Ancient and recent evidence of endemic fluorosis in the Naples area

Journal of Geochemical Exploration 131 (2013) 14–27

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Ancient and recent evidence of endemic fluorosis in the Naples area Pierpaolo Petrone a,⁎, Fabio M. Guarino b, Stefano Giustino b, Fernando Gombos c a b c

Università degli Studi di Napoli Federico II, via Mezzocannone 8, 80134 Naples, Italy Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi di Napoli Federico II, via Cintia, Complesso Monte S. Angelo, 80126, Naples, Italy Via Generale Orsini 42, Naples, Italy

a r t i c l e

i n f o

Article history: Received 13 February 2012 Accepted 20 November 2012 Available online 30 November 2012 Keywords: Environment and human health Health risk assessment Endemic fluorosis Epidemiology Herculaneum victims 79 AD Vesuvius eruption

a b s t r a c t Endemic fluorosis induced by high concentrations of natural fluoride in groundwater and soils is a major health problem in several countries, particularly in volcanic areas. The early stages of skeletal fluorosis, a chronic metabolic bone and joint disease rarely considered in palaeopathological diagnoses, are often misdiagnosed in endemic areas. In this paper, morphological, radiological, histological and chemical skeletal and dental features of the 79 AD Herculaneum population show that in this area fluorosis has been endemic since Roman times. Long-term exposure to high levels of environmental fluoride is revealed by intense calcification of the ligaments, tendons and cartilage, diffuse axial and appendicular osteosclerosis, spine osteophytosis and spondyloarthritis, bone histopathological alterations and bone fractures. High levels of fluoride found in the skeleton, as well as dental features such as mottling and hypomineralization, confirm the endemicity of fluorosis, which still occurs today. When merged with the results of a recent clinical–epidemiological investigation in schoolchildren from the Vesuvian towns, our findings reveal for the resident population a permanent fluoride hazard whose health and socio-economic impact is currently underestimated. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Ecological, hydrogeological and geochemical factors affect the physical environment in which people live. Several potentially toxic elements may contaminate the environment, and their biological and physiological effects are associated to the prevalence and severity of various endemic diseases (Cao et al., 2004). Fluorine is present in its ionic form of fluoride in soil, water, plants, foods and even air (D'Alessandro, 2006). During weathering and circulation of water in rocks and soils, fluorine can be leached out and dissolved in groundwater and thermal gases. In groundwater, the natural concentration of fluoride depends on the geological, chemical and physical characteristics of the aquifer, the porosity and acidity of the soil and rocks, the temperature and the action of other chemical elements. Potentially fluoride-rich environments are mainly linked with Precambrian basement areas and those affected by recent volcanism (Bocanegra et al., 2005). Because of the large number of variables, fluoride concentrations in groundwater can range from well under 1 mg/L to more than 35 mg/L. Therefore, high concentrations of naturally occurring fluoride in groundwater can be found in different countries as well as locally in most parts of the world (WHO, 2008). In several countries, fluoride is also added to public drinking water supplies due to the significant effects of mitigating dental caries and strengthening bones if the concentration intake is approximately 1 mg/L (Littleton, 1999; Ozsvath, 2009). Fluoride can enter public water systems from ⁎ Corresponding author. Tel./fax: +39 081 2535211, +39 339 5807057(mobile). E-mail address: [email protected] (P. Petrone). 0375-6742/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gexplo.2012.11.012

natural sources (Ayoob and Gupta, 2006; D'Alessandro, 2006), particularly in volcanic areas, where high amounts of fluoride in drinking water are typically found due to contamination from ash deposits (Ozsvath, 2009; U.S. NRC, 2006). This is the case of the Somma–Vesuvius surroundings, repeatedly covered by pyroclastic products since prehistoric times (Mastrolorenzo et al., 2006). Long-term intake of high doses of fluoride can have adverse effects on human health, including dental, musculoskeletal, reproductive, developmental, renal, endocrine, neurological, and genotoxic effects (Ozsvath, 2009; U.S. NRC, 1993). Bones and teeth are the target organs of fluoride, and tend to accumulate it with age (Ayoob and Gupta, 2006; Littleton, 1999; U.S. NRC, 1993). Dose and duration of fluoride intake, age, sex, nutritional status and diet, climate and renal efficiency in fluoride excretion are the main variable factors affecting the risk of developing fluorosis, an increasingly disabling disease. This disease usually affects older adults, and men more frequently than women (Ayoob and Gupta, 2006; Ozsvath, 2009). Skeletal fluorosis is a chronic metabolic bone and joint disease caused by prolonged, excessive ingestion of fluoride, mostly through water in endemic areas. Increased fluoride content in the bone is the main indicator of fluoride poisoning (Krishnamachari, 1986). Primarily, fluoride acts as a cumulative toxin altering accretion and resorption of bone tissue and affecting the homeostasis of bone mineral metabolism, causing functional adverse effects (Littleton, 1999). Chronic fluorine intoxication may also induce endemic dental hypoplasia, which has effects ranging from mild tooth discoloration (mottling) to severe staining, pitting and loss of enamel (Thylstrup and Fejerskov, 1978; U.S. NRC, 2006; WHO, 1970). Enamel hypoplasias

P. Petrone et al. / Journal of Geochemical Exploration 131 (2013) 14–27

are caused by a wide range of factors, including malnutrition, febrile diseases, infections during pregnancy or infancy, trauma to the teeth and jaws, exposure to toxic chemicals, and a variety of hereditary disorders (Aoba and Fejerskov, 2002). Hypoplastic defects of fluorotic origin may have a different etiology than linear enamel hypoplasia (LEH). Linear enamel hypoplasias, a deficiency in enamel formation visible on mammal tooth crowns, are unspecific stress markers commonly seen in several economically less developed present-day countries as well as in children of malnourished ancient communities. Therefore, LEH are mostly utilized in palaeopathology as a systemic physiological stress indicator (Skinner and Goodman, 1992). In bones and teeth, fluorine ion exchange takes place via recrystallization of hydroxyapatite into the more stable fluoroapatite (WHO, 1970). The physiological effects of fluoride intake on the adult skeleton are the result of effects on the chemistry, gross morphology, histopathology, X-ray density, and integrity of structure of bone and teeth (Aoba and Fejerskov, 2002; WHO, 1970). Skeletal fluorosis is characterized by periosteal thickening, calcification of tendons and ligaments, and abnormal production of multiple hypertrophic bony exostoses (osteophytes) at ligamentous and muscular attachments to bone (Resnick and Niwayama, 1983). The clinical condition exhibits bone, joint and muscle pains due to early restrictions in spine movements and at a later stage progressive ankylosis of the vertebral joints induced by ligamentous calcification. Vertebrae, ribs and pelvis are more prone to osteophyte formation than long bones, even if increasing immobilization also spreads to the major joints of the chest and knees. In advanced stages the entire skeleton may be involved by crippling deformities, which can be found in the pediatric age group too (Weidmann et al., 1963). Radiological and histological findings closely parallel macroscopic changes. Extensive production of new bone, usually associated with bone resorption, may result in an overall increase in bone thickness and radio-opacity, besides a low degree of mineralization (Chavassieux et al., 2007). The outcome is a combination of osteosclerosis, osteoporosis and osteomalacia of different degrees (Krishnamachari, 1986; Littleton, 1999; Weidmann et al., 1963). An altered organic matrix, reduced mineralization and osteosclerosis are also apparent from histopathological examinations (Krishnamachari, 1986). Despite the increase in bone tissue mass but not in density, fluorotic bones are thus brittle, of poorer mechanical quality and easier to break (Chavassieux et al., 2007). Even if skeletal fluorosis has been widely studied, because some of the early clinical symptoms resemble those of osteoarthritis, the first clinical phases of skeletal fluorosis could be easily misdiagnosed (Ayoob and Gupta, 2006). In its advanced stage it becomes a crippling disability that has a major public health and socio-economic impact, affecting tens of millions of people in Africa, India and China, and being endemic in at least 25 countries across the globe (Ayoob and Gupta, 2006; D'Alessandro, 2006). In studies of ancient skeletal populations, this condition has rarely been considered in differential diagnoses of palaeopathological lesions, mostly concerning specific single cases showing excessive ossification and joint ankylosis (Ortner, 2003). Dental fluorosis was first reported for Neolithic and Chalcolithic skeletal samples from Pakistan (Lukacs, 1984; Lukacs et al., 1985). Later reports for historical times (Littleton, 1999; Yoshimura et al., 2006) refer to arid regions and other areas in which the disease still occurs today due to high fluoride concentrations in drinking water. Here we further discuss the pathological condition of 76 Herculaneum victims of the 79 AD Vesuvius eruption (Mastrolorenzo et al., 2001), making comparisons with unpublished data and clinical findings from schoolchildren in the Vesuvian area (Gombos et al., 1994). Being a unique cross section of the entire living population, the Herculaneum skeletal sample is particularly suited to palaeoepidemiologic investigation, which also has important implications for present-day populations. All skeletons were in an extraordinary good state of preservation as a result of the unusual death and burial conditions involved: instant death

