severe dental fluorosis in juvenile deer linked to a recent volcanic

SEVERE DENTAL FLUOROSIS IN JUVENILE DEER LINKED TO A

RECENT VOLCANIC ERUPTION IN PATAGONIA

Werner T. Flueck1,2,3,4 and Jo Anne M. Smith-Flueck1

1 DeerLab, C.C. 592, 8400 San Carlos de Bariloche, Argentina2 National Council of Scientific and Technological Research (CONICET), Buenos Aires, Argentina3 Swiss Tropical and Public Health Institute, University Basel, Basel, Switzerland4 Corresponding author (email: [email protected])

ABSTRACT: The Puyehue–Cordon Caulle volcanic eruption deposited large amounts of tephra(ashes) on about 36 million ha of Argentina in June of 2011. Tephra was considered chemicallyinnoxious based on water leachates, surface water fluoride levels were determined to be safe, andlivestock losses were attributable to inanition and excessive tooth wear. To evaluate effects on wildungulates, we sampled wild red deer (Cervus elaphus) at 100 km from the volcano in September–November 2012. We show that tephra caused severe dental fluorosis, with bone fluoride levels upto 5,175 ppm. Among subadults, tephra caused pathologic development of newly emerging teethtypical of fluorosis, including enamel hypoplasia, breakages, pitting, mottling, and extremely rapidablation of entire crowns down to underlying pulp cavities. The loss of teeth functionality affectedphysical condition, and none of the subadults was able to conceive. Susceptibility to fluorosisamong these herbivores likely resides in ruminant food processing: 1) mastication and tephra sizereduction, 2) thorough and repeated mixing with alkaline saliva, 3) water-soluble extraction in therumen, and 4) extraction in the acidic abomasum. Although initial analyses of water and tephrawere interpreted not to present a concern, ruminants as a major component of this ecosystem areshown to be highly susceptible to fluorosis, with average bone level increasing over 38-fold duringthe first 15.5 mo of exposure to tephra. This is the first report of fluorosis in wild ungulates from avolcanic eruption. The described impact will reverberate through several aspects of the ecology ofthe deer, including effects on population dynamics, morbidity, predation susceptibility, and othercomponents of the ecosystem such as scavenger and plant communities. We anticipate furtherimpact on livestock production systems, yet until now, existence of fluorosis had not beenrecognized.

Key words: Cervids, Cervus elaphus, dental fluorosis, fluoride, pathology, teeth, tephra,volcanic eruption.

INTRODUCTION

The Puyehue–Cordon Caulle volcaniceruption (PCCVE, June 2011) producedplumes 15 km high and deposited largeamounts of tephra (ejected solid matter) inChile and over about 36 million ha inArgentina (Fig. 1; Gaitan et al., 2011; MohrBell and Siebert, 2011). Winds depositedlayers of tephra 5 cm thick up to 240 kmdistant, but it also reached Buenos Aires,1,400 km away (Wilson et al., 2012).Volcanic eruptions may emit toxic levelsof fluoride, impacting animals in thesurroundings (Cronin et al., 2000, 2003),yet fluorosis in wildlife raised concerns onlyas recently as 1967, principally regardingpollution or use of fertilizers (Kierdorf et al.,1996b; Richter et al., 2010). A recent localexample was the Lonquimay volcano erup-tion (1988, 200 km north of PCCVE),

where fluorosis in livestock occurred withinweeks (Araya et al., 1990). Immediatelyafter the PCCVE, analyses of tephrarevealed mainly O, Si, Al, Fe, Na, and K(Buteler et al., 2011; Hufner and Osuna,2011). Although initial concern was aboutfluorosis, based on incidences from otherChilean volcanoes, only fluorine-containingmicrobubbles that may turn into fluoro-hydric acid upon contact with water werementioned in tephra from Argentina (Ber-mudez and Delpino, 2011). Moreover,water-soluble extracts from tephra revealedlow fluoride levels (0.7 ppm; Hufner andOsuna, 2011), surface water analyses fromboth countries revealed low fluoride levels,and overall water consumption was consid-ered without risk for humans and animals(DGA, 2012; Wilson et al., 2012). Still,livestock losses in the region of our studysite were high, but they were attributed to

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DOI: 10.7589/2012-11-272 Journal of Wildlife Diseases, 49(2), 2013, pp. 355–366# Wildlife Disease Association 2013

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Sitio Argentino de Producción Animal

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inanition, rumen blockage, and excessivetooth wear, rather than to known toxicelements (Wilson et al., 2012). We areaware of only Chilean analyses conductedon fluoride levels in forage plants andanimal tissues from the PCCVE: Foragefluoride levels were more than threefoldhigher than accepted levels, while levels intephra and blood from livestock were alsoelevated (Araya, pers. comm.). Given theseobservations in Chile and overt fluorosisdocumented from the nearby 1988 erup-tion, reasons for absent fluorotic cases fromPCCVE remained elusive.

