication of the average elementary concentrations in the bones), and the study of the

(27)

27 soil of arrangement (assessing the possible ion exchange between bone and surrounding land). Furthermore, it may be desirable to relate each element analyzed with calcium (chosen because the main constituent of the bone matrix), in order to standardize the data and to mitigate the influence of any contaminants [63,65,66]. The report Element/Ca assumes that each enrichment or loss of bone Ca corresponds enrichment or a loss in the other elements. In reality, this condition is not always verified, due to the predisposition of calcium to diagenesis [67], especially if we consider the relationship with the elements undergo less diagenetic effect. The use of compact bone instead of spongy bone is a good way to mitigate the diagenesis phenomena. Compact tissue presents a lower outer surface (the point at which the effects are more concentrated diagenetic), reducing the extension of the exchange with the ground. Another way to reduce diagenesis effect is take advantage of the greater solubility, in weak acid solution, of the bone that has undergone to diagenesis, thus this part can be removed with a series of washes with a weakly acidic solution. This method takes the name of "profile of solubility" [68,69] and has been especially developed for the study of Strontium. An example of a profile of solubility tract from the literature is shown in Figure 1.3.3.4.1.

Figure 1.3.3.4.1 solubility profile

The porosity of the bone can be also used as a measure of the dissolution of the hydroxyapatite crystals occurred due to diagenetic effects [70]. Soil sample analysis can be help-full to exclude the possibility of migration of elements from the bone to the soil [71]. Other studies measured the content of copper, calcium, iron, zinc and lead in human bones from the Cartagena region. The bone samples were from different historical times and recent times. Their results showed an increased amount of elements in the bones through time [72].

(28)

28

1.5 ANALYTICAL TECHNIQUES FOR ELEMENTAL ANALYSIS IN

BONES

The analysis of the trace element in bones found in archaeological field mainly aims at reconstructing ancient peoples dietary habits. This is demonstrated by the numerous studies reported in the literature. The techniques most frequently used for this purpose are: the atomic emission spectroscopy with inductively coupled plasma (ICP-AES) [10,11], atomic absorption spectroscopy (AAS) [8,9], instrumental neutron activation analysis (INAA) [16-19] and inductively coupled plasma – mass spectrometry (ICP-MS) [12-15]. The most important feature of these four techniques is in the possibility of realizing multi-elemental analysis, in order to determine more elements simultaneously and thus, considering the high number of components often searched within the bones, to reduce the time of analysis and the risk of errors or contamination. Among the proposed methods, the ICP -AES supplanted in time the AAS. ICP-AES is a robust technique with a relatively low cost, and most of all allows numerous elements to be analyzed simultaneously, with good detection limits, better than those of the atomic absorption, as can be seen by comparing the values reported in Table 1.5.1

Element AAS ICP-AAS ICP-MS INAA

Al (ppm) 2 0.7 0.1 1 Ba (ppm) 10 0.01 0.02 1 Ca (ppm) 1 0.005 5 100 Cu (ppm) 1 0.3 0.03 0.01 Fe (ppm) 1 0.2 0.2 100 Na (ppm) 2 0.3 0.06 0.1 Mg (ppm) 0.03 0.02 0.1 10 Mn (ppm) 0.2 0.04 0.04 0.01 Pb (ppm) 0.5 0.8 0.02 1000 Sr (ppm) 1 0.02 0.02 10 Zn (ppm) 0.02 0.06 0.08 10

Table 1.5.1 Comparison between detection limit of the main techniques used for bone elemental analysis

Similar results and, indeed, even better in many cases can be obtained through the use of ICP-MS, which has a still higher sensitivity compared to ICP-AES for almost all the elements of interest.

Due to high sensitivity of the ICP-MS technique, proper dilution of the sample becomes crucial, in order to obtain the best results for all the elements searched, avoid signal saturation. The INAA technique, finally, differs from the other in many respects. First, unlike the above described ones that require the dissolution of the

(29)