15

caused by emplacement of hot pyroclastic surge (ca. 500 °C), followed by rapid vaporization of soft tissues replaced by ash (Mastrolorenzo et al., 2010). This anoxic fluoride-rich ash bed deposit was permanently saturated by groundwater (Capasso, 2001). Further analysis of the skeletal and dental evidences for fluoride exposure at Herculaneum available to date (Petrone et al., 2011), merged with currently available epidemiologic data on school-children, strongly supports, as expected, the hypothesis of a long-lasting fluoride health hazard for the Vesuvius area population. 2. Materials and methods This study was approved by the Ethics Committee for Biomedical Activities of the University of Naples, Azienda Ospedaliera Universitaria “Federico II”, Naples (Protocol 154/10, 9.08.10). The Superintendency of Pompeii granted field investigation and study of the human skeletal materials unearthed in the 1997–99 excavations of the water-front chambers at Herculaneum. 2.1. Morphological, X-ray and histological bone analysis We analyzed 76 human skeletons aged 0 to 52 years old, excavated within the water-front chambers 5, 10 and 12 of the Herculaneum suburban area (Table 1) (Mastrolorenzo et al., 2001). The composition by age of the entire sample shows about 62% of adults vs. 38% of sub adults (24% infants and 14% juveniles), with a sex-ratio of 1.89 (36 males vs. 19 females) assessed on individuals > 15 years old. Sex and age at death, as well as the prevalence of linear enamel hypoplastic defects (LEH) and dental caries were assessed according to standard diagnostic procedures (Buikstra and Ubelaker, 1994; Hillson, 2005; Resnick and Niwayama, 1983). Enamel fluorosis was scored according to Dean's classification (Dean, 1942), that we simplified by adopting a four-value scoring system. The chest bones, spine, pelvis and long bones of each individual were examined for the calcification of ligaments, cartilage and tendons (Rogers et al., 1997), as well as the presence of healed fractures. Hypertrophic osteosclerosis (osteophytosis) and spondyloarthritis of the spine, and osteoarthritic lesions of the appendicular skeleton were scored both individually and by single joint following standardized scoring criteria (Jurmain, 1977, 1990). The most severe cases were also evaluated adopting digital radiography (Villa Mercury 332, Kodak Direct View CR 850, Naples, IT) and histological analysis. We examined the undecalcified and unstained bone ground sections (80–100 μm thick), obtained after embedding in LY-554 araldite resin (Vantico) and observed under a transmitted ordinary and polarized light microscope. Due to the peculiar burial conditions of these skeletons, specific attention has been paid to discriminate pathological vs. diagenetic alteration. Thus the bone histology concerning alterations of the microstructure and its birefringence were also investigated (Guarino et al., 2006). Bone histology, porosity and enrichment of chemical elements are diagenetic parameters that quantify the post-mortem osteoalteration. Bones buried for long periods absorb and accumulate fluoride from soil, and the extent of bone diagenesis is particularly related to fluctuating hydrological regimes (Hedges, 2002; WHO, 1970). Furthermore, permanently waterlogged environments are anoxic, and inhibit microbial attack and related diagenetic processes (Hedges, 2002; Tressaud, 2006). Chemical analyses were carried out on a random group of victims' skeletons. The amount of fluoride in their bones and in the volcanic sediments were measured in order to test the pathologic diagnosis derived from the preliminary morphological and radiographic analyses of the skeletons, and also to avoid possible diagenetic processes. Additional analysis of the content of oligoelements in the human bone was also carried out in order to define the nutritional status at Herculaneum. Bone samples of herbivores (Equus caballus, Ovis capra) found in the water-front area were tested for standardization

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P. Petrone et al. / Journal of Geochemical Exploration 131 (2013) 14–27

Table 1 Sex and age at death estimates of 76 victim skeletons of the AD 79 eruption of Vesuvius. The skeletons analyzed in the present study have been unearthed by one of the authors (P. Petrone) in 1997–1999, during archeological excavations in the Herculaneum suburban area (boat-chambers N5, N10, N12) on behalf of the Archaeological Superintendency of Pompeii. N

Ind.

Sex

Age at death

N

Ind.

Sex

Age at death

N

Ind.

Sex

Age at death

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

5:1 5:2 5:3 6:15 10:1 10:2 10:3 10:4 10:5 10:6 10:7 10:8 10:9 10:10 10:11A 10:11B 10:12 10:13 10:14 10:15 10:16 10:17 10:18 10:19 10:20 10:21

M F M ? M M M F M M M M M M F M M M M F F M F M M M

13–16 18–22.5 18–22.5 7 iu-m 36–42 14–16 45–55 26–32 18–25 28–33.5 33–38 11–15.5 13–16.5 33–38 28–34.5 31–37.5 31–37 31–37 34–40 26–31 34–38.5 32–39 35–41 27–33 40–48 34–41

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

10:22 10:23 10:24 10:25 10:25B 10:26 10:27 10:28 10:29 10:30 10:32 10:33 10:34 10:35 10:36 10:38 10:39 10:40 10:41 12:1 12:2 12:3 12:4 12:5 12:6 12:7

M M F M ? M?* F?* F F M?* M?* F* F? M M ? M ? F?* M?* F F M M F?* M

18–23 33–40 37–45 20–23 19–24 8–10.5 4–6.5 33–40 26–32 2–3 8–11 11–13 17–22 20–39 15–17 12–15 12–15 11–14 0.5–1.5 3–5 23–29 25–30 24–30 15–18 7–10 16–18.5

53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76

12:8 12:9 12:10 12:11 12:12 12:13 12:14 12:15 12:16 12:17 12:18 12:19 12:20 12:21 12:22 12:23 12:24 12:25 12:26 12:27 12:28 12:29 12:30 12:31

M F M?* M M?* F M* F M F? F?* M M F M M M?* M* M M F F?* F F

34–41.5 22–28 8–10 47–57 2–4 33–41.5 11–13 30–35.5 33–41.5 5–6 3–4 31–37 16–20 29–35 18–23 38–46 8–11 9–11 28–34 33–39 33–39 11–13 33–37.5 20–39

of the Sr/Ca ratio in human bones vs. herbivore bones from the same site (Bisel, 1980). Samples of sediment collected within the town and on the ancient beach were utilized for comparative diagenetic tests.

2.2. Instrumental neutron activation analysis (INAA) The amounts of fluorine (F), sodium (Na) and calcium (Ca) in the bone were measured in the iliac crest and/or rib bones of 27 Herculaneum victims aged 0 to 52 years old of both sexes, by Instrumental Neutron Activation Analysis (INAA) at the University of Missouri Research Reactor Centre (MURR) (Cheng et al., 1997). As infant bones are more easily prone to post-mortem taphonomic and diagenetic changes because of their porosity and lower rate of calcification (Wittmers et al., 2008), as expected, the fluorine amounts found in the infants aged 0–10 years (N: 7; average: 16,371 ppm) exceeded those recorded for the 12–30 age class (N: 7; average: 16,164 ppm) (Table 6). Accordingly, only the results from the latter group were considered in statistical analyses.

2.3. Ion-selective electrode (ISE) The fluoride content in volcanic ash was determined at the University of Notre Dame Fluoride Dating Service, by using an ion-selective electrode (ISE) according to Schurr (1989).

2.4. Atomic absorption spectroscopy (AAS) The contents of zinc (Zn), strontium (Sr), magnesium (Mg), copper (Cu) and calcium (Ca) in the bone were quantified by atomic absorption spectroscopy (AAS) following the standard pre-treatment technique (Hughes et al., 1976). Bone element concentration was measured with a Perkin-Elmer atomic absorption spectroscope (mod. 2100). In order to evaluate the palaeonutritional status of the sample, the results were compared with available data from other ancient skeletal populations (Petrone et al., 2002).

