Journal of Geochemical Exploration 131 (2013) 14–27

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Journal of Geochemical Exploration

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

⁎ Corresponding author. Tel./fax: +39 081 2535211,E-mail address: pipetron@unina.it (P. Petrone).

0375-6742/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.gexplo.2012.11.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 February 2012Accepted 20 November 2012Available online 30 November 2012

Keywords:Environment and human healthHealth risk assessmentEndemic fluorosisEpidemiologyHerculaneum victims79 AD Vesuvius eruption

Endemic fluorosis induced by high concentrations of natural fluoride in groundwater and soils is a major healthproblem in several countries, particularly in volcanic areas. The early stages of skeletal fluorosis, a chronic met-abolic bone and joint disease rarely considered in palaeopathological diagnoses, are often misdiagnosed in en-demic areas. In this paper, morphological, radiological, histological and chemical skeletal and dental features ofthe 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 asdental features such as mottling and hypomineralization, confirm the endemicity of fluorosis, which still occurstoday. When merged with the results of a recent clinical–epidemiological investigation in schoolchildren fromthe Vesuvian towns, our findings reveal for the resident population a permanent fluoride hazard whose healthand socio-economic impact is currently underestimated.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Ecological, hydrogeological and geochemical factors affect thephysical environment in which people live. Several potentially toxicelements may contaminate the environment, and their biologicaland physiological effects are associated to the prevalence and severityof 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 circu-lation of water in rocks and soils, fluorine can be leached out anddissolved in groundwater and thermal gases. In groundwater, the natu-ral concentration of fluoride depends on the geological, chemical andphysical characteristics of the aquifer, the porosity and acidity of thesoil and rocks, the temperature and the action of other chemical ele-ments. Potentially fluoride-rich environments are mainly linked withPrecambrian basement areas and those affected by recent volcanism(Bocanegra et al., 2005). Because of the large number of variables, fluo-ride concentrations in groundwater can range from well under 1 mg/Lto more than 35 mg/L. Therefore, high concentrations of naturallyoccurring fluoride in groundwater can be found in different countriesas well as locally in most parts of the world (WHO, 2008). In severalcountries, fluoride is also added to public drinking water supplies dueto the significant effects of mitigating dental caries and strengtheningbones if the concentration intake is approximately 1 mg/L (Littleton,1999; Ozsvath, 2009). Fluoride can enter public water systems from

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natural sources (Ayoob and Gupta, 2006; D'Alessandro, 2006), particu-larly in volcanic areas, where high amounts offluoride in drinkingwaterare typically found due to contamination from ash deposits (Ozsvath,2009; U.S. NRC, 2006). This is the case of the Somma–Vesuvius sur-roundings, repeatedly covered by pyroclastic products since prehistorictimes (Mastrolorenzo et al., 2006).

Long-term intake of high doses of fluoride can have adverse effectson 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 or-gans of fluoride, and tend to accumulate it with age (Ayoob andGupta, 2006; Littleton, 1999; U.S. NRC, 1993). Dose and duration offluoride intake, age, sex, nutritional status and diet, climate andrenal efficiency in fluoride excretion are the main variable factorsaffecting the risk of developing fluorosis, an increasingly disablingdisease. This disease usually affects older adults, and men more fre-quently than women (Ayoob and Gupta, 2006; Ozsvath, 2009). Skel-etal fluorosis is a chronic metabolic bone and joint disease causedby prolonged, excessive ingestion of fluoride, mostly through waterin endemic areas. Increased fluoride content in the bone is the mainindicator of fluoride poisoning (Krishnamachari, 1986). Primarily,fluoride acts as a cumulative toxin altering accretion and resorptionof bone tissue and affecting the homeostasis of bone mineral metab-olism, causing functional adverse effects (Littleton, 1999). Chronicfluorine 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 andFejerskov, 1978; U.S. NRC, 2006; WHO, 1970). Enamel hypoplasias

15P. Petrone et al. / Journal of Geochemical Exploration 131 (2013) 14–27

are caused by a wide range of factors, including malnutrition, febrilediseases, infections during pregnancy or infancy, trauma to theteeth and jaws, exposure to toxic chemicals, and a variety of heredi-tary disorders (Aoba and Fejerskov, 2002). Hypoplastic defects offluorotic origin may have a different etiology than linear enamelhypoplasia (LEH). Linear enamel hypoplasias, a deficiency in enamelformation visible on mammal tooth crowns, are unspecific stressmarkers commonly seen in several economically less developedpresent-day countries as well as in children of malnourished ancientcommunities. Therefore, LEH are mostly utilized in palaeopathologyas a systemic physiological stress indicator (Skinner and Goodman,1992). In bones and teeth, fluorine ion exchange takes place via re-crystallization of hydroxyapatite into the more stable fluoroapatite(WHO, 1970). The physiological effects of fluoride intake on theadult skeleton are the result of effects on the chemistry, gross mor-phology, histopathology, X-ray density, and integrity of structure ofbone and teeth (Aoba and Fejerskov, 2002; WHO, 1970).

Skeletal fluorosis is characterized by periosteal thickening, calcifi-cation of tendons and ligaments, and abnormal production of multi-ple hypertrophic bony exostoses (osteophytes) at ligamentous andmuscular attachments to bone (Resnick and Niwayama, 1983). Theclinical condition exhibits bone, joint and muscle pains due to earlyrestrictions in spine movements and at a later stage progressive anky-losis of the vertebral joints induced by ligamentous calcification. Ver-tebrae, ribs and pelvis are more prone to osteophyte formation thanlong bones, even if increasing immobilization also spreads to themajor joints of the chest and knees. In advanced stages the entireskeleton may be involved by crippling deformities, which can befound in the pediatric age group too (Weidmann et al., 1963). Radio-logical and histological findings closely parallel macroscopic changes.Extensive production of new bone, usually associated with boneresorption, may result in an overall increase in bone thickness andradio-opacity, besides a low degree of mineralization (Chavassieuxet al., 2007). The outcome is a combination of osteosclerosis, osteopo-rosis and osteomalacia of different degrees (Krishnamachari, 1986;Littleton, 1999; Weidmann et al., 1963). An altered organic matrix,reduced mineralization and osteosclerosis are also apparent fromhistopathological examinations (Krishnamachari, 1986). Despite theincrease in bone tissue mass but not in density, fluorotic bonesare thus brittle, of poorer mechanical quality and easier to break(Chavassieux et al., 2007).