Soon after the PCCVE, livestock be-came weak, and thousands died; causeswere attributed to emaciation from lack offorage. In surviving individuals, excessivetooth wear, attributed to abrasive tephra,was noted within 2 mo, and this currentlyevokes the application of artificial den-tures to extend the life of livestock.

Given the effects on livestock and alsobecause we did not observe unusualmortality rates among wild red deer(Cervus elaphus), we asked if wild ungu-lates were similarly affected by tephra. Toinvestigate possible impacts of tephra onwildlife, and being unaware of any pub-lished fluoride concentrations in terrestrialanimal or plant tissues from the studyregion, we began examining deer at 100 kmfrom PCCVE. We provide the firstevidence of tephra producing severedental fluorosis in wild deer, which canserve as highly sensitive bioindicators(Walton, 1988; Kierdorf et al., 1996b).Tooth enamel is especially suitable as anindicator because it does not undergoremodeling, and, therefore, any distur-bance during its development leaves apermanent record (Kierdorf and Kierdorf,1999). The low water fluoride levelsinitially reported (,1 ppm) indicate that,


FIGURE 1. Site of eruption of Puyehue–Cordon Caulle volcano, 4 June 2011, in Patagonia. Map shows thewestern portion of the region with major tephra deposition (shaded area), and the study area.

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for organisms like ruminants, there areother factors responsible for the observedfluorosis. Considering the severity ofdescribed pathologies and anticipatingfurther chronic responses due to contin-ued exposure to physical and chemicalproperties of tephra, various biologic andecologic parameters will be affected at theindividual, population, and communitylevels. This is the first report of fluorosisin wild ungulates due to volcanic eruption(Shupe et al., 1984; Walton, 1988; web-of-knowledge search, November 2012).

MATERIALS AND METHODS

Study area

The study area is in the National Reserve ofthe Nahuel Huapi National Park, province ofNeuquen, Argentina (41u169120S, 71u99250W).The topography is primarily mountainouswith most features formed by glacial process-es. The majority of soils originated fromvolcanic processes and are young. The dom-inant climate is temperate, with main precip-itation occurring April–September and snowfalling frequently during June–September.There is an abrupt precipitation gradient fromwest to east due to the Andean orography,resulting in a strongly defined vegetationstructure and floristic composition. The studysite is between 900 m and 1,200 m in elevationand represents the ecotone between forestsand steppe. Patches of forests are character-ized by Nothofagus antarctica and Austroce-drus chilensis at lower elevations, replaced byNothofagus pumilio at higher elevations. For-est patches at lower elevations alternate withwet grasslands with abundant growth ofherbaceous plants, whereas at high elevationthey are replaced by grass-dominated steppeof Stipa speciosa var. major and Festucapallescens, with variable occurrence of brushspecies including Mulinum spinosum, Berberisspp., and Colletia spinosissima. Riparian areasalso contain galleries of trees including Loma-tia hirsuta, Maytenus boaria, and Schinuspatagonicus. The principal area sampled forour study is ecotonal, though dominated byrangeland.

Deer population

This deer population has been monitoredannually since 1991. For our study, deer(n526) were shot using rifles between 13September and 23 November 2012 (spring

equinox), necropsies were performed in thefield, and animals were evaluated for mor-phometry, reproductive status, physical condi-tion, pathology, and age (Flueck, 2002).

Two subadult males (,2 yr of age) camefrom an area with substantial brush and treecover, whereas the remainder of collecteddeer were females and calves of the previousyear from a predominantly treeless area about8 km away. For aging, we used jaws of deercollected from the same area, which had fullyerupted permanent teeth and for which ageswere determined via cementum annuli analy-sis (Matson’s Laboratory, Milltown, Montana,USA; n5137). Ages of prime-aged femalescurrently sampled were based on wear ofpremolars and molars in comparison to thereference jaws, and by deducting 1 yr assumedto have resulted from excessive wear since thePCCVE of June 2011. For the purpose of thisstudy, some aging error among these middle-aged individuals is not considered relevant,given that ages through the teeth-replacementphase were accurate, and no old individualswere sampled. Fetuses were identified to sexand weighed, and their morphometry wasrecorded. Studies done regularly during therutting period and spring, plus data on fetusescollected since 1991 (n5348), support theconclusion that most calves were born within a4–6-wk period.