2.5. Skeletal lesion index The degree of lesion involving spine and peripheral joints was evaluated by an ordinal scaling system. The degenerative changes were scored following the four-value scoring classification adopted by Jurmain (1977, 1990) and then standardized by means of our own lesion index (Petrone et al., 2011). 3. Results The majority of the Herculaneum population (73.5% of individuals ≥ 15 years old) was affected by intense calcification of the ligaments, tendons, cartilage and interosseous membranes, associated with diffuse axial and appendicular osteosclerosis. Severe calcification along with proliferative bone abnormalities particularly involves costochondral and costosternal junctions, ribs, spine anterior longitudinal ligaments, iliac crest and sacrotuberous ligaments. Gross and radiographic examinations of the long bones reveal massive cortical thickening, increased bone matrix density, narrowed medullary cavity and increased radio-opacity. In addition, most of the individuals (91.8%) display at least one long or flat bone affected by abnormal growth of osteophytes, including several juveniles and children. The topographic frequency distribution of ligament and tendon calcification and ankylosis in the postcranial skeleton of specimens aged 1 to 52 years old are summarized in Fig. 1. The microscopic examination of ground cross sections of the juvenile and adult long bones from both sexes show several histopathological alterations, including lost or poorly formed Haversian lamellar systems (Fig. 2A and B) and extensive mottled bone matrix and enlarged Haversian canals (Fig. 2B and C). No evidence of structural diagenetic change by microorganism activity can be observed, as predictable due to the anoxic burial environment of the skeletons. Within the investigated sample, a distinctive osteoalteration consists in widespread microcracking (Fig. 2C), due to exposure of the victims' corpses to the high temperature of the pyroclastic surge (Mastrolorenzo et al., 2001, 2010). Hypertrophic osteosclerosis (osteophytosis) and spondyloarthritis increase towards the lumbar spine, with an overall prevalence of

P. Petrone et al. / Journal of Geochemical Exploration 131 (2013) 14–27

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Fig. 1. Assessment of ligaments and tendons calcification and ankylosis in the postcranial skeleton. 91.8% of the individuals aged 1 to 52 years old show ossification processes in at least one of the long or flat bones (femur, tibia, clavicle, pelvis), with clavicle as the most involved bone (88.2%). Ankylosis, mainly detectable in spine, foot toe distal interphalangeal joint and manubriosternal joint, affects at least one of these three anatomical sites in 39.2% of the individuals.

27.6% and 18.5%, respectively (Fig. 3). Spine ankylosis mainly involves thoraco-lumbar (3.1%) and sacroiliac (4.6%) joints, affecting males and females equally (18.2% vs. 17.6% of individuals, z = 0.0523, P > 0.05). In these severe cases the vertebral body contours are largely uneven or fused, and bones have a chalky white appearance. In the appendicular skeleton, a 47.2% overall occurrence of osteoarthritic-like alterations of the joints appears particularly severe given the mean age of about 30 years of individuals ≥ 15 years old. The coxofemoral, knee, sacroiliac, elbow, and sternoclavicular joints and pedal phalanges are the most noticeably affected anatomical districts (Fig. 4). In general, ankylosis affects at least one

anatomical site in 39.2% of the individuals. In addition, nearly one individual out of three (32.1%) shows one or more pathological fractures involving mostly the spine or os coxae, as well as the long bones, while osteomalacia affects 8.2% of the individuals. Evaluating the cases of spondylolysis (L5 vertebrae, 8.9% vs. 3–7% of the general population) as stress fractures (Capasso et al., 1999; Haettich et al., 1991), the susceptibility to bone fractures is particularly high at Herculaneum (35.7%) (Table 2) (for further details, see Petrone et al., 2011). The degree of individual osteoarthritic-like lesions involving the post-cranial skeleton was also assessed by means of a skeletal lesion

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P. Petrone et al. / Journal of Geochemical Exploration 131 (2013) 14–27

Fig. 3. Occurrence of osteophytosis and spondyloarthritis in spine joints. Osteophytic lesions (moderate + severe + ankylosis) of specimens aged ≥15 years old increase towards lumbar joints (17.5% cervical, 26.5% thoracic, 38.8% lumbar), with 27.6% overall occurrence. Spondyloarthritic lesions (moderate + severe + ankylosis) occur in 18.5% of the joints, with lumbar vertebrae as the most affected (22.8%).

Fig. 2. Histopathological bone features. Representative bone ground sections observed under a transmitted light microscope: A. Phalanx showing extensive mottling of bone matrix and disordered lamellar architecture, 10 years old male; B. Mid shaft of femur with widespread deficiency of the Haversian lamellar systems and several enlarged Haversian canals, 15 years old male. Note the presence of several linear formation defects (arrows); C. Mid shaft of tibia showing mottling of bone matrix and enlarged Haversian canals, 37 years old male. Note the irregular cracking (arrows) induced by victim corpses exposure to the hot pyroclastic surge. Images A, B and C are of the same magnification.

index (SLI). The SLI index was adopted to better evaluate and compare the expression and variability of pathological lesions as to anatomical location, age and gender. This index, calculated by considering both appendicular skeleton and spine joints of the individuals aged ≥15 years old (Table 3), was regressed by individual age, separately for males and females (Fig. 5). In both sexes the linear regression shows that nearly 90% of the SLI index variability is age-related (males: R2 = 0.895, P b 0.0001; females: R2 = 0.877, P b 0.0001), but the slope differs significantly between males and females (test for non-equality of regression coefficients, t = 7, P b 0.0001). Males ≤30 years old are on average more affected than females, while females over 30 seem more prone to be

involved. SLI indexes calculated on 10 post-cranial large joints, show that the spine is by far the most affected (0.616) (z= 2.15, P b 0.05), compared with the knee (0.356), shoulder (0.317), coxo-femural (0.306), sacro-iliac (0.284) and other appendicular joints. Analysis of the permanent dentitions reveals 96.1% of the individuals (47.3% of teeth) affected by linear hypoplastic defects (LEH) (Fig. 6A). In addition, mottling, pitting and/or staining of the enamel (Fig. 6B, C and D) was found in 53.1% of the sample (54.9% of teeth), with moderate to severe enamel alterations involving 34.4% of the individuals (27.6% of teeth) (Table 4). In the cases of marked hypomineralization (25.0%, 17.8% of teeth), a corroded-like appearance and alterations of the tooth form are evident (Fig. 6D). Mottled enamel, associated with chronic dental fluorosis since prehistoric times (Lukacs et al., 1985), may also affect well nourished people. The healthy diet of the Herculanenses is testified by historical and archeological evidences (Feemster Jashemski and Meyer, 2002), as well as from trace-element analyses of a previously excavated group of victims (Herc2) (Bisel, 1991; Capasso, 2001). Their well-balanced diet in terms of protein and carbohydrate intake also emerges from the Zn/Ca ratio (0.48 ± 0.11) and site-corrected Sr/Ca ratio (0.73 ± 0.10) derived from the amounts of bone oligoelements that we determined by atomic absorption spectrophotometry (AAS) (Table 5). These values, compared with those found in ancient skeletal populations, suggest the consumption of red meat, crustaceans, oysters, dry fruit and legumes, and point to a diet also rich in sea fish and proteins of plant origin, as well as in carbohydrates (Petrone et al., 2002). Carious lesions, collected as an additional test of dental pathological status particularly with regard to dental fluorosis, were found in 20% of permanent teeth (N: 1358) and 78.6% of individuals. Even if this finding confirms as expected that, in Roman times, caries follows

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Fig. 4. Occurrence of osteoarthritic lesions in 737 joints of the appendicular skeleton. The joints of individuals aged ≥15 years old show 14.9% incidence of osteoarthritic major (severe + ankylosis) lesions (47.2%, total lesions), with foot toes (37.1%) and coxofemoral (22.2%), knee (19.1%), sacroiliac (18.8%), sternoclavicular and elbow (10.8%) joints as the most affected districts.

a modern epidemiologic pattern, it also appears to conflict with an expected low frequency of carious lesions in the case of naturally fluoridated water intake, since daily moderate fluoride ingestion is widely supposed to mitigate dental caries (Littleton, 1999; Ozsvath, 2009). A few cases of root hypercementosis have also been detected, given its possible correlation to high fluoride intake during teeth growth (WHO, 2002). The fluorine ( 19F) bone concentration was measured in a group of victims selected by anatomical district, age and state of preservation (Table 6). Volcanic ash samples from the three investigated chambers were also tested by ion-selective electrode (ISE). Adjusted values of μ fluoride (160 ± 33, 190 ± 18, 200 ± 6, ppm) do not diverge statistically, being within the normal score limits (±1.96). Average bone fluorine concentrations varying between 14,000 and 23,300 ppm are a function of age as shown by the equation [F] = 11,958.5 + 200.4 · age. The intercept (b0 = 11,958.5 ± 1120; P b 0.001)

represents the mean amount of fluorine at age 0 (Fig. 7A). These values clearly exceed the range of normal–physiological fluorine content in the bone (Eble et al., 1992; Sastri et al., 2001) as well as the maximum expected pathological levels (Ayoob and Gupta, 2006; U.S. NRC, 2006). This finding is consistent with the particular burial conditions of the skeletons. The ash deposit was permanently waterlogged (Capasso, 2001), and the bones were therefore enriched with fluoride leaching from the groundwater. In this area the maximum concentration of present-day groundwater fluorine is 3.6 mg/L. Thus, in order to discriminate the post-mortem from intra-vitam fluoride bone enrichment, we assumed a 0 fluorine concentration at age 0 (Fig. 7B), considering that new-born bone usually contains nearly 50 ppm fluoride (WHO, 1970). The slope (b1 = 200.4± 9; P b 0.001) from the resulting straight-line equation [F] = 200.4 · age represents the rate of physiological intake per individual per year. This model shows that 99% of fluorine concentration variability is age-related (R2 = 0.961).