29 sample in solution, INAA is a non-destructive technique that does not require any pre-treatment stage (except for the removal of soft tissue from the bone). This is a crucial feature, considered the high value that certain artifacts may have, especially those of the archaeological origin. In this technique the sample is irradiated with neutrons, giving rise to radioactive isotopes of the elements present, which decay by emitting γ radiation having a wavelength characteristic of each element. Therefore the measurement of the energy of the emitted radiation allows to obtain qualitative information on the elements. Moreover, the intensity is related to the amount of the element that decays, from which one can derive the concentration of the starting element . Unfortunately, the cost associated with this type of analysis is very high , as it is required a highly specialized structure to employ neutron-based spectroscopies. In recent times, for the elemental analyses methods based on the use of laser sources are proving more and more successful, mainly including the LIBS (Laser Induced Breakdown Spectroscopy) [73] and LA- ICP -MS ( laser ablation ICP-MS) [74-76]. Compared to traditional techniques, the latter require a minimal amount of sample for analysis and do not require special pretreatments of the sample. In this way it is possible to obtain analytical data less affected by errors, in less time and with a minimum consumption of sample. Furthermore, since the analysis is only focused on a small spot on the sample (the diameter of the crater created by the laser is of the order of tens of µm), it is possible to obtain information on elemental distribution both along the entire surface (space scanning) and along the sample thickness (depth profile) [77,78]. This type of data is very useful in the analysis of archaeological bones, where the spatial profile allows to detect possible signs of diagenesis.

Mercury is usually measured by cold vapour atomic absorption on a dedicated mercury analyzer. This system features better detection limits for Hg than the ICP-MS [79].

(30)

30

CHAPTER 2 EXPERIMENTAL

2.1 SAMPLES

The samples analyzed in this work come from two different countries, Italy and Denmark. The samples from Italy come from two different burials in the Franciscan friary in Montella, Iannelli’s chapel and internal garden sidewalk respectively. The Danish samples were collected in the Hardenberg’s chapel in the Franciscan friary in Svendborg. All the samples were taken from long bones, mainly from femur when it was possible, otherwise humerus or tibia, only few samples come from skull bones. For each individual, when possible, a sample of both compact and spongy tissue was taken.

2.1.1

Iannelli chapel’s samples

This set of samples was collected from individuals who belong to the same family, the Iannellis, a noble and rich family that lived in Montella. All the individuals died in the period between 17-18th century, and the documents shows that the burial was used from 1678 to 1740.

The samples analyzed were kindly provided by Marielva Torino, and they consist in bones fragments cut from long bones. A team of anthropologists studied the individuals before chemical analysis, in order to collect information about sex, age, diseases, etc…

(31)

31

Sample Number age min age max Bone sex Tissue Diseases

KLR-9429 Ian 1 15 16 male compact

KLR-9430 a Ian 2 50 70 femur male compact

KLR-9430 a Ian 2 50 70 femur male spongy

KLR-9430 b skull male compact

KLR-9431 Ian 4 20 25 female compact

KLR-9432 Ian 5 45 58 male compact TBC

KLR-9432 Ian 5 45 58 male spongy TBC

KLR-9433 Ian 7 45 53 male spongy TBC

KLR-9434 Ian 8 35 50 male spongy

KLR-9435 a Ian 10 21 26 male compact

KLR-9435 a Ian 10 21 26 male spongy

KLR-9435 b skull male compact

KLR-9437 a Ian 13 40 50 male compact Siph.

KLR-9437 a Ian 13 40 50 male spongy Siph.

KLR-9437 b skull male spongy

KLR-9438 Ian 14 40 55 skull male compact

KLR-9438 Ian 14 40 55 skull male spongy

KLR-9439 Ian 15 male compact

KLR-9439 Ian 15 male spongy

KLR-9440 a Ian 16 45 55 male compact Siph.

KLR-9440 a Ian 16 45 55 male spongy Siph.

KLR-9440 b skull male

KLR-9443 Ian 22 25 38 phalanx male compact

KLR-9443 Ian 22 25 38 phalanx male spongy

KLR-9444 Ian 23 30 40 female compact

KLR-9446 Ian 25 female compact

KLR-9446 Ian 25 female spongy

Table 2.1.1.1 For each individual it is shown: sample name; the number used in data analysis; the age range at the moment of death; kind of bone sampled; sex; bone tissue sampled; diseases visible on the bones. When both compact and spongy bones were available, they are labeled with the same name.

(32)

32

Figure 2.1.1 The Iannellis family tree, dating from the 17th to the 18th century, and the location of the chapel where the individuals were buried

(33)

33

2.1.2

Montella friary sidewalk’s samples

These samples, as Iannelli’s ones, were kindly provided by Prof. Marielva Torino. They were collected from the Franciscan friary of Montella, more exactly from the inner sidewalk of the monastery. The individuals were excavated in two different times. Nothing is know about these individuals but it is supposed that they were or monks form the monastery or Montella villagers who made a donation to the monastery to be buried there [24,25]. The samples analyzed were long bones fragments, and both compact and spongy tissue for each individuals were taken when possible. The interest in the study of this set of samples was to compare them with Iannelli’s ones, in order to see differences in people come from the same geographical area but with different social status.