Even if skeletal fluorosis has been widely studied, because some ofthe early clinical symptoms resemble those of osteoarthritis, the firstclinical phases of skeletal fluorosis could be easily misdiagnosed(Ayoob and Gupta, 2006). In its advanced stage it becomes a cripplingdisability that has a major public health and socio-economic impact, af-fecting tens of millions of people in Africa, India and China, and beingendemic 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 ofpalaeopathological lesions, mostly concerning specific single casesshowing excessive ossification and joint ankylosis (Ortner, 2003). Den-tal fluorosis was first reported for Neolithic and Chalcolithic skeletalsamples from Pakistan (Lukacs, 1984; Lukacs et al., 1985). Later reportsfor historical times (Littleton, 1999; Yoshimura et al., 2006) refer to aridregions and other areas in which the disease still occurs today due tohigh fluoride concentrations in drinking water.

Here we further discuss the pathological condition of 76 Herculane-um victims of the 79 AD Vesuvius eruption (Mastrolorenzo et al., 2001),making comparisons with unpublished data and clinical findings fromschoolchildren in the Vesuvian area (Gombos et al., 1994). Being aunique cross section of the entire living population, the Herculaneumskeletal sample is particularly suited to palaeoepidemiologic investiga-tion, which also has important implications for present-day populations.All skeletonswere in an extraordinary good state of preservation as a re-sult of the unusual death and burial conditions involved: instant death

caused by emplacement of hot pyroclastic surge (ca. 500 °C), followedby rapid vaporization of soft tissues replaced by ash (Mastrolorenzoet al., 2010). This anoxic fluoride-rich ash bed deposit was permanentlysaturated by groundwater (Capasso, 2001).

Further analysis of the skeletal and dental evidences for fluoride ex-posure at Herculaneum available to date (Petrone et al., 2011), mergedwith currently available epidemiologic data on school-children, strong-ly supports, as expected, the hypothesis of a long-lasting fluoride healthhazard for the Vesuvius area population.

2. Materials and methods

This studywas approvedby the Ethics Committee for Biomedical Ac-tivities of the University of Naples, Azienda Ospedaliera Universitaria“Federico II”, Naples (Protocol 154/10, 9.08.10). The Superintendencyof Pompeii granted field investigation and study of the human skeletalmaterials unearthed in the 1997–99 excavations of the water-frontchambers at Herculaneum.

2.1. Morphological, X-ray and histological bone analysis

We analyzed 76 human skeletons aged 0 to 52 years old, excavat-ed within the water-front chambers 5, 10 and 12 of the Herculaneumsuburban area (Table 1) (Mastrolorenzo et al., 2001). The composi-tion 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 of1.89 (36 males vs. 19 females) assessed on individuals >15 yearsold. Sex and age at death, as well as the prevalence of linear enamelhypoplastic defects (LEH) and dental caries were assessed accordingto standard diagnostic procedures (Buikstra and Ubelaker, 1994;Hillson, 2005; Resnick and Niwayama, 1983). Enamel fluorosis wasscored according to Dean's classification (Dean, 1942), that we sim-plified by adopting a four-value scoring system. The chest bones,spine, pelvis and long bones of each individual were examined forthe calcification of ligaments, cartilage and tendons (Rogers et al.,1997), as well as the presence of healed fractures. Hypertrophicosteosclerosis (osteophytosis) and spondyloarthritis of the spine,and osteoarthritic lesions of the appendicular skeleton were scoredboth individually and by single joint following standardized scoringcriteria (Jurmain, 1977, 1990). The most severe cases were also eval-uated adopting digital radiography (Villa Mercury 332, Kodak DirectView CR 850, Naples, IT) and histological analysis. We examinedthe undecalcified and unstained bone ground sections (80–100 μmthick), obtained after embedding in LY-554 araldite resin (Vantico)and observed under a transmitted ordinary and polarized light micro-scope. Due to the peculiar burial conditions of these skeletons, specif-ic attention has been paid to discriminate pathological vs. diageneticalteration. Thus the bone histology concerning alterations of themicrostructure and its birefringence were also investigated (Guarinoet al., 2006). Bone histology, porosity and enrichment of chemicalelements are diagenetic parameters that quantify the post-mortemosteoalteration. Bones buried for long periods absorb and accumulatefluoride from soil, and the extent of bone diagenesis is particularlyrelated to fluctuating hydrological regimes (Hedges, 2002; WHO,1970). Furthermore, permanently waterlogged environments areanoxic, 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 volcanicsediments were measured in order to test the pathologic diagnosisderived from the preliminary morphological and radiographic analy-ses of the skeletons, and also to avoid possible diagenetic processes.Additional analysis of the content of oligoelements in the humanbone was also carried out in order to define the nutritional statusat Herculaneum. Bone samples of herbivores (Equus caballus, Oviscapra) found in the water-front area were tested for standardization

Table 1Sex 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 ofPompeii.

N Ind. Sex Age at death N Ind. Sex Age at death N Ind. Sex Age at death

1 5:1 M 13–16 27 10:22 M 18–23 53 12:8 M 34–41.52 5:2 F 18–22.5 28 10:23 M 33–40 54 12:9 F 22–283 5:3 M 18–22.5 29 10:24 F 37–45 55 12:10 M?* 8–104 6:15 ? 7 iu-m 30 10:25 M 20–23 56 12:11 M 47–575 10:1 M 36–42 31 10:25B ? 19–24 57 12:12 M?* 2–46 10:2 M 14–16 32 10:26 M?* 8–10.5 58 12:13 F 33–41.57 10:3 M 45–55 33 10:27 F?* 4–6.5 59 12:14 M* 11–138 10:4 F 26–32 34 10:28 F 33–40 60 12:15 F 30–35.59 10:5 M 18–25 35 10:29 F 26–32 61 12:16 M 33–41.510 10:6 M 28–33.5 36 10:30 M?* 2–3 62 12:17 F? 5–611 10:7 M 33–38 37 10:32 M?* 8–11 63 12:18 F?* 3–412 10:8 M 11–15.5 38 10:33 F* 11–13 64 12:19 M 31–3713 10:9 M 13–16.5 39 10:34 F? 17–22 65 12:20 M 16–2014 10:10 M 33–38 40 10:35 M 20–39 66 12:21 F 29–3515 10:11A F 28–34.5 41 10:36 M 15–17 67 12:22 M 18–2316 10:11B M 31–37.5 42 10:38 ? 12–15 68 12:23 M 38–4617 10:12 M 31–37 43 10:39 M 12–15 69 12:24 M?* 8–1118 10:13 M 31–37 44 10:40 ? 11–14 70 12:25 M* 9–1119 10:14 M 34–40 45 10:41 F?* 0.5–1.5 71 12:26 M 28–3420 10:15 F 26–31 46 12:1 M?* 3–5 72 12:27 M 33–3921 10:16 F 34–38.5 47 12:2 F 23–29 73 12:28 F 33–3922 10:17 M 32–39 48 12:3 F 25–30 74 12:29 F?* 11–1323 10:18 F 35–41 49 12:4 M 24–30 75 12:30 F 33–37.524 10:19 M 27–33 50 12:5 M 15–18 76 12:31 F 20–3925 10:20 M 40–48 51 12:6 F?* 7–1026 10:21 M 34–41 52 12:7 M 16–18.5