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Table 2 Occurrence of healed bone fractures in 56 individuals aged ≥15 years old. Spine and pelvis are the most involved anatomical districts, followed by large and small long bones. The 32.1% of males and females are equally involved, at all ages. Considering also the cases of spondylolysis of LV vertebra, the total occurrence of fractures is 35.7%. The number of fractures (1 to 4) per person significantly increases with advancing age (Spearman's rank correlation r = 0.647; P b 0.005). Individual

Sex

Age at death

Bone

5:3 10:22 12:9 10:15 10:35 12:SP1 12:26 10:11A

M M F F M M? M F

18–22.5 18–23 22–28 26–31 20–39 20–39 28–34 28–34.5

12:19 12:30 10:16 10:23 12:16

M F F M M

31–37 33–37.5 34–38.5 33–40 33–41.5

12:13 12:8 10:1 10:24

F M M F

33–41.5 34–41.5 36–42 37–45

12:23

M

38–46

10:3

M

45–55

12:11

M

47–57

Clavicle (right) Hand phalanx (right) Foot phalanx (right) Rib 11 (left) Fibula (left) Tibia (right) Spondylolysis L5 vertebra Ox coxae Head of femur (right) Foot phalanx (right) T11 vertebra Clavicle (right) Spondylolysis L5 vertebra Clavicle (right) Foot phalanx (left) Rib 7 (right) Foot phalanx (right) Clavicle (left) T8 vertebra Foot phalanx (right) T12 vertebra Spondylolysis L5 vertebra Spondylolysis L5 vertebra Foot phalanx (left) Radius (left) T10 vertebra Spondylolysis L5 vertebra Sacrum Rib 6 (left)

Taking into account this last correlation and that the skeletal lesion index is fluorine concentration-related (R2 = 0.923), we obtained a new equation combining ½F ¼ 200:347⋅age

ð1Þ

and SLI ¼ 0:000058696⋅½F

ð2Þ

thus yielding SLI ¼ 0:000058696⋅ð200:347⋅ageÞ ¼ 0:011759⋅age:

ð3Þ

This equation, applied to the available data, shows the correlation (R 2 = 0.81) of the observed data with those expected (Fig. 8). This indicates that the SLI index suitably describes the degree of joint lesions shown by the inhabitants of Herculaneum as a result of the fluorine accumulating in their bones. 4. Discussion The overall evidence at Herculaneum clearly shows the shape of an endemic system-disease, affecting both adults and subadults, characterized by diffuse osteosclerosis and enthesopathy. Although these conditions may be associated to other bone disorders, the concomitant aberrant growth of new bone, ligamentous calcification and osteosclerosis, along with osteoarthritic-like lesions and ankylosis of spine and appendicular joints, strongly suggest skeletal fluorosis (Littleton, 1999; Ozsvath, 2009; WHO, 2002). In addition, histopathological bone features like increased cortical thickness, abnormal lamellar texture, disordered lamellar orientation, extensive mottling of the bone matrix, enlarged and poorly formed Haversian systems, are highly characteristic of skeletal fluorosis (Aggarwal, 1973; Boivin

et al., 1989; Chavassieux et al., 2007). The high occurrence of bone fractures and a few cases of osteomalacia are also typical of fluoro-osteoporotic bones, likely resulting from calcium deficiency (Chavassieux et al., 2007; Teotia et al., 1998; WHO, 2002) and other mineral abnormalities induced by fluoride (Boivin et al., 1989). Furthermore, a major result is the significant correlation between the number of bone fractures (1 to 4) per individual and age (Spearman's rank correlation, r = 0.647, P b 0.005). The widespread calcification of the sacrotuberous ligament and a few cases of tooth hypercementosis also confirm the diagnosis of skeletal fluorosis (Littleton, 1999; WHO, 2002). A distinctive result is the high levels of fluoride found in the victims' bones, whose corrected average values ranging from 2042 to 11,342 ppm (mean value ± SE: 6672 ± 570 ppm) clearly indicate fluoride poisoning. Fluorine concentration as a function of age (Fig. 7B) shows that the majority of individuals belong to all three clinical phases of skeletal fluorosis. Higher values (>9000 ppm) observed in mature adults (≥40 years old) can be ascribable to the crippling phase III (Ayoob and Gupta, 2006), as currently seen in endemic regions (Choubisa, 2001). The regression line describing the annual variation rate of fluoride (ppm/years) shows a significant increase in fluoride concentration with age (Fig. 7B), and a correlation with the degree of pathological involvement of the spine and appendicular joints, as assessed by the SLI index evaluation (Fig. 5). This correlation of bone fluoride concentration with both duration of exposure and extent of bone lesions has been demonstrated in present-day patients affected by skeletal fluorosis, the severity of which was found to be related to the amount of fluoride incorporated into bones. Usually, fluorine can range from ca. 500 to ca. 3000 ppm in unaffected people, exposed to optimal fluoride intakes ≤ 1 mg/L (Eble et al., 1992; Sastri et al., 2001). Instead, extreme high values of ca. 10,000 or 12,000 ppm are typically associated with crippling fluorosis due to exposure to fluoride intakes ≥ 4 mg/L (Ozsvath, 2009; U.S. NRC, 2006). The widespread occurrence of osteoarthritis, osteophytosis, enthesopathy and fractures is particularly high in comparison with other Roman and pre-Roman communities (Robb et al., 2001; Vargiu et al., 2007), even with those of low social status (Sperduti, 1997). Palaeopathological investigation of the Herc2 specimens confirms the high occurrence of degenerative joint disease, long bone osteosclerosis, enthesopathy and trauma (Bisel, 1991; Capasso, 1998, 2001; Capasso et al., 1999). In contrast, an analogous pattern of skeletal changes, but with a lower occurrence and involving older individuals has been reported in ancient Arabic people, also affected by dental fluorosis. These arid regions are characterized by medium-high concentrations (0.5 to 3.0 mg/L) of fluoride in water (Littleton, 1999; Yoshimura et al., 2006). Dental fluorosis was first reported for the early Neolithic at Mehrgarh, Pakistan. Groundwater samples from this arid area show 1.9–2.0 mg/L of natural fluoride (Lukacs, 1984; Lukacs et al., 1985). At Herculaneum, where the concentration of fluoride in groundwater was found to be as high as 3.6 mg/L, the occurrence of dental fluorosis is analogous to the degree of fluorotic lesions exhibited by the victims' skeletons. Thus, the occurrence of endemic dental hypoplasia appears correlated to high fluoride intake during life, inferred from the high amount of fluoride that we determined in the bones. In ancient Herculaneum enamel mottling is associated with high levels of linear enamel hypoplasia (LEH), which occurs commonly in most ancient populations (Resnick and Niwayama, 1983; Robb et al., 2001). Also other Roman communities, including the Herc2 sample, show constantly high rates of LEH, independently of socioeconomic status (Capasso, 2001; Cucina et al., 2006; Manzi et al., 1999; Robb et al., 2001; Sperduti, 1997; Vargiu et al., 2007). Given the possible benefits of low fluoride intake in preventing dental decay, caries occurrence in the investigated sample of Herculaneum appears unusually high if compared with other Roman Imperial age communities (Cucina et al., 2006; Manzi et al., 1999). According to

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21

Table 3 Skeletal lesion index calculated on post-cranial joints of 48 individuals aged ≥15 years old. Osteoarthritic lesions involving spine and peripheral joints were evaluated assigning to each joint a 0-to-3 score on an ordinal scale (0 = absent, 1 = moderate, 2 = severe, 3 = ankylosis). The total measured score (I) was divided by the maximum measurable score (Imax) assigned to each individual. The obtained relative and normalized skeletal lesion index (SLI) ranged from 0 to 1 (0 ≤ SLI ≤ 1, with 0 = absence of lesion and 1 = maximum degree of joint lesion). The unpreserved joints were not considered in the index calculation. N

Ind

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

12:2 12:3 12:4 12:5 12:7 12:8 12:9 12:11 12:13 12:15 12:16 12:19 12:20 12:21 12:22 12:23 12:26 12:27 12:28 12:30 5:01 5:02 5:03 10:1 10:2 10:3 10:4 10:5 10:6 10:7 10:10 10:11 10:12 10:13 10:14 10:15 10:16 10:17 10:18 10:19 10:20 10:21 10:22 10:23 10:24 10:25 10:28 10:29

St-Cl

Shou

Elbow

Wrist

Sa-Il

Co-Fe

1 0

1 0

0

0 1 0.5

0 1 1 0 0 2 0 2 2 1 1 0 0 2 0 1 2 0 1 1 0 0 1 2 0 2 1 0 1 1 0 2 1 1 2 1 1 1 0 0 1 2 1 1 2 0 2 1

1

1 2 1 2 2 0 0 0 2 2 2 2 2 0 1 1 1 0 3 0.5 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 0 2

1 1

2 0 1 1 2 0 2 0.5 0 1 1 2 1 1 1 1 1 1 2 0 2 2 1 0 1 1

0 1 1 3 1 1 1 1 0 1 0 1 0 1 1 1 0 0 0 2 0 0.5 1 0 0.5 1 0 1 1 2 2 1 1 0 1 1 2 2 1 1 1