Determination of fluorine concentrations

Samples were obtained from mandibles byremoving 0.5 g from the ventral ridge at thelevel of molar M1. Recently shed antlers, orthose removed at necropsy, were sampled bydrilling into the antler base parallel to the shaftand near the outer border such that corticalantler provided the shaving. Fluorine concen-trations were determined in the Laboratoriode Biologıa Osea (Universidad Nacional deRosario, Rosario, Argentina; Rigalli et al.,2007). Samples were ashed at 550 C, acidlabile fluorine was isolated from 50 mg ofsample by isothermal distillation, and thesample was treated with phosphoric acid 98w/w% at 60 C for 1 day. During this time,hydrofluoric acid released from samples wasrecovered by sodium hydroxide placed in thecup of the distillation chamber. Subsequently,the sodium hydroxide trap was adjusted topH 5.5 with 17.5 mol/L acetic acid. Standardsranging from 1023 to 1026 mol/L weresimultaneously processed. Total fluorine wasmeasured by direct potentiometry using an ionselective electrode ORION 94-09 (OrionResearch Inc, Cambridge, Massachusetts,USA) and a reference electrode of Ag/AgCl.


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Duplicate samples were analyzed, resulting incoefficients of variation of ,6%, and theresults are presented as the mean, expressedas ppm in dry bone. In addition, rat boneswere analyzed simultaneously: ash of ratstreated with NaF averaged 2,317 ppm, where-as control rats averaged 11 ppm.

RESULTS

Effects on red deer

As described for local livestock, deeralso exhibited excessive tooth wear follow-ing the PCCVE, being notable in cheekteeth, but particularly in incisive teeth.Prime-aged females (6–9 yr) had incisorsworn down to the gums (Fig. 2A). Com-paratively, a similar physiognomy of inci-sive teeth prior to PCCVE would havecorresponded to 15–20 yr of age. Accord-ingly, these females had essentially no fatreserves, although the winter had beenmild with very little snow. Foraging effi-ciency is reduced by a diminished incisivearcade, but tephra deposits may have alsoreduced food availability. Adult femalescarried predominately male fetuses (70%,

n510), as previously observed here duringyears of drought and reduced forageconditions, which resulted in 71% malefetuses versus 42% in good years (n5167;Flueck, 2002). Subadult females also ex-hibited extreme tooth wear. Thus, whereasdeciduous premolars p3 and p4 wouldnormally shed as complete teeth, thecurrent anomalous wear involved grindingteeth right down to the root stubs, such thatonly the two p3 and the three p4 root stubsremained exposed before casting (Fig. 2B).Moreover, permanent molars M1, whichwould have emerged only about 14 moearlier and are unaffected by fluorideduring development (see following), al-ready showed pronounced wear (Fig. 2C).

We sought to understand if exposure totephra elicited additional effects besideexcessive mechanical abrasion of teeth. Toexplore systemic effects, we focused onsubadults in the process of deciduousteeth eruption, which therefore had beendeveloping under conditions influencedby tephra. The subadults (n510) showedwidely varying stages of permanent teeth


FIGURE 2. Excessive tooth wear in red deer (Cervus elaphus) due to abrasion from volcanic tephra depositsfollowing 2011 volcano eruption in Patagonia. (A) Incisor wear below the level of the gum of a prime age female.(B) Deciduous premolars on a juvenile (,2 yr old), with only root stubs remaining (two of p3, three of p4). (C)Molars M1 and M2 of juveniles, with wide area of exposed dentine (black arrows) and reduction in molar height.Extensive abnormal wear of emerging molars M2, with grooves and pitting (white arrows).