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of the Sr/Ca ratio in human bones vs. herbivore bones from the samesite (Bisel, 1980). Samples of sediment collected within the town andon 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 thebone were measured in the iliac crest and/or rib bones of 27 Hercula-neum victims aged 0 to 52 years old of both sexes, by InstrumentalNeutron Activation Analysis (INAA) at the University of Missouri Re-search Reactor Centre (MURR) (Cheng et al., 1997).

As infant bones are more easily prone to post-mortem taphonomicand diagenetic changes because of their porosity and lower rate of cal-cification (Wittmers et al., 2008), as expected, the fluorine amountsfound 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 lattergroup were considered in statistical analyses.

2.3. Ion-selective electrode (ISE)

Thefluoride content in volcanic ashwas determined at theUniversityof Notre Dame Fluoride Dating Service, by using an ion-selective elec-trode (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 bonewere quantified by atomic absorptionspectroscopy (AAS) following the standard pre-treatment technique(Hughes et al., 1976). Bone element concentration was measured witha Perkin-Elmer atomic absorption spectroscope (mod. 2100). In orderto evaluate the palaeonutritional status of the sample, the results werecompared 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 wasevaluated by an ordinal scaling system. The degenerative changeswere scored following the four-value scoring classification adoptedby Jurmain (1977, 1990) and then standardized by means of ourown lesion index (Petrone et al., 2011).

3. Results

The majority of the Herculaneum population (73.5% of individ-uals ≥15 years old) was affected by intense calcification of the liga-ments, tendons, cartilage and interosseous membranes, associatedwith diffuse axial and appendicular osteosclerosis. Severe calcificationalong with proliferative bone abnormalities particularly involvescostochondral and costosternal junctions, ribs, spine anterior longitudi-nal ligaments, iliac crest and sacrotuberous ligaments. Gross and radio-graphic examinations of the long bones reveal massive corticalthickening, increased bone matrix density, narrowed medullary cavityand increased radio-opacity. In addition, most of the individuals(91.8%) display at least one long or flat bone affected by abnormalgrowth of osteophytes, including several juveniles and children. The to-pographic frequency distribution of ligament and tendon calcificationand ankylosis in the postcranial skeleton of specimens aged 1 to52 years old are summarized in Fig. 1.

The microscopic examination of ground cross sections of the juve-nile and adult long bones from both sexes show several histopatholog-ical alterations, including lost or poorly formed Haversian lamellarsystems (Fig. 2A and B) and extensive mottled bone matrix and en-larged Haversian canals (Fig. 2B and C). No evidence of structural diage-netic change by microorganism activity can be observed, as predictabledue to the anoxic burial environment of the skeletons.Within the inves-tigated sample, a distinctive osteoalteration consists in widespreadmicrocracking (Fig. 2C), due to exposure of the victims' corpses to thehigh temperature of the pyroclastic surge (Mastrolorenzo et al., 2001,2010).

Hypertrophic osteosclerosis (osteophytosis) and spondyloarthritisincrease towards the lumbar spine, with an overall prevalence of

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 atleast 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 interphalangealjoint and manubriosternal joint, affects at least one of these three anatomical sites in 39.2% of the individuals.

17P. Petrone et al. / Journal of Geochemical Exploration 131 (2013) 14–27

27.6% and 18.5%, respectively (Fig. 3). Spine ankylosis mainly involvesthoraco-lumbar (3.1%) and sacroiliac (4.6%) joints, affecting malesand females equally (18.2% vs. 17.6% of individuals, z=0.0523,P>0.05). In these severe cases the vertebral body contours are largelyuneven or fused, and bones have a chalky white appearance.

In the appendicular skeleton, a 47.2% overall occurrence ofosteoarthritic-like alterations of the joints appears particularly se-vere given the mean age of about 30 years of individuals ≥15 yearsold. The coxofemoral, knee, sacroiliac, elbow, and sternoclavicularjoints and pedal phalanges are the most noticeably affected anatom-ical districts (Fig. 4). In general, ankylosis affects at least one

anatomical site in 39.2% of the individuals. In addition, nearly one in-dividual out of three (32.1%) shows one or more pathological frac-tures involving mostly the spine or os coxae, as well as the longbones, while osteomalacia affects 8.2% of the individuals. Evaluatingthe cases of spondylolysis (L5 vertebrae, 8.9% vs. 3–7% of the generalpopulation) as stress fractures (Capasso et al., 1999; Haettich et al.,1991), the susceptibility to bone fractures is particularly high atHerculaneum (35.7%) (Table 2) (for further details, see Petroneet al., 2011).

The degree of individual osteoarthritic-like lesions involving thepost-cranial skeleton was also assessed by means of a skeletal lesion

Fig. 2. Histopathological bone features. Representative bone ground sections observedunder a transmitted light microscope: A. Phalanx showing extensive mottling of bonematrix and disordered lamellar architecture, 10 years old male; B. Mid shaft of femurwith widespread deficiency of the Haversian lamellar systems and several enlargedHaversian canals, 15 years old male. Note the presence of several linear formation de-fects (arrows); C. Mid shaft of tibia showing mottling of bone matrix and enlargedHaversian canals, 37 years old male. Note the irregular cracking (arrows) induced byvictim corpses exposure to the hot pyroclastic surge. Images A, B and C are of thesame magnification.