2 1

2 1

1 1 0.5 1 1 1 1 0 0 0 1 1

0 0 0 1 0.5 1 1 0.5 0 0 0 1 1 1 0 0 0.5 1 0 1 0.5 0 1 0.5 0.5 1 2 0 1 1 1 0 1 1 0 1 0.5 0 1

0 2 2 0 0 0 0 0 0 1 0 0 0 1 0.5 0 0.5 1 0 3 1 0 0 2 2 1 2 2 1 1 2 1 1 1 1 1 1 2 2 1

Knee 2 0 0 0 3 1 2 1 1 1 2 0 2 1 1 1 0 0 1 0.5 1 0.5 1 1 0 0 1 1 1 1 2 2 1 1 1 2 1 2 1 1 1 2 2 1 1

Ankle

Foot

Spine

I

Imax

Inorm

0 0 0.5 0 0 1 1 2 2 1 1 1 0 1 1 1 0 1 0 1 0 2 0 1 1 2 1 0.5 1 1 1 0.5 1 1 1 0 1 1 2 1 1 1 0 1 2 0 1 1

0 0 1

1 1 2 1

3 5 6 1 3 13 10 14.5 14 10.5 14 10 0.5 11 2 14 11 9 11 12 0.5 6 6.5 14 2.5 20.5 9.5 3.5 7.5 14 9 10 13.5 15 18 8.5 14 10 14 10 16 12.5 6 12 14.5 3 13 11

21 27 21 12 18 24 30 24 27 30 30 30 27 30 30 24 30 27 27 30 30 30 30 30 30 30 30 30 27 30 24 27 30 30 30 30 30 27 30 30 30 27 27 30 27 12 30 30

0.143 0.185 0.286 0.083 0.167 0.542 0.333 0.604 0.519 0.350 0.467 0.333 0.019 0.367 0.067 0.583 0.367 0.333 0.407 0.400 0.017 0.200 0.217 0.467 0.083 0.683 0.317 0.117 0.278 0.467 0.375 0.370 0.450 0.500 0.600 0.283 0.467 0.370 0.467 0.333 0.533 0.463 0.222 0.400 0.537 0.250 0.433 0.367

3 3 3 0 0 2 3 1 0 3 0 3 2 3 1 0 0 0 0 0 3 1 1 2 3

3 2 2 0.5 2 3 1 2 0.5 1 2 1 0 1

2 2 3 3 2 3 2 0 2 1 3 3 2 2 2 0 1 2 3 1 3 2 1 1 2 2 2 2 2 3 1 2 1 2 1 2 2 2 2 2 2 2

Ind = individual; St-Cl = sternoclavicular; Shou = shoulder; Sa-Il = sacroiliac; Co-Fe = coxofemoral; I = total measured score; Imax = maximum measurable score; Inorm = skeletal lesion index.

recent epidemiological studies on the adverse effects of high fluoride intake on dental caries (Ijaz et al., 2010; U.S. NRC, 2006; Wondwossen et al., 2004), the estimated high fluoride intake and the resulting hypomineralization detected in the tooth enamels of the Herculaneum residents seem strictly related to an increased risk of caries. This palaeoepidemiologic scenario has likely remained unchanged for the population in the Vesuvius area till today. Currently, the maximum fluorine concentration of the water-bearing stratum is close to the current maximum contaminant level (MCL) of 4 mg/L of drinking water, and within the range of concentrations able to induce crippling skeletal fluorosis (Connet, 2004; U.S. Environmental Protection Agency, 2002). We calculated a fluoride intake of 10.8–18.0 mg/day per person at the time of the AD 79 eruption, which is equivalent to the intake, commonly associated with crippling skeletal involvement, of 10–20 mg/day over a 10–20 year period (Connet, 2004; U.S. Environmental Protection Agency, 2002; U.S. NRC, 2006) and able to

increase the risk of bone fractures (Ayoob and Gupta, 2006; WHO, 2002). At present, in volcanic and other areas where groundwater is contaminated with fluoride of natural origin, communities with normal nutritional intake exposed to fluoride water concentrations of ca. 4 mg/L and daily total intake of ca. 14.0 mg/day show a prevalence of skeletal and dental fluorosis equivalent to those observed for the Herculaneum residents (Choubisa, 2001; Grimaldo et al., 1995; Jolly et al., 1968; WHO, 2002). Notably, lower concentrations of ca. 2.0 mg/L of fluoride present in the Bolan and Nari rivers in Baluchistan, Pakistan, have been found associated with severe cases of dental fluorosis in modern and ancient peoples of this arid region (Lukacs, 1984; Lukacs et al., 1985). Furthermore, extensive research from India (Ayoob and Gupta, 2006) provides evidence that endemic skeletal fluorosis can occur at water-borne fluoride concentrations of 2–3 mg/L or even as low as 1.1–1.5 mg/L, with crippling deformities appearing at 2.8 mg/L, given

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P. Petrone et al. / Journal of Geochemical Exploration 131 (2013) 14–27

Fig. 5. Skeletal lesion index related to age by gender. The linear regressions obtained by comparing skeletal lesion index (SLI) vs. age, separately for males and females, shows that nearly 90% of SLI variability is age related (males: R2 = 0.895, P b 0.0001; females: R2 = 0.877, P b 0.0001). However, males aged ≤ 30 years old are in average more affected than females, while females over the thirties are more frequently involved (test for no equality of regression coefficients, t = 7, P b 0.0001).

the presence of a favorable environmental context (geology, soil, climate, groundwater chemistry). The clinical and analytical scenario of endemic fluorosis detected at ancient Herculaneum emerge as still occurring today. A clinical– epidemiological investigation in 845 schoolchildren from the Vesuvian towns, where the maximum fluoride content in tap water is 2.8 mg/L according to local guidelines (Regione Campania, 2011), found 81.6% prevalence of dental fluorosis (Gombos et al., 1994), which is commonly considered a biomarker for fluoride exposure (WHO, 1970). The children, 6 to 11 years old, showed moderate to severe tooth discolorations (mottling) and brown staining (Fig. 9A, B and C), pitting (Fig. 9C) and extensive loss of enamel (Fig. 9D). In addition to dental fluorosis, significant clinical features were apparent. Mottled teeth were frequently accompanied by certain skin complaints (angiectasia, 51.0%) of different degrees and location (Fig. 10A, B, C and D), and hair loss (alopecia, 15.7%). Stomachache, articular pains and mottled finger-nails were also recurrent. In those children who exhibited dental fluorosis, chemical, clinical and hematological analyses also revealed that both free fluorine and total fluorine content in the serum greatly exceeded the normal (0.05 ppm) and pathological (0.22 ppm) maximum levels recommended by the World Health Organization (WHO) (WHO, 2008). It should be noted that pathological values of serum fluoride of 0.22 ppm or higher are typical of areas in which fluorosis is endemic (WHO, 1984). Furthermore, as regards thyroid activity, in several children affected by dental fluorosis the levels of serum thyroid parameters such as FT4 (free thyroxine hormone) and TSH (thyroid-stimulating hormone) suggested borderline hyperthyroidism. The recurrent cases of mottled teeth frequently accompanied by changes in skin, hair and nails, coupled with pathological levels of serum fluoride appear to be evident features of chronic fluorine poisoning. The common manifestation of these pathological features in the present-day school-age population shows the real extent of fluorosis around Vesuvius. A recent report by the National Academy's National Research Council (NRC) (U.S. NRC, 2006) concluded that the maximum contaminant level (MCL) of 4 mg/L of fluoride allowed by the U.S. Environmental Protection Agency in drinking water does not protect Fig. 6. Dental pathological features. A. Marked linear enamel hypoplasia (LEH), maxillary anterior teeth, 19 year old male. The right lateral incisor and canine are fused into one single dental element; B. Brown stains and mottling, upper central incisors, 36 year old male; C. Severe and confluent pitting, and yellow-brown staining, lower canines, 44 year old male. Note the longitudinal hypoplastic alteration of the enamel surface (arrows) in the right canine; D. Discrete and confluent pitting (black arrows), upper left central incisor, male 20 years old. Note the severe enamel hypomineralization in the form of corroded-like appearance (white arrow).