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replacement (I1–3, C, P2–4, M2–3). Thenewly emerged incisors of all subadultfemales clearly showed additional patho-physiognomy typical of fluorosis. Centralincisors I1 showed enamel hypoplasia,some even to the degree of entirelylacking enamel on about 70% of theanterior aspect, along with breakages(Fig. 3A). Extreme wear on I1 was notablein all subadult females, with much damageto the enamel and different degrees ofwear, resulting in highly uneven incisorarcades with a reduction in functionality(Fig. 3B). Not only were large portions ofthe crowns without enamel, but muchdentine was also eroded away. Additional-ly, 88% showed substantial prematurewear on I2, plus pitting, mottling, and

breakage (Fig. 3A–C). In five cases, thewear of new I1/I2 (including next to milki3/c) was so advanced that practically allposterior enamel was ablated, and the under-lying pulp cavity was exposed (Fig. 3D).Two subadults with newly emergedfull-sized I1 (Fig. 3C) compared withindividuals already more advanced indevelopment (Fig. 3B, bottom) showedthat the latter had lost up to 90% ofthe crown height, and within 2–4 mo,based on observed periods of rutting andcalving, fetal development, and individualvariation.

Damage from fluorosis was furtherevidenced on emerging molars. Whereaswear of mandibular M1 was substantialin all subadult females, 88% of recently


FIGURE 3. Mandibular teeth of red deer (Cervus elaphus) affected by fluorosis following 2011 volcaniceruption in Patagonia. (A) Damaged incisors I1, lacking enamel in large portions (arrows) and with breakages(star). (B) Uneven incisor arcades with a reduction in functionality. Note: lacking enamel (arrows) anddeciduous teeth c and i3. (C) Full-sized I1 in two subadults due to recent emergence (upper photo, female,note breakage and pitting), and due to habitat used with abundant leafy forage (bottom photo, male, note onlyslight pitting). (D) Wear of the new permanent incisors is so advanced that all of the posterior enamel is wornoff and the dental pulp cavity is exposed (arrow).



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emerged M2 not only showed extensiveabnormal wear, but major pitting andgrooves (Fig. 2C). Inspection of maxillaryteeth revealed additional profound patho-logic changes. Thus, p4 remained only asroot stubs, M1 showed advanced wear,and recently emerged M2 had grosslyenlarged infundibula of about 25% themolar width and 10 mm in depth, withbroken tooth fragments trapped inside(Fig. 4). Additionally, enamel wear wasadvanced, with much dentine exposed,and molar height was reduced.

Loss of functional tooth shape in thesenulliparous subadults also explains theirpoor average physical condition with prac-tically no fat reserves (sternal fat: 0.13 mm,SE50.13; omental fat: 0%), and lack ofpregnancies in all these females. In previ-ous years, 46% of subadult females werepregnant (n539), with 69% pregnant inone year (n513). Mineralized in utero, themandibular M1 is established in 4-mo-oldcalves. There appears to be some barrier tofluoride transport in utero as well as duringlactation (Kierdorf et al., 1996a; Cronin etal., 2000; Richter et al., 2011). As expected,calves showed unaffected M1; solely thedeciduous incisive teeth showed substantialwear, given that permanent incisors wouldnot emerge for another 6–10 mo.

Bone fluoride levels corroborate thepathophysiognomy (Table 1). In livestock,normal levels are 200–600 mg/kg of fluoridein dry bone (Aiello and Moses, 2012). Severecases in our study averaged 2,362 ppm(SE5268.1, range 1,067–5,175 ppm),whereas a calf had 1,611 ppm. Two subadultmales from the brushy area averaged756 ppm in mandibles and 494 ppm inantlers, whereas three antlers shed recentlyin that area averaged 657 ppm. In contrast,antlers from old deer hunted in 2009 in theseverely affected area averaged 63 ppm(SE510.7, range 34–92 ppm).

DISCUSSION

Effects on red deer

In our study area, premature tooth wearwas omnipresent and due to the abrasive-ness of tephra. This effect was exacerbatedin subadults as shown by extremely rapidwear of new teeth, indicating that thebiophysical properties of teeth have beenaffected during their development. Asthese deer were not even 2 yr old, suchadvanced wear would likely have consider-ably reduced morphometric developmentand health condition, shortened life expec-tancy, and reduced lifetime reproductivesuccess. In particular, incisive teeth worndown to the gums would impair feeding.Developmental variability among subadultswith delayed eruption of permanent incisorteeth is also considered diagnostic offluorosis (Krook et al., 1983), and newlyemerged incisors of all subadult femalesclearly showed additional pathophysiog-nomy typical of fluorosis, similar to otherdeer (Suttie et al., 1985; Vikoren and Stuve,1996). In addition to fluoride-induceddental lesions, concomitant occurrence ofmarked periodontal disease and tooth losswas an important factor responsible for areduction of life expectancy in severelyfluorotic wild red deer (Schultz et al.,1998). According to the classification ofdental fluorosis in wild ungulate incisors,female subadults in this study fall into themost severe category (Shupe et al., 1984).