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

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

index (SLI). The SLI index was adopted to better evaluate and comparethe expression and variability of pathological lesions as to anatomicallocation, age and gender. This index, calculated by considering both ap-pendicular skeleton and spine joints of the individuals aged ≥15 yearsold (Table 3), was regressed by individual age, separately for males andfemales (Fig. 5). In both sexes the linear regression shows that nearly90% of the SLI index variability is age-related (males: R2=0.895,Pb0.0001; females: R2=0.877, Pb0.0001), but the slope differs signif-icantly between males and females (test for non-equality of regressioncoefficients, t=7, Pb0.0001).Males≤30 years old are on averagemoreaffected than females, while females over 30 seem more prone to be

involved. SLI indexes calculated on 10 post-cranial large joints, showthat the spine is by far the most affected (0.616) (z=2.15, Pb0.05),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 individ-uals (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% ofthe individuals (27.6% of teeth) (Table 4). In the cases of markedhypomineralization (25.0%, 17.8% of teeth), a corroded-like appear-ance and alterations of the tooth form are evident (Fig. 6D). Mottledenamel, associated with chronic dental fluorosis since prehistorictimes (Lukacs et al., 1985), may also affect well nourished people.The healthy diet of the Herculanenses is testified by historical andarcheological evidences (Feemster Jashemski and Meyer, 2002), aswell as from trace-element analyses of a previously excavated groupof victims (Herc2) (Bisel, 1991; Capasso, 2001). Their well-balanceddiet in terms of protein and carbohydrate intake also emerges fromthe 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 deter-mined by atomic absorption spectrophotometry (AAS) (Table 5).These values, compared with those found in ancient skeletal popu-lations, suggest the consumption of red meat, crustaceans, oysters,dry fruit and legumes, and point to a diet also rich in sea fish and pro-teins of plant origin, as well as in carbohydrates (Petrone et al., 2002).

Carious lesions, collected as an additional test of dental patholog-ical status particularly with regard to dental fluorosis, were found in20% of permanent teeth (N: 1358) and 78.6% of individuals. Even ifthis finding confirms as expected that, in Roman times, caries follows

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 asthe most affected districts.

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a modern epidemiologic pattern, it also appears to conflict with anexpected low frequency of carious lesions in the case of naturallyfluoridated water intake, since daily moderate fluoride ingestion iswidely 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 teethgrowth (WHO, 2002).

The fluorine (19F) bone concentration was measured in a group ofvictims selected by anatomical district, age and state of preservation(Table 6). Volcanic ash samples from the three investigated chamberswere also tested by ion-selective electrode (ISE). Adjusted values of μfluoride (160±33, 190±18, 200±6, ppm) do not diverge statistical-ly, being within the normal score limits (±1.96).

Average bone fluorine concentrations varying between 14,000 and23,300 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; Pb0.001)

represents the mean amount of fluorine at age 0 (Fig. 7A). These valuesclearly exceed the range of normal–physiological fluorine content in thebone (Eble et al., 1992; Sastri et al., 2001) as well as the maximumexpected pathological levels (Ayoob and Gupta, 2006; U.S. NRC,2006). This finding is consistent with the particular burial conditionsof the skeletons. The ash deposit was permanently waterlogged(Capasso, 2001), and the bones were therefore enriched with fluorideleaching from the groundwater. In this area the maximum concentra-tion of present-day groundwater fluorine is 3.6 mg/L. Thus, in order todiscriminate the post-mortem from intra-vitam fluoride bone enrich-ment, we assumed a 0 fluorine concentration at age 0 (Fig. 7B), consid-ering that new-born bone usually contains nearly 50 ppm fluoride(WHO, 1970). The slope (b1=200.4±9; Pb0.001) from the resultingstraight-line equation [F]=200.4·age represents the rate of physiolog-ical intake per individual per year. This model shows that 99% of fluo-rine concentration variability is age-related (R2=0.961).

Table 2Occurrence of healed bone fractures in 56 individuals aged ≥15 years old. Spine andpelvis are the most involved anatomical districts, followed by large and small longbones. The 32.1% of males and females are equally involved, at all ages. Consideringalso the cases of spondylolysis of LV vertebra, the total occurrence of fractures is35.7%. The number of fractures (1 to 4) per person significantly increases with advanc-ing age (Spearman's rank correlation r=0.647; Pb0.005).

Individual Sex Age at death Bone

5:3 M 18–22.5 Clavicle (right)10:22 M 18–23 Hand phalanx (right)12:9 F 22–28 Foot phalanx (right)10:15 F 26–31 Rib 11 (left)10:35 M 20–39 Fibula (left)12:SP1 M? 20–39 Tibia (right)12:26 M 28–34 Spondylolysis L5 vertebra10:11A F 28–34.5 Ox coxae

Head of femur (right)12:19 M 31–37 Foot phalanx (right)12:30 F 33–37.5 T11 vertebra10:16 F 34–38.5 Clavicle (right)10:23 M 33–40 Spondylolysis L5 vertebra12:16 M 33–41.5 Clavicle (right)

Foot phalanx (left)12:13 F 33–41.5 Rib 7 (right)12:8 M 34–41.5 Foot phalanx (right)10:1 M 36–42 Clavicle (left)10:24 F 37–45 T8 vertebra

Foot phalanx (right)12:23 M 38–46 T12 vertebra

Spondylolysis L5 vertebra10:3 M 45–55 Spondylolysis L5 vertebra

Foot phalanx (left)Radius (left)

12:11 M 47–57 T10 vertebraSpondylolysis L5 vertebraSacrumRib 6 (left)

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

Taking into account this last correlation and that the skeletal lesionindex is fluorine concentration-related (R2=0.923), we obtained anew equation combining

F½ � ¼ 200:347⋅age ð1Þ

and

SLI ¼ 0:000058696⋅ F½ � ð2Þ

thus yielding

SLI ¼ 0:000058696⋅ 200:347⋅ageð Þ ¼ 0:011759⋅age: ð3Þ

This equation, applied to the available data, shows the correlation(R2=0.81) of the observed data with those expected (Fig. 8). This in-dicates that the SLI index suitably describes the degree of joint lesionsshown by the inhabitants of Herculaneum as a result of the fluorineaccumulating in their bones.