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Table 4 Evaluation of hypoplastic mottling in enamel of permanent teeth. The occurrence of enamel mottling, staining and pitting, along with alterations of the form of permanent teeth are symptomatic of dental fluorosis. Moderate to severe fluoroses involves 34.4% of the individuals (27.6% of teeth), with 25.0% occurrence of marked hypomineralization (17.8% of teeth). In the present work, according to Dean's classification (1942) of fluorosis, authors adopted a simplified four-value scoring system. Individual

M-WA

BS

P

CP

CLA

Minimum ind. score

Maximum ind. score

Average ind. score

General evaluation

5.1 5.3 10.2 10.3 10.5 10.6 10.7 10.12 10.13 10.14 10.15 10.17 10.19 10.20 10.22 10.23 10.24 10.26 10.28 10.SPD1 10.SPD2 10.SPD3 12.5 12.15 12.16 12.19 12.22 12.23 12.26 12.27 12.28 12.30

2.0 1.7 0.7 1.8 0 0.3 1.0 0.7 0.4 1.0 0.9 1.4 1.3 1.6 1.0 1.6 0.4 0.5 2.0 3.0 1.0 1.0 0.8 1.2 0.6 0.6 1.8 2.5 0 0.7 1.6 0

1.0 1.2 0.8 2.6 0.3 0.4 0.3 0.3 0.4 2.0 0 1.9 2.3 1.4 0.8 1.6 0.4 0 0 3.0 0 / 0.3 0.5 0.3 0 1.5 2.8 0 0.4 1.2 0

2.0 1.6 0.8 1.9 0.9 1.0 1.1 0.8 0.7 1.0 1.1 1.3 2.0 1.4 2.0 1.6 0.1 0.3 1.5 3.0 1.0 0 0.6 1.6 0.8 0.9 2.3 1.8 0 0.7 1.2 0

1.0 1.4 0.6 1.3 0.1 0.4 0.7 0.1 0.4 1.0 0.5 0.5 1.7 1.1 1.6 1.3 0 0 0.5 1.0 0 0 0.3 1.5 0.2 0.5 2.1 1.3 0 0.3 0.6 0

2.0 1.3 0.6 1.0 0 0.2 0.7 0.2 0.3 1.0 0.4 0.7 1.7 0.8 1.6 1.3 0 0 0 / 0 0 0.3 1.6 0.2 0.5 2.3 0.9 0 0.1 0.4 0

1.0 1.2 0 1.0 0 0.2 0.3 0.1 0.3 1.0 0 0.7 1.3 0.8 0.8 1.3 0 0 0 1.0 0 0 0.3 0.5 0.2 0 1.5 0.9 0 0.1 0.4 0

2.0 1.7 0.8 2.6 0.9 1.0 1.1 0.8 0.7 2.0 1.1 1.9 2.3 1.6 2.0 1.6 0.4 0.5 2.0 3.0 1.0 1.0 0.8 1.6 0.8 0.9 2.3 2.9 0 0.7 1.6 0

1.4 1.4 0.7 1.9 0.3 0.5 0.8 0.4 0.4 1.2 0.6 1.2 1.8 1.3 1.4 1.5 0.2 0.2 0.8 2.0 0.4 0.3 0.5 1.3 0.4 0.5 2.0 1.9 0 0.4 1.0 0

Moderate Mild Normal Severe Normal Normal Mild Normal Normal Moderate Mild Moderate Severe Mild Moderate Moderate Normal Normal Moderate Severe Normal Normal Normal Mild normal Normal Severe Severe Normal Normal Mild Normal

M-WA = milky-white appearance. BS = yellow-brown stains. P = pitting. CP = confluent pitting. CLA = corroded-like appearance. 0 = normal (translucent and smooth enamel, glossy appearance). 1 = mild (scattered small, opaque, milky-white patches; faint brown stains are sometimes apparent). 2 = moderate (diffuse white opaque areas, minute pitting; brown stain is frequent; surfaces subject to attrition show marked wear). 3 = severe (pits deeper and confluent, widespread stains; the tooth shows a corroded-like appearance). / = indefinable.

against adverse health effects, particularly in children. Even the so-called secondary MCL of 2 mg/L proved inadequate (Ayoob and Gupta, 2006). Evidence from modern Pakistan confirms that even this modest level of fluoride in drinking water can have toxic effects in children. In those seasonally hot and arid environments, greater water consumption required to prevent dehydration and the use of fluoridated water to irrigate crops and prepare food, as well as malnutrition, can significantly elevate the fluoride intake, exacerbating its Table 5 Oligoelement contents in human bones. The amounts of zinc (Zn), strontium (Sr), magnesium (Mg), copper (Cu) and calcium (Ca) in the human bone were assessed by atomic absorption spectroscopy. The oligoelement concentration was measured in herbivore bones (Equus caballus, Ovis capra) and sediment as well, in order to exclude inaccurate data as a result of possible diagenetic processes. Element

Human bone (N = 10)

Herbivore bone (N = 3)

Sediment

Estimation

Ca (mg/g) Sr (ppm) Zn (ppm) Mg (ppm) Cu (ppm) Sr/Ca Sr/Ca (*) Zn/Ca

319.51 ± 16.70 131.00 ± 18.36 151.46 ± 31.53 4309.20 ± 1137.52 184.06 ± 79.67 0.41 ± 0.06 0.73 ± 0.10 0.48 ± 0.11

266.33 ± 68.90 148.17 ± 9.09 137.90 ± 39.67 3368.67 ± 817.59 175.97 ± 88.10 / / /

40.8 85.1 68.7 8545.0 44.9 / / /

Not defiled Not defiled Not defiled Defiled Not defiled / / /

adverse physiological effects (Lukacs, 1984; Lukacs et al., 1985). In addition, NRC model predictions show that bone fluoride concentrations resulting from lifetime exposure to fluoride in drinking water at 2 or 4 mg/L fall within or exceed the ranges associated with stage II and stage III of skeletal fluorosis, and may increase the risk of overall fractures (U.S. NRC, 2006). Furthermore, the NRC report concluded that fluoride could start or promote cancer, and osteosarcoma is of a particular concern, together with other types of bone cancer. A recent large hospital-based case-control study of age-specific fluoride exposure in drinking water and the incidence of osteosarcoma in the United States found a seven-fold increased risk of bone cancer in young boys due to fluorosilicates (Bassin et al., 2006), the most widely used form of fluoride added to drinking water. 5. Conclusions Our findings on the pathological skeletal and dental features of the ancient residents of Herculaneum indicate that fluorosis in the Neapolitan area was endemic already during Roman times. The close – and somewhat disturbing – parallels that we found between fluoride exposure in the ancient population of AD 79 Herculaneum and that in present-day schoolchildren in the Vesuvian area show a permanent fluoride health hazard for the entire population living around Vesuvius. At present, the major public health and socio-economic impact of this

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Table 6 Human bone samples tested for determination of fluorine concentration. For each sample are reported specimen, age at death, tested skeletal element, measured sodium-corrected fluorine concentration (ppm), fluorine corrected values (ppm), and interval for expected single value (95%). Fluorine concentration of infants aged 0 to 10 years old were not utilized for statistical elaboration, since the amounts detected for infants (16,371 ppm on average) exceeded those of 12 to 30 year old individuals (16,164 ppm on average). Individual

Average age

Skeletal element

Na-corrected μ F (ppm)

Corrected F values

±E.S.V.I. 95%

11:15 10:41 12:12 12:18 12:17 12:6 12:25 12:14 5:1 12:20 12:9 12:4 10:29 10:19 12:26 10:13 10:12 12:28 12:27 10:14 10:21 10:18 10:1 12:23 10:20 10:3 12:11

0 (7 iu-m) 0.5–1.5 2.5–3.5 3–4 5–6 8–9 9–11 11–13 13–16 16–20 23–28 24–30 26–32 27–33 28–34 31–37 31–37 32–39 32–39 34–40 34.5–40.5 35–41 36–42 38–46 40–48 45–55 47–57

Rib Ilium Ilium Ilium Ilium Ilium Ilium Ilium Ilium Ilium Rib Rib Rib Rib Ilium Ilium Ilium Ilium Ilium Ilium Ilium Rib Ilium Ilium Ilium Rib Ilium

18,400 ± 1275 16,600 ± 540 16,900 ± 450 15,200 ± 793 16,500 ± 476 16,100 ± 1803 14,900 ± 1340 14,000 ± 1267 14,400 ± 372 16,000 ± 624 16,300 ± 467 17,200 ± 381 16,350 ± 166 18,900 ± 435 17,300 ± 597 19,000 ± 495 19,250 ± 311 19,300 ± 271 20,800 ± 197 18,200 ± 398 19,200 ± 213 19,850 ± 221 22,100 ± 363 19,900 ± 620 23,050 ± 508 23,300 ± 95 18,200 ± 196

/ / / / / / / 2042 2442 4042 4342 5242 4392 6942 5342 7042 7292 7342 8842 6242 7242 7892 10,142 7942 11,092 11,342 6242

/ / / / / / / 3392 3328 3249 3138 3118 3103 3098 3094 3090 3090 3095 3095 3099 3102 3105 3113 3142 3169 3279 3325

iu-m = intra-uterine months; E.S.V.I. = interval for expected single value.

hazard is underestimated. In the past 25 years, the local authorities have reduced the maximum permitted fluoride content in tap water throughout the area from 4.0 to 3.0 mg/L and again to its current level of 2.5 mg/L. However, this lower value still far exceeds WHO guidelines for maximum fluoride content (1.5 mg/L). WHO drinking water guidelines are based on internationally agreed procedures for risk assessment and aim to ensure that drinking water does not represent any significant risk to health over a lifetime of consumption. Effects on the skeleton are the best indicators of the toxic responses to fluoride and are considered to have direct public health