Sample Number Bone Tissue

KLR-9447 USD 374 Ibur 1 left femur compact

KLR-9448 USD 377 Ibur 2 right femur compact

KLR-9449 USD 382 Ibur 3 right femur spongy

KLR-9458 USD 408 Ibur 12 right humerus compact

KLR-9460 USD 410 Ibur 14 left femur spongy

KLR-9462 USD 416 Ibur 16 left humerus compact

(34)

34

KLR-9471 USD 449 Ibur 25 left humerus spongy

KLR-9472 USD 450 Ibur 26 left tibia compact

KLR-9474 USD 459 Ibur 28 left femur spongy

Table 2.1.2.1 For each individual it is shown: sample name; the number used in data analysis; kind of bone sampled; tissue sampled. When both compact and spongy bone were available they are labeled with the same name

Sample Number Bone Tissue

KLR-9475 US 372 II 1 left femur compact

KLR-9476 US 372 II 2 right femur compact

KLR-9476 US 372 II 2 right femur spongy

KLR-9477 USD 380 II 3 left tibia compact

KLR-9478 USD 399 II 4 right tibia compact

KLR-9479 USD 400 II 5 skull compact

KLR-9480 USD 438 II 6 left femur compact

KLR-9480 USD 438 II 6 left femur spongy

KLR-9481 USD 440 II 7 right tibia compact

Table 2.1.1.2 For each individual it is shown: sample name; the number used in data analysis; kind of bone sampled; tissue sampled. When both compact and spongy bone were available they are labeled with the same name

2.1.3

Hardenberg chapel’s samples

The individuals were buried in a private chapel, the Hardenberg chapel in connection with the Franciscan friary of Svendborg. Seven individuals were found: 4 females and 2 males, while for one individual it was not possible to determinate the sex. These individuals belong to a Danish noble family, the Hardenbergs.

Sample Number Age min Age max sex bone tissue

HB 76/75 HB 76/75 40 50 female right femur compact

HB 76/75 HB 76/75 40 50 female right femur spongy

HB 86/75 HB 86/75 33 40 male left femur compact

HB 86/75 HB 86/75 33 40 male left femur spongy

(35)

35

HB 125/75 HB 125/75 right femur spongy

HB 128/75 HB 128/75 30 50 female left humerus compact

HB 128/75 HB 128/75 30 50 female left humerus spongy

HB 65/75 HB 65/75 45 55 female right femur compact

HB 65/75 HB 65/75 45 55 female right femur spongy

HB 127/75 HB 127/75 35 60 male right humerus compact

HB 127/75 HB 127/75 35 60 male right humerus spongy

HB 137/75 HB 137/75 35 50 female right tibia compact

HB 137/75 HB 137/75 35 50 female right tibia spongy

Table 2.1.3.1 For each individual it is shown: sample name; the number used in data analysis; the age range at the moment of death; kind of bone sampled; sex; bone tissue sampled. When both compact and spongy bone were available they are labeled with the same name

2.2 CHEMICALS AND INSTRUMENTS

2.2.1

Chemicals and materials

 Drill: Dremel model 3000 F013300045 Ø 38,1 mm max Ø 3,2 mm 230V, 50 Hz 130 W n0 33000/min

 Tube 15 mL, 120X17 mm, PP

 Tube 50 mL, 114X28 mm, PP

 Single use syringe, 12 mL NORM-JECT®, filter Q-Max PVDF 0.45µm

 Steel spoon

 Electronics pipette RAININ E4XLS, LTS 500µL-5mL, precision pipette tips RC-L5000, 5000µL LTS TIPS

 Electronics pipette RAININ E4XLS, LTS 20-300µL, 300µL pipette tips Chemicals

 HNO3: Fluka analytical traceSELECT®, for trace analysis, ≥ 69%

 HCl: PlasmaPURE Plus 32-35%

 H2O2: : Fluka analytical traceSELECT®, for trace analysis, ≥ 30%

 KMnO4: Sigma-Aldrich

 NaBH4: Aldrich chemistry

Standard solutions

 Tuning solution: Inorganic Ventures, VAR-TS-MS, 7% HNO3 (v/v).

 Internal standard: Inorganic Ventures, IV-ICPMS-71D, 2-5% HNO3 (v/v).

 Multielemental standard: AccuTrace™ Reference Standard, ICP multi-element standard solution XXI for MS, plasma emission standard (ICP), 2-5% HNO3 (v/v).

(36)

36

 Hg-standard: PerkinElmer Pure, atomic spettroscopy, Mercury 10% HNO3

(v/v).

 Sn standard: Fulka analytical, Tin standard for ICP

 Sb standard: Fulka analytical , Antimony ICP/DCP standard solution

 Au standard: goodfellow LS10335 Au rod 99,99% Reference materials

 Together with the samples, as referenced material for ICP-MS analysis, NIST SRM-1486 bone meal was run. This referenced material is a modern bone sample (NIST, Gaithersburg, MD, USA). The preparation of this sample is a bit different from the other sample preparation, because NIST SRM-1486 bone meal have more organic matter than the ancient bones.