4. Discussion

The overall evidence at Herculaneum clearly shows the shape ofan endemic system-disease, affecting both adults and subadults, char-acterized by diffuse osteosclerosis and enthesopathy. Although theseconditions may be associated to other bone disorders, the concomi-tant aberrant growth of new bone, ligamentous calcification andosteosclerosis, along with osteoarthritic-like lesions and ankylosis ofspine and appendicular joints, strongly suggest skeletal fluorosis(Littleton, 1999; Ozsvath, 2009; WHO, 2002). In addition, histopatho-logical bone features like increased cortical thickness, abnormallamellar texture, disordered lamellar orientation, extensive mottlingof 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 bonefractures and a few cases of osteomalacia are also typical offluoro-osteoporotic bones, likely resulting from calcium deficiency(Chavassieux et al., 2007; Teotia et al., 1998; WHO, 2002) andother mineral abnormalities induced by fluoride (Boivin et al.,1989). Furthermore, a major result is the significant correlation be-tween the number of bone fractures (1 to 4) per individual and age(Spearman's rank correlation, r=0.647, Pb0.005). The widespreadcalcification of the sacrotuberous ligament and a few cases of toothhypercementosis also confirm the diagnosis of skeletal fluorosis(Littleton, 1999; WHO, 2002).

A distinctive result is the high levels of fluoride found in the vic-tims' bones, whose corrected average values ranging from 2042 to11,342 ppm (mean value±SE: 6672±570 ppm) clearly indicatefluoride poisoning. Fluorine concentration as a function of age(Fig. 7B) shows that the majority of individuals belong to all threeclinical phases of skeletal fluorosis. Higher values (>9000 ppm) ob-served in mature adults (≥40 years old) can be ascribable to the crip-pling phase III (Ayoob and Gupta, 2006), as currently seen in endemicregions (Choubisa, 2001).

The regression line describing the annual variation rate of fluoride(ppm/years) shows a significant increase in fluoride concentrationwith age (Fig. 7B), and a correlation with the degree of pathologicalinvolvement of the spine and appendicular joints, as assessed by theSLI index evaluation (Fig. 5). This correlation of bone fluoride concen-tration with both duration of exposure and extent of bone lesions hasbeen demonstrated in present-day patients affected by skeletal fluo-rosis, the severity of which was found to be related to the amountof fluoride incorporated into bones. Usually, fluorine can range fromca. 500 to ca. 3000 ppm in unaffected people, exposed to optimalfluoride intakes≤1 mg/L (Eble et al., 1992; Sastri et al., 2001). In-stead, extreme high values of ca. 10,000 or 12,000 ppm are typicallyassociated with crippling fluorosis due to exposure to fluorideintakes≥4 mg/L (Ozsvath, 2009; U.S. NRC, 2006).

The widespread occurrence of osteoarthritis, osteophytosis,enthesopathy and fractures is particularly high in comparison withother Roman and pre-Roman communities (Robb et al., 2001; Vargiuet al., 2007), even with those of low social status (Sperduti, 1997).Palaeopathological investigation of the Herc2 specimens confirms thehigh occurrence of degenerative joint disease, long bone osteosclerosis,enthesopathy and trauma (Bisel, 1991; Capasso, 1998, 2001; Capassoet al., 1999). In contrast, an analogous pattern of skeletal changes, butwith a lower occurrence and involving older individuals has beenreported 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; Yoshimuraet al., 2006). Dental fluorosis was first reported for the early Neolithicat Mehrgarh, Pakistan. Groundwater samples from this arid area show1.9–2.0 mg/L of natural fluoride (Lukacs, 1984; Lukacs et al., 1985). AtHerculaneum, where the concentration of fluoride in groundwaterwas found to be as high as 3.6 mg/L, the occurrence of dental fluorosisis analogous to the degree of fluorotic lesions exhibited by the victims'skeletons. Thus, the occurrence of endemic dental hypoplasia appearscorrelated to high fluoride intake during life, inferred from the highamount of fluoride that we determined in the bones.

In ancient Herculaneum enamel mottling is associated with highlevels of linear enamel hypoplasia (LEH), which occurs commonlyin most ancient populations (Resnick and Niwayama, 1983; Robbet al., 2001). Also other Roman communities, including the Herc2sample, show constantly high rates of LEH, independently of socio-economic status (Capasso, 2001; Cucina et al., 2006; Manzi et al.,1999; Robb et al., 2001; Sperduti, 1997; Vargiu et al., 2007). Giventhe possible benefits of low fluoride intake in preventing dentaldecay, caries occurrence in the investigated sample of Herculaneumappears unusually high if compared with other Roman Imperial agecommunities (Cucina et al., 2006; Manzi et al., 1999). According to

Table 3Skeletal lesion index calculated on post-cranial joints of 48 individuals aged ≥15 years old. Osteoarthritic lesions involving spine and peripheral joints were evaluated assigning toeach 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=maximumdegree of joint lesion). The unpreserved joints were not considered in the index calculation.