Fig. 7. Fluorine (19F) bone concentration (ppm) as a function of age. The linear regression resulting from (A) the fluorine mean amount of 18,400 to 23,300 ppm measured by INAA (intercept = 11,958.5 ± 1120, P b 0.001) is compared with an equivalent regression (B) obtained considering a 0 fluorine concentration at age 0 (slope = 200.4 ± 9, P b 0.001). The last model, representing the physiological rate of individual intake per year cleansed of the fraction of fluorine contamination by soil ash deposit, shows an evident age-dependent increase of fluorine (R2 = 0.961). Children aged ≤10 years old were not included in this model, due to the high diagenetic amount of fluorine released by the ash deposit. The resulting corrected mean values of 2042 to 11,342 ppm show a minority of individuals matching the normal–physiological (b3500 ppm) and preclinical (b5500 ppm) ranges of fluorine bone concentration, while the majority belongs to all the three clinical phases of skeletal fluorosis, with several mature (≥40 year old) individuals in the crippling phase III.

relevance. According to WHO recommendations, in areas with high fluoride levels and a warm climate it would be appropriate to lower the maximum value of 1.5 mg/L established for naturally occurring fluoride in drinking water. Therefore in setting guidelines for fluoride

Fig. 8. Skeletal lesion index related to age by observed vs. expected data. The equation SLI = 0.000058696 ·(200,347 · age) = 0.011759 · age applied to the available data, shows the correlation (R2 = 0.81) of the observed data with those expected, thus indicating that the SLI index suitably describes the degree of joint lesions shown by the Herculaneum residents.

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Fig. 9. Dental fluorosis in schoolchildren from Vesuvian towns. Moderate to severe discoloration, staining and pitting, and hypomineralization in permanent anterior teeth. A: light dyschromic linear patches (male child); B: moderate brown mottling and milky patches (male child); C: severe dyschromia due to linear and pitted brown mottling (female child); D: severe hypoplasia in the form of extensive enamel loss of (male child).

in the densely populated Vesuvius area, the following predisposing factors for fluorosis should be better evaluated: ambient temperature, volume of water intake, other trace elements in the water, a diet based on beverages and food preparation in naturally fluoridated boiled water, and water storage methods. Bearing in mind that progressively higher fluoride intakes lead to increasing risks of dental and skeletal fluorosis, as clearly demonstrated by the Herculaneum

Fig. 10. Skin complaints in schoolchildren from Vesuvian towns affected by dental fluorosis. Different appearance and location of angiectasia (blood vessel dilatation). A: “microangioma-like” type (female child); B: “reticulus-like” type (female child); C: marked blood vessel dilatation in the form of “reticulus-like” type (male child); D: angiectasia in the form of both “microangioma-like” and “reticulus-like” types (female child).

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victims, the adoption of low-cost defluoridation methods should be seriously considered and encouraged. In evaluating all the possible health consequences of exposure to fluoride concentrations higher than the established WHO parameters, it should also be taken into account that the maximum fluoride content of 5.0 mg/L accepted in natural mineral waters protects only the population over 15 years old and only if there is no exposure to fluoride from other sources, as experienced by communities living in volcanic and other fluoride-rich areas. Author contributions PP designed the site and laboratory research. PP performed the bioanthropological and palaeopathological research. GFM performed the histological research and wrote the histopathological part. GF performed the epidemiological research. PP GS GFM GF analyzed the data. GS GFM did statistics. PP wrote the paper. Acknowledgments We thank the Superintendency of Pompeii for granting the field investigation and the study of the human skeletal material. We also thank Vincenzo Monetti for the water analysis and Emanuele Minelli for the digital X-ray images. We are also indebted to Mark Walters for the final text editing. References Aggarwal, N.D., 1973. Structure of human fluorotic bone. Journal of Bone and Joint Surgery 55, 331–334. Aoba, T., Fejerskov, O., 2002. Dental fluorosis: chemistry and biology. Critical Reviews in Oral Biology and Medicine 13, 155–170. Ayoob, S., Gupta, A.K., 2006. Fluoride in drinking water: a review on the status and stress effects. Critical Reviews in Environmental Science and Technology 36, 433–487. Bassin, E.B., Wypij, D., Davis, R.B., Mittleman, M.A., 2006. Age-specific fluoride exposure in drinking water and osteosarcoma (United States). Cancer Causes & Control 17, 481–482. Bisel, C., 1980. A pilot study in aspects of human nutrition in the ancient eastern Mediterranean, with particular attention to trace mineral in several populations from different time periods, PhD University of Minnesota, Minneapolis. Bisel, C., 1991. The human skeletons of Herculaneum. International Journal of Anthropology 6, 1–20. Bocanegra, E.M., Hernández, M.A., Usunoff, E., 2005. Groundwater and human development. IAH Selected Papers on Hydrogeology, vol. 6. Taylor & Francis, London, p. 278. Boivin, G., Chavassieux, P., Chapuy, M.C., Baud, C.A., Meunier, P.J., 1989. Skeletal fluorosis: histomorphometric analysis of bone changes and bone fluoride in 29 patients. Bone 10, 89–99. Buikstra, J.E., Ubelaker, D.H., 1994. Standards for Data Collection from Human Skeletal Remains Research Series, no. 44. Arkansas Archaeological Survey, Fayetteville. Cao, Y.Q., Yang, Y.S., Hu, K.R., Kalin, R.M., 2004. Ecological and geochemical modelling of hydrogeological system with particular connection to human health. Ecological Modelling 174, 375–385. Capasso, L., 1998. Work-related syndesmoses on the bones of children who died at Herculaneum. Lancet 352, 1634. Capasso, L., 2001. I fuggiaschi di Ercolano. Paleobiologia delle vittime dell'eruzione vesuviana del 79 d.C. “L'ERMA” di Bretschneider, Roma, p. 1089. Capasso, L., Kennedy, K.A.R., Wilczak, C.A. 1999. Atlas of occupational markers of human skeletal remains Edigraphital S.P.A., Teramo, p. 24. Chavassieux, P., Seeman, E., Delmas, P.D., 2007. Insights into material and structural basis of bone fragility from diseases associated with fractures: how determinants of the biomechanical properties of bone are compromised by disease. Endocrine Rewievs 28, 151–164. Cheng, T.P., Anderson, H.D., Mills, D.S., Spate, V.L., Baskett, C.K., Morris, J.S., 1997. Determination of the fluoride distribution in rabbit bone using instrumental neutron activation analysis. Journal of Radioanalytical and Nuclear Chemistry 217, 171–174. Choubisa, S.L., 2001. Endemic fluorosis in southern Rajasthan. India Fluoride 34, 61–70. Connet, M., 2004. Fluoride & Bone Damage: Published Data. http://www.fluoridealert. org/bone-data.pdf (9.22.2011). Cucina, A., Vargiu, R., Mancinelli, D., Ricci, R., Santandrea, A., Catalano, P., Coppa, A., 2006. The necropolis of Vallerano (Rome, 2nd–3rd century AD): an anthropological perspective on the ancient Romans in the suburbium. International Journal of Osteoarchaeology 16, 104–117. D'Alessandro, W., 2006. Human fluorosis related to volcanic activity: a review. In: Kungolos, A.G., Brebbia, C.A., Samaras, C.P., Popov, V. (Eds.), Transactions on