 Every time an CV-AAS analysis was carried on, a reference material for Hg NIST SRM 1515 Apple Leaves was also analyzed.

 Indoor bone standard material was prepared, in the same way as normal samples, and analyzed every time both with ICP-MS and CV-AAS. This bone powder come from a Danish woman died around 16th century. This indoor made standard was used because of its similarity with medieval bones that has been analyzed in this work. The elements value in the bone, shows in the table, was obtained after many analysis on the bone after years.

2.2.2

ICP-MS

The analyses were carried out using a Bruker ICPMS 820 instrument equipped with a frequency matching RF generator and a collision reaction interface (CRI), the latter operated with either helium or hydrogen (Bruker Daltronics, Solna, Sweeden). The samples were introduced into the system using a Bruker SPS3 autosampler and a OneFast flow injection inlet system (Elemental Scientific, Omaha, NE, USA). The basic parameters were as follows: radiofrequency power 1.40 kW, plasma gas (Ar) flow rate 15.50 L/min; auxiliary gas (Ar) flow rate 1.65 L/ min; sheath gas (Ar) flow rate 0.12 L/min; nebuliser gas (Ar) flow rate 1.00 L/min. The CRI reaction system was activated for Cu and Fe because of interferences from polyatomic species produced by a combination of isotopes coming from the argon plasma, reagents and the bone matrix. 56Fe was measured with hydrogen as the skimmer gas and 63Cu with helium as the skimmer gas. A mixture of 45Sc, 89Y and 159 Tb was used as the internal standard and added continuously to all samples. The dwell time on each peak was 30 ms. Five replicate analyses were made of each of the dissolved bone samples and each replicate consisted of 30 full scan spectra, which were averaged. All ICP-MS analysis were performed using the software Quantum

(37)

37

23

Na 24Mg 27Al 44Ca 55Mn 88Sr 107Ag 118Sn 121Sb 137Ba 197Au Pb (206Pb, 207Pb and

208

Pb) 238U 56Fe 66Zn 52Cr 59Co 60Ni 63Cu 69Ga 75As

2.2.2.1

Preparation of calibration solutions

An ICP multi-element standard solution XXI for MS was used to prepare six calibration standard solutions in 1% HNO3 at six different concentrations: 0.1, 1, 10,

100 and 200 µg/L. For each element only three standards were selected to fit the appropriate concentration range in the samples. For the main element Ca three standards solutions in 1% HNO3 of concentrations 100, 200 and 250 mg/L were used.

For Sn and Sb three standard solutions in 1% HNO3 at three different concentrations,

1, 10 and 20 µg/L were used. For Au two different standard solutions in 1% HNO3, 1

and 5 µg/L were used.

Nine standard solutions, prepared as described below, were analyzed:

Standard nr. Standard solution type Final elemental concentration Original standard solution concentration Standard solution volume 1% HNO3 volume 1 Multi 0.1 ppb 100 ppb 40 µL 39.96 mL 2 Multi 1 ppb 100 ppb 100 µL 9.9 mL 3 Multi 10 ppb 100 ppb 1 mL 9 mL 4 Multi 20 ppb 100 ppb 2 mL 8 mL 5 Multi 100 ppb 10 ppm 100 µL 9.9 mL 6 Multi 200 ppb 10 ppm 200 µL 9.8 mL 7 Ca 100 ppm 10000 ppm 100 µL 9.7 mL Au 1 ppb 100 ppb 100 µL Sn/Sb 1 ppb 100 ppb 100 µL 8 Ca 200 ppm 10000 ppm 200 µL 8.3 mL Au 5 ppb 100 ppb 500 µL Sn/Sb 10 ppb 100 ppb 1 mL 9 Ca 250 ppm 10000 ppm 250 µL 7.75 mL Sn/Sb 20 ppb 100 ppb 2 mL

Table 2.2.2.1.1 In this Table are reported the solutions used to build the calibration curves for ICP-MS analysis.

A stock solution, 100 ppb, was prepared by dilution, in 1% HNO3, from the mother

stock solution ICP.multi-element standard solution XXI for MS. This diluited solution was used to obtain the first four solutions 1,2,3 and 4. Standard solution 5 and 6 was created by direct dilution in 1% HNO3 of ICP.multi-element standard solution XXI for

MS. Solutions 7, 8 and 9 was obtained by dilution of Ca, Au and Sn/Sb standard solutions in 1% HNO3.