N Ind St-Cl Shou Elbow Wrist Sa-Il Co-Fe Knee Ankle Foot Spine I Imax Inorm

1 12:2 1 1 0 0 0 0 1 3 21 0.1432 12:3 0 0 0 1 1 2 0 0 1 5 27 0.1853 12:4 1 0.5 1 0 0.5 1 2 6 21 0.2864 12:5 0 0 0 1 1 12 0.0835 12:7 0 0 0 0 0 3 3 18 0.1676 12:8 1 1 0 2 3 1 3 2 13 24 0.5427 12:9 1 1 1 0 0 0 1 1 3 2 10 30 0.3338 12:11 2 0.5 3 2 2 2 0 3 14.5 24 0.6049 12:13 1 1 1 2 2 2 2 0 3 14 27 0.51910 12:15 1 1 1 0.5 0 1 1 1 2 2 10.5 30 0.35011 12:16 2 1 1 1 0 1 1 1 3 3 14 30 0.46712 12:19 2 1 1 1 0 0 1 1 1 2 10 30 0.33313 12:20 0 0 0 0.5 0 0 0 0 0 0.5 27 0.01914 12:21 0 0 1 0 0 2 2 1 3 2 11 30 0.36715 12:22 0 0 0 0 0 0 0 1 0 1 2 30 0.06716 12:23 2 1 1 1 2 1 3 3 14 24 0.58317 12:26 2 1 0 0 0 2 1 0 2 3 11 30 0.36718 12:27 2 1 1 1 0 0 1 1 2 9 27 0.33319 12:28 2 1 1 0 1 1 0 3 2 11 27 0.40720 12:30 2 2 1 1 1 1 0 1 1 2 12 30 0.40021 5:01 0 0 0 0 0.5 0 0 0 0 0 0.5 30 0.01722 5:02 1 1 0 0 0 0 1 2 0 1 6 30 0.20023 5:03 1 1 0 0.5 0.5 1 0.5 0 0 2 6.5 30 0.21724 10:1 1 2 2 1 1 2 1 1 0 3 14 30 0.46725 10:2 0 0 0 0 0 0 0.5 1 0 1 2.5 30 0.08326 10:3 3 2 0.5 1 3 2 1 2 3 3 20.5 30 0.68327 10:4 0.5 0.5 1 0.5 1 1 1 1 1 2 9.5 30 0.31728 10:5 1 0 0 0 0 0 0 0.5 1 1 3.5 30 0.11729 10:6 1 1 0.5 0 1 0 1 2 1 7.5 27 0.27830 10:7 1 1 1 1 2 1 1 1 3 2 14 30 0.46731 10:10 1 2 0 2 0 1 1 2 9 24 0.37532 10:11 1 1 1 0.5 1 2 1 0.5 2 10 27 0.37033 10:12 1 1 1 0.5 2 1 1 1 3 2 13.5 30 0.45034 10:13 1 1 2 1 2 1 2 1 2 2 15 30 0.50035 10:14 2 1 2 2 1 2 2 1 2 3 18 30 0.60036 10:15 2 1 1 0 1 1 1 0 0.5 1 8.5 30 0.28337 10:16 2 1 1 1 2 1 1 1 2 2 14 30 0.46738 10:17 2 2 0 1 1 1 1 1 1 10 27 0.37039 10:18 2 0 1 1 1 0 2 2 3 2 14 30 0.46740 10:19 2 2 1 0 1 0 1 1 1 1 10 30 0.33341 10:20 2 2 2 1 1 1 2 1 2 2 16 30 0.53342 10:21 2 1 2 1 2 1 1 0.5 2 12.5 27 0.46343 10:22 0 0 1 0 1 1 1 0 2 6 27 0.22244 10:23 2 1 1 1 1 1 1 1 1 2 12 30 0.40045 10:24 1 1 0.5 2 2 2 2 2 2 14.5 27 0.53746 10:25 0 2 0 1 3 12 0.25047 10:28 1 2 2 0 2 2 1 1 0 2 13 30 0.43348 10:29 1 1 1 1 1 1 1 1 1 2 11 30 0.367

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

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recent epidemiological studies on the adverse effects of high fluorideintake on dental caries (Ijaz et al., 2010; U.S. NRC, 2006; Wondwossenet al., 2004), the estimated high fluoride intake and the resultinghypomineralization detected in the tooth enamels of the Herculaneumresidents seem strictly related to an increased risk of caries.

This palaeoepidemiologic scenario has likely remained unchangedfor the population in the Vesuvius area till today. Currently, the max-imum fluorine concentration of the water-bearing stratum is close tothe current maximum contaminant level (MCL) of 4 mg/L of drinkingwater, and within the range of concentrations able to induce cripplingskeletal fluorosis (Connet, 2004; U.S. Environmental ProtectionAgency, 2002). We calculated a fluoride intake of 10.8–18.0 mg/dayper person at the time of the AD 79 eruption, which is equivalent tothe 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 con-taminatedwith fluoride of natural origin, communitieswith normal nu-tritional intake exposed to fluoride water concentrations of ca. 4 mg/Land daily total intake of ca. 14.0 mg/day show a prevalence of skeletaland dental fluorosis equivalent to those observed for the Herculaneumresidents (Choubisa, 2001; Grimaldo et al., 1995; Jolly et al., 1968;WHO, 2002). Notably, lower concentrations of ca. 2.0 mg/L of fluoridepresent in the Bolan and Nari rivers in Baluchistan, Pakistan, havebeen found associated with severe cases of dental fluorosis in modernand 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 atwater-borne fluoride concentrations of 2–3 mg/L or even as low as1.1–1.5 mg/L, with crippling deformities appearing at 2.8 mg/L, given

Fig. 5. Skeletal lesion index related to age by gender. The linear regressions obtained bycomparing skeletal lesion index (SLI) vs. age, separately for males and females, showsthat nearly 90% of SLI variability is age related (males: R2=0.895, Pb0.0001; females:R2=0.877, Pb0.0001). However, males aged≤30 years old are in average more affect-ed than females, while females over the thirties are more frequently involved (test forno equality of regression coefficients, t=7, Pb0.0001).

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the presence of a favorable environmental context (geology, soil, cli-mate, groundwater chemistry).

The clinical and analytical scenario of endemic fluorosis detected atancient Herculaneum emerge as still occurring today. A clinical–epidemiological investigation in 845 schoolchildren from the Vesuviantowns, where the maximum fluoride content in tap water is 2.8 mg/Laccording to local guidelines (Regione Campania, 2011), found 81.6%prevalence of dental fluorosis (Gombos et al., 1994), which is common-ly considered a biomarker for fluoride exposure (WHO, 1970). The chil-dren, 6 to 11 years old, showedmoderate to severe tooth discolorations(mottling) and brown staining (Fig. 9A, B and C), pitting (Fig. 9C) andextensive loss of enamel (Fig. 9D). In addition to dental fluorosis, signif-icant clinical features were apparent. Mottled teeth were frequentlyaccompanied by certain skin complaints (angiectasia, 51.0%) of differ-ent degrees and location (Fig. 10A, B, C and D), and hair loss (alopecia,15.7%). Stomachache, articular pains and mottled finger-nails werealso recurrent.