Biomedicine and Health: Environmental Toxicology, vol. 10. Witpress, Southampton, Boston, pp. 21–30. Dean, H.T., 1942. The investigation of physiological effects by the epidemiological method. In: Moulton, F.R. (Ed.), Fluorine and Dental Health, 19. American Association for the Advancement of Science, Washington, DC, pp. 23–31. Eble, D.M., Deaton, T.G., Wilson Jr., F.C., Bawden, J.W., 1992. Fluoride concentrations in human and rat bone. Journal of Public Health Dentistry 52, 288–291. Feemster Jashemski, W., Meyer, F.G. (Eds.), 2002. The Natural History of Pompeii. Cambridge University Press, Cambridge, p. 502. Gombos, F., Mangoni Di Stefano, C., Ruggiero, M., 1994. Investigation into fluorosis in school children of two Vesuvian towns. Stomatologic Archives 35, 141–168. Grimaldo, M., Turrubiartes, F., Milan, J., Pozos, A., Alfaro, C., Diaz-Barriga, F., 1995. Endemic fluorosis in San Luis Potosi, Mexico. I. Identification of risk factors associated with human exposure to fluoride. Environmental Research 68, 25–30. Guarino, F.M., Angelini, F., Vollono, C., Orefice, C., 2006. Bone preservation in human remains from the Terme del Sarno at Pompeii using light microscopy and scanning electron microscopy. Journal of Archaeological Science 33, 513–520. Haettich, B., Lebreton, C., Prier, A., Kaplan, G., 1991. Magnetic resonance imaging of fluorosis and stress fractures due to fluoride. Revue du Rhumatisme et des Maladies Ostéo-Articulaires 58, 803–808. Hedges, R.E.M., 2002. Bone diagenesis: an overview of processes. Archaeometry 44, 319–328. Hillson, S., 2005. Teeth Manuals in Archaeology, 2nd edition. Cambridge University Press, Cambridge, p. 373. Hughes, M.J., Cowell, M.R., Craddock, P.T., 1976. Atomic absorption techniques in archaeology. Archaeometry 18, 19–37. Ijaz, S., Croucher, R.E., Marinho, V.C.C., 2010. Systematic reviews of topical fluorides for dental caries: a review of reporting practice. Caries Research 44, 579–592. Jolly, S.S., Singh, B.M., Mathur, O.C., Malhotra, K.C., 1968. Epidemiological, clinical, and biochemical study of endemic dental and skeletal fluorosis in Punjab. British Medical Journal 4, 427–429. Jurmain, R.D., 1977. Stress and etiology of osteoarthritis. American Journal of Physical Anthropology 46, 353–366. Jurmain, R.D., 1990. Paleoepidemiology of a central California prehistoric population from CA-ALA-329: II. Degenerative disease. American Journal of Physical Anthropology 83, 83–94. Krishnamachari, K.A., 1986. Skeletal fluorosis in humans. A review of recent progress in the understanding of the disease. Progress in Food & Nutrition Science 10, 279–314. Littleton, J., 1999. Paleopathology of skeletal fluorosis. American Journal of Physical Anthropology 109, 465–483. Lukacs, J.R., 1984. Dental fluorosis in early Neolithic Pakistan. American Journal of Physical Anthropology 63, 188. Lukacs, J.R., Retief, D.H., Jarrige, J.-F., 1985. Dental disease in prehistoric Baluchistan. National Geographic Research 1, 184–197. Manzi, G., Salvadei, L., Vienna, A., Passarello, P., 1999. Discontinuity of life conditions at the transition from the Roman Imperial Age to the Early Middle Ages: example from Central Italy evaluated by pathological dento-alveolar lesions. American Journal of Human Biology 11, 327–341. Mastrolorenzo, G., Petrone, P.P., Pagano, M., Incoronato, A., Baxter, P.J., Canzanella, A., Fattore, L., 2001. Herculaneum victims of Vesuvius in AD 79. Nature 410, 769–770. Mastrolorenzo, G., Petrone, P., Pappalardo, L., Sheridan, M.F., 2006. The Avellino 3780-yrB.P. catastrophe as a worst-case scenario for a future eruption at Vesuvius. Proceedings of the National Academy of Sciences of the United States of America 103, 4366–4370. Mastrolorenzo, G., Petrone, P., Pappalardo, L., Guarino, F.M., 2010. Lethal thermal impact at periphery of pyroclastic surges: evidences at Pompeii. PLoS One 5 (1-12), e11127 http://dx.doi.org/10.1371/journal.pone.0011127. Ortner, D.J., 2003. Identification of Pathological Conditions in Human Skeletal Remains, 2nd edition. Academic Press, Elsevier, San Diego, pp. 406–410. Ozsvath, D.L., 2009. Fluoride and environmental health: a review. Reviews in Environmental Science and Biotechnology 8, 59–79. Petrone, P., Fattore, L., Monetti, V., 2002. Alimentazione e malattie ad Ercolano Vesuvio 79 AD. Vita e morte ad Ercolano. In: Petrone, P., Fedele, F. (Eds.), Fridericiana Editrice Universitaria, Napoli, pp. 75–83. Petrone, P., Giordano, M., Giustino, S., Guarino, F.M., 2011. Enduring fluoride health hazard for the Vesuvius area population: the case of AD 79 Herculaneum. PLoS One 5 http://dx.doi.org/10.1371/journal.pone.0021085 (1-12 6:6 e21085). Regione Campania, 2011. Deliberazione N. 988 del 30/12/2010 Deroga al parametro del fluoro ai sensi della direttiva 98/83/CE concernente la qualità delle acque destinate al consumo umano in quattordici Comuni dell'Area Vesuviana, Anno. Resnick, D., Niwayama, G., 1983. Entheses and enthesopathy. Anatomical, pathological, and radiological correlation. Radiology 146, 1–9. Robb, J., Bigazzi, R., Lazzarini, L., Scarsini, C., Sonego, F., 2001. Social “status” and “biological” status: a comparison of grave goods and skeletal indicators from Pontecagnano. American Journal of Physical Anthropology 115, 213–222. Rogers, J., Shepstone, L., Dieppe, P., 1997. Bone formers: osteophyte and enthesophyte formation are positively associated. Annals of the Rheumatic Diseases 56, 85–90. Sastri, C.S., Iyengar, V., Blondiaux, G., Tessier, Y., Petri, H., Hoffman, P., Aras, N.K., Zaichick, V., Ortner, H.M., 2001. Fluorine determination in human and animal bones by particle-induced gamma-ray emission. Fresenius' Journal of Analytical Chemistry 370, 924–929. Schurr, M.R., 1989. Fluoride dating of prehistoric bones by ion selective electrode. Journal of Archaeological Science 16, 265–270. Skinner, M.F., Goodman, A.H., 1992. Anthropological uses of developmental defects of enamel. In: Saunders, S.R., Katzenberg, M.A. (Eds.), Skeletal Biology of Past Peoples: Research Methods. Wiley-Liss, New York, pp. 153–174.

P. Petrone et al. / Journal of Geochemical Exploration 131 (2013) 14–27 Sperduti, A., 1997. Life conditions of a Roman Imperial Age population: occupational stress markers and working activities in Lucus Feroniae (Rome, lst–2nd cent. AD). Human Evolution 12, 253–267. Teotia, M., Teotia, S.P.S., Singh, K.P., 1998. Endemic chronic fluoride toxicity and dietary calcium deficiency interaction syndromes of metabolic bone disease and deformities in India: year 2000. Indian Journal of Pediatrics 65, 371–381. Thylstrup, A., Fejerskov, O., 1978. Clinical appearance of dental fluorosis in permanent teeth in relation to histologic changes. Community of Dental Oral Epidemiology 6, 315–328. Tressaud, A., 2006. Fluorine and the Environment, Agrochemicals, Archaeology, Green Chemistry and Water, vol. 2. Elsevier, Amsterdam, Oxford, p. 286. U.S. Environmental Protection Agency, 2002. National primary drinking water regulations, drinking water contaminants. Fluoride 56–80 http://water.epa.gov/drink/ contaminants/index.cfm#Inorganic (11.23.2011). U.S. NRC, 1993. Health Effects of Ingested Fluoride. United States National Research Council, National Academies Press, Washington DC . http://books.nap.edu/openbook.php? isbn=030904975X&page=R1 (11.11.2011). U.S. NRC, 2006. Fluoride in Drinking Water: A Scientific Review of EPA's Standards. United States National Research Council, National Academies Press, Washington DC . http://www.nap.edu/catalog/11571.html (11.11.2011). Vargiu, R., Bellini, G.R., Mancinelli, D., Santoro, P., Miranda, G., Paine, R.R., Russo, I., Coppa, A., 2007. Condizioni di vita e stato di salute della comunità di Aquinum (Castrocielo, Frosinone) Lazio e Sabina. Scoperte, scavi e ricerche “L'Erma” di Bretschneider, Roma, pp. 476–481.

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Weidmann, S.M., Weatherell, J.A., Jackson, D., 1963. The effect of fluoride on bone. Proceedings of the Nutrition Society 22, 105–110. WHO, 1970. Fluorides and human health. Monograph Series, No. 59. World Health Organization, Geneva, p. 364. WHO, 1984. Environmental Health Criteria 36. Fluorine and Fluorides World Health Organization, Geneva . http://www.inchem.org/documents/ehc/ehc/ehc36.htm (12.15.2011). WHO, 2002. Environmental Health Criteria 227: Fluorides. World Health Organization, Geneva, pp. 104–114. http://www.inchem.org/documents/ehc/ehc/ehc227.htm#1.2 (11.11.2011). WHO, 2008. Guidelines for drinking-water quality. Incorporating 1st and 2nd Addenda to Third Edition. : Recommendations, vol. 1. World Health Organization, Geneva, pp. 375–377. http://www.who.int/water_sanitation_health/dwq/ gdwq3rev/en/ (11.11.2011). Wittmers, L.E., Aufderheide, A.C., Pounds, J.G., Jones, K.V., Angel, J.L., 2008. Problems in determination of skeletal lead burden in archaeological samples: an example from the first African Baptist Church population. American Journal of Physical Anthropology 136, 379–386. Wondwossen, F., Nordrehaug Ǻstrøm, A., Kjell Bjorvatn, K., Bårdsen, A., 2004. The relationship between dental caries and dental fluorosis in areas with moderate- and highfluoride drinking water in Ethiopia community. Dentistry and Oral Epidemiology 32, 337–344. Yoshimura, K., Nakahashi, T., Saito, K., 2006. Why did the ancient inhabitants of Palmyra suffer fluorosis? Journal of Archaeological Science 33, 1411–1418.