In those childrenwho exhibited dentalfluorosis, chemical, clinical andhematological analyses also revealed that both free fluorine and totalfluorine content in the serum greatly exceeded the normal (0.05 ppm)and pathological (0.22 ppm) maximum levels recommended by theWorld Health Organization (WHO) (WHO, 2008). It should be notedthat pathological values of serum fluoride of 0.22 ppm or higher aretypical of areas inwhich fluorosis is endemic (WHO, 1984). Furthermore,as regards thyroid activity, in several children affected by dental fluorosisthe levels of serum thyroid parameters such as FT4 (free thyroxinehormone) and TSH (thyroid-stimulating hormone) suggested borderlinehyperthyroidism. The recurrent cases of mottled teeth frequently accom-panied by changes in skin, hair and nails, coupledwith pathological levelsof serumfluoride appear to be evident features of chronicfluorine poison-ing. The common manifestation of these pathological features in thepresent-day school-age population shows the real extent of fluorosisaround Vesuvius.

A recent report by the National Academy's National ResearchCouncil (NRC) (U.S. NRC, 2006) concluded that the maximum con-taminant level (MCL) of 4 mg/L of fluoride allowed by the U.S. Envi-ronmental Protection Agency in drinking water does not protect

Fig. 6. Dental pathological features. A. Marked linear enamel hypoplasia (LEH), maxil-lary anterior teeth, 19 year old male. The right lateral incisor and canine are fused intoone single dental element; B. Brown stains and mottling, upper central incisors, 36 yearold 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), upperleft central incisor, male 20 years old. Note the severe enamel hypomineralization inthe form of corroded-like appearance (white arrow).

Table 4Evaluation 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 aresymptomatic 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% ofteeth). 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 Minimumind. score

Maximumind. score

Averageind. score

Generalevaluation

5.1 2.0 1.0 2.0 1.0 2.0 1.0 2.0 1.4 Moderate5.3 1.7 1.2 1.6 1.4 1.3 1.2 1.7 1.4 Mild10.2 0.7 0.8 0.8 0.6 0.6 0 0.8 0.7 Normal10.3 1.8 2.6 1.9 1.3 1.0 1.0 2.6 1.9 Severe10.5 0 0.3 0.9 0.1 0 0 0.9 0.3 Normal10.6 0.3 0.4 1.0 0.4 0.2 0.2 1.0 0.5 Normal10.7 1.0 0.3 1.1 0.7 0.7 0.3 1.1 0.8 Mild10.12 0.7 0.3 0.8 0.1 0.2 0.1 0.8 0.4 Normal10.13 0.4 0.4 0.7 0.4 0.3 0.3 0.7 0.4 Normal10.14 1.0 2.0 1.0 1.0 1.0 1.0 2.0 1.2 Moderate10.15 0.9 0 1.1 0.5 0.4 0 1.1 0.6 Mild10.17 1.4 1.9 1.3 0.5 0.7 0.7 1.9 1.2 Moderate10.19 1.3 2.3 2.0 1.7 1.7 1.3 2.3 1.8 Severe10.20 1.6 1.4 1.4 1.1 0.8 0.8 1.6 1.3 Mild10.22 1.0 0.8 2.0 1.6 1.6 0.8 2.0 1.4 Moderate10.23 1.6 1.6 1.6 1.3 1.3 1.3 1.6 1.5 Moderate10.24 0.4 0.4 0.1 0 0 0 0.4 0.2 Normal10.26 0.5 0 0.3 0 0 0 0.5 0.2 Normal10.28 2.0 0 1.5 0.5 0 0 2.0 0.8 Moderate10.SPD1 3.0 3.0 3.0 1.0 / 1.0 3.0 2.0 Severe10.SPD2 1.0 0 1.0 0 0 0 1.0 0.4 Normal10.SPD3 1.0 / 0 0 0 0 1.0 0.3 Normal12.5 0.8 0.3 0.6 0.3 0.3 0.3 0.8 0.5 Normal12.15 1.2 0.5 1.6 1.5 1.6 0.5 1.6 1.3 Mild12.16 0.6 0.3 0.8 0.2 0.2 0.2 0.8 0.4 normal12.19 0.6 0 0.9 0.5 0.5 0 0.9 0.5 Normal12.22 1.8 1.5 2.3 2.1 2.3 1.5 2.3 2.0 Severe12.23 2.5 2.8 1.8 1.3 0.9 0.9 2.9 1.9 Severe12.26 0 0 0 0 0 0 0 0 Normal12.27 0.7 0.4 0.7 0.3 0.1 0.1 0.7 0.4 Normal12.28 1.6 1.2 1.2 0.6 0.4 0.4 1.6 1.0 Mild12.30 0 0 0 0 0 0 0 0 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.

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against adverse health effects, particularly in children. Even theso-called secondary MCL of 2 mg/L proved inadequate (Ayoob andGupta, 2006). Evidence from modern Pakistan confirms that eventhis modest level of fluoride in drinking water can have toxic effectsin children. In those seasonally hot and arid environments, greaterwater consumption required to prevent dehydration and the use offluoridated water to irrigate crops and prepare food, as well as malnu-trition, can significantly elevate the fluoride intake, exacerbating its

Table 5Oligoelement contents in human bones. The amounts of zinc (Zn), strontium (Sr), mag-nesium (Mg), copper (Cu) and calcium (Ca) in the human bone were assessed byatomic absorption spectroscopy. The oligoelement concentration was measured in her-bivore bones (Equus caballus, Ovis capra) and sediment as well, in order to exclude in-accurate data as a result of possible diagenetic processes.

Element Human bone(N=10)

Herbivore bone(N=3)

Sediment Estimation

Ca (mg/g) 319.51±16.70 266.33±68.90 40.8 Not defiledSr (ppm) 131.00±18.36 148.17±9.09 85.1 Not defiledZn (ppm) 151.46±31.53 137.90±39.67 68.7 Not defiledMg (ppm) 4309.20±1137.52 3368.67±817.59 8545.0 DefiledCu (ppm) 184.06±79.67 175.97±88.10 44.9 Not defiledSr/Ca 0.41±0.06 / / /Sr/Ca (*) 0.73±0.10 / / /Zn/Ca 0.48±0.11 / / /

adverse physiological effects (Lukacs, 1984; Lukacs et al., 1985). Inaddition, NRC model predictions show that bone fluoride concentra-tions resulting from lifetime exposure to fluoride in drinking waterat 2 or 4 mg/L fall within or exceed the ranges associated with stageII and stage III of skeletal fluorosis, and may increase the risk of over-all fractures (U.S. NRC, 2006). Furthermore, the NRC report concludedthat fluoride could start or promote cancer, and osteosarcoma is of aparticular concern, together with other types of bone cancer. A recentlarge hospital-based case-control study of age-specific fluoride expo-sure in drinking water and the incidence of osteosarcoma in the UnitedStates found a seven-fold increased risk of bone cancer in young boysdue to fluorosilicates (Bassin et al., 2006), the most widely used formof fluoride added to drinking water.

5. Conclusions

Our findings on the pathological skeletal and dental features of theancient residents of Herculaneum indicate that fluorosis in the Neapol-itan area was endemic already during Roman times. The close – andsomewhat disturbing – parallels that we found between fluoride expo-sure in the ancient population of AD 79 Herculaneum and that inpresent-day schoolchildren in the Vesuvian area show a permanentfluoride health hazard for the entire population living around Vesuvius.At present, the major public health and socio-economic impact of this

Table 6Human bone samples tested for determination of fluorine concentration. For each sam-ple are reported specimen, age at death, tested skeletal element, measuredsodium-corrected fluorine concentration (ppm), fluorine corrected values (ppm), andinterval for expected single value (95%). Fluorine concentration of infants aged 0 to10 years old were not utilized for statistical elaboration, since the amounts detectedfor infants (16,371 ppm on average) exceeded those of 12 to 30 year old individuals(16,164 ppm on average).

Individual Averageage

Skeletalelement

Na-correctedμ F (ppm)

CorrectedF values

±E.S.V.I.95%

11:15 0 (7 iu-m) Rib 18,400±1275 / /10:41 0.5–1.5 Ilium 16,600±540 / /12:12 2.5–3.5 Ilium 16,900±450 / /12:18 3–4 Ilium 15,200±793 / /12:17 5–6 Ilium 16,500±476 / /12:6 8–9 Ilium 16,100±1803 / /12:25 9–11 Ilium 14,900±1340 / /12:14 11–13 Ilium 14,000±1267 2042 33925:1 13–16 Ilium 14,400±372 2442 332812:20 16–20 Ilium 16,000±624 4042 324912:9 23–28 Rib 16,300±467 4342 313812:4 24–30 Rib 17,200±381 5242 311810:29 26–32 Rib 16,350±166 4392 310310:19 27–33 Rib 18,900±435 6942 309812:26 28–34 Ilium 17,300±597 5342 309410:13 31–37 Ilium 19,000±495 7042 309010:12 31–37 Ilium 19,250±311 7292 309012:28 32–39 Ilium 19,300±271 7342 309512:27 32–39 Ilium 20,800±197 8842 309510:14 34–40 Ilium 18,200±398 6242 309910:21 34.5–40.5 Ilium 19,200±213 7242 310210:18 35–41 Rib 19,850±221 7892 310510:1 36–42 Ilium 22,100±363 10,142 311312:23 38–46 Ilium 19,900±620 7942 314210:20 40–48 Ilium 23,050±508 11,092 316910:3 45–55 Rib 23,300±95 11,342 327912:11 47–57 Ilium 18,200±196 6242 3325

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

Fig. 7. Fluorine (19F) bone concentration (ppm) as a function of age. The linear regres-sion resulting from (A) the fluorine mean amount of 18,400 to 23,300 ppm measuredby INAA (intercept=11,958.5±1120, Pb0.001) is compared with an equivalent re-gression (B) obtained considering a 0 fluorine concentration at age 0 (slope=200.4±9, Pb0.001). The last model, representing the physiological rate of individual intakeper year cleansed of the fraction of fluorine contamination by soil ash deposit, showsan evident age-dependent increase of fluorine (R2=0.961). Children aged ≤10 yearsold were not included in this model, due to the high diagenetic amount of fluorine re-leased by the ash deposit. The resulting corrected mean values of 2042 to 11,342 ppmshow a minority of individuals matching the normal–physiological (b3500 ppm) andpreclinical (b5500 ppm) ranges of fluorine bone concentration, while the majority be-longs to all the three clinical phases of skeletal fluorosis, with several mature (≥40 yearold) individuals in the crippling phase III.

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hazard is underestimated. In the past 25 years, the local authoritieshave reduced the maximum permitted fluoride content in tap waterthroughout the area from 4.0 to 3.0 mg/L and again to its current levelof 2.5 mg/L. However, this lower value still far exceedsWHO guidelinesfor maximum fluoride content (1.5 mg/L). WHO drinking water guide-lines are based on internationally agreed procedures for risk assessmentand aim to ensure that drinking water does not represent any signifi-cant risk to health over a lifetime of consumption.

Effects on the skeleton are the best indicators of the toxic re-sponses to fluoride and are considered to have direct public health

Fig. 8. Skeletal lesion index related to age by observed vs. expected data. The equation SLI=correlation (R2=0.81) of the observed data with those expected, thus indicating that theresidents.

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

0.000058696·(200,347·age)=0.011759·age applied to the available data, shows theSLI index suitably describes the degree of joint lesions shown by the Herculaneum

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

Fig. 10. Skin complaints in schoolchildren from Vesuvian towns affected by dentalfluorosis. 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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in the densely populated Vesuvius area, the following predisposingfactors for fluorosis should be better evaluated: ambient temperature,volume of water intake, other trace elements in the water, a dietbased on beverages and food preparation in naturally fluoridatedboiled water, and water storage methods. Bearing in mind that pro-gressively higher fluoride intakes lead to increasing risks of dentaland skeletal fluorosis, as clearly demonstrated by the Herculaneum

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

victims, the adoption of low-cost defluoridation methods should beseriously considered and encouraged.

In evaluating all the possible health consequences of exposure tofluoride concentrations higher than the establishedWHO parameters,it should also be taken into account that the maximum fluoride con-tent of 5.0 mg/L accepted in natural mineral waters protects onlythe population over 15 years old and only if there is no exposure tofluoride from other sources, as experienced by communities livingin volcanic and other fluoride-rich areas.

Author contributions

PP designed the site and laboratory research. PP performed thebioanthropological and palaeopathological research. GFM performedthe histological research and wrote the histopathological part. GFperformed the epidemiological research. PP GS GFM GF analyzedthe data. GS GFM did statistics. PP wrote the paper.

Acknowledgments

We thank the Superintendency of Pompeii for granting the fieldinvestigation and the study of the human skeletal material. We alsothank Vincenzo Monetti for the water analysis and Emanuele Minellifor the digital X-ray images. We are also indebted to MarkWalters forthe final text editing.

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