FIGURE 4. Maxillary teeth of a red deer (Cervuselaphus) affected by fluorosis following 2011 volcaniceruption in Patagonia. M2 has grossly enlargedinfundibula of about 25% the molar width and10 mm depth (arrow at distal cusps). Note: brokentooth fragments trapped inside the infundibulumbetween the mesial cusps of M2; excessive wear onM1 and p4, which remains as two root stubs.



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Effects on population dynamics havebeen described for C. elaphus subjected tofluorosis from exposure to geothermalcontamination. Compared to the averagebone fluoride levels in this study (2,1722,362 ppm), Garrott et al. (2002) foundbones averaging 1,711 ppm in deer withsevere dental fluorosis. Whereas the onsetof survival senescence in nonaffectedherds occurred at about 16 yr of age witha life expectancy of 25 yr, none of the deerexposed to fluoride survived beyond 16 yr.Accordingly, there was a 24% reduction inthe potential annual population growthrate (Garrott et al., 2002). Consideringthat our study population in addition to

fluorosis is subjected to mechanical wearfrom tephra, the survival senescence willlikely begin at an even earlier age.

Skeletal bone fluoride content common-ly increases continuously with age inmammals (Walton, 1988). Whereas sub-adult red deer in unpolluted Spain aver-aged 79 ppm of dry bone fluoride, adults 8to 15 yr old averaged 310 ppm (Azorit et al.,2012). Antlers constitute an unusual formof bone in that they are shed and regrowneach year. Although antlers grow onlyduring a few months, skeletal material ismobilized and incorporated into antlers.Accordingly, no significant differences influoride concentration were found among


TABLE 1. Fluoride concentration (ppm) in dry bone and antler from red deer (Cervus elaphus) exposed totephra from the Puyehue–Cordon Caulle volcanic eruption of 4 June 2011 in Patagonia, Chile, and antlersfrom males hunted in 2009.

Sex Age (yr)aDegree of fluorosis in

mandiblesb Tissue sampled Fluoride (ppm)

F 7.5 None detected Mandible 3,720F 6.5 None detected Mandible 5,175F 5.5 None detected Mandible 1,076F 3.5 None detected Mandible 1,875F 2.5 Some staining Mandible 2,191F 2.5 None detected Mandible 1,830F 0.5 None detected Mandible 1,611F 1.5 Severe Mandible 2,302F 1.5 Severe Mandible 2,154F 1.5 Severe Mandible 2,464F 1.5 Severe Mandible 1,895F 1.5 Severe Mandible 1,990F 1.5 Severe Mandible 1,551F 1.5 Severe Mandible 2,340F 1.5 Severe Mandible 2,513M 1.5 Mild Mandible 704

Antler (spike 20 cm) 445M 1.5 Mild Mandible 807

Antler (spike 29 cm) 542M Mature NAc Antler, shed 2012 696M Mature NA Antler, shed 2012 633M Mature NA Antler, shed 2012 641M Mature NA Antler, shed 2009 92M Mature NA Antler, shed 2009 53M Mature NA Antler, shed 2009 83M Mature NA Antler, shed 2009 52M Mature NA Antler, shed 2009 34

a Mature: maximal antler length in this population was 135 cm; antlers from 2009 averaged 82 cm (SE52.16), whichcorresponds to about 5–7 yr old; antlers from 2012 averaged 71 cm (SE52.73).

b Severe cases exhibited one of the following: overt enamel hypoplasia, uneven tooth wear, extreme tooth wear, brokenteeth. Mild cases exhibited only pitting, mottling, and minor breakage.

c NA 5 not applicable.



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skull, antler base, or antler top, whichcorroborates that antler material is in equi-librium with fluoride elsewhere in theskeleton (Walton and Ackroyd, 1988). InEurope, fluoride levels in antlers collectedbefore 1860 (i.e., prior to anthropogenicinfluences) ranged from about 18 to 50 ppmdry weight and were considered naturalbaseline levels (Kierdorf and Kierdorf,2000). This compares well with backgroundlevels measured here (63 ppm) for a regionwithout industry. Therefore, fluoride levels inantlers before the 2011 PCCVE not onlyindicate an environment with normally verylow fluoride levels, but that these levelsincreased by .38-fold from merely 15.5 moof exposure to tephra.

Studies on fluoride content in plants and animalsfollowing the 2011 eruption

Although lichens are well suited tomonitor aerial immission of fluoride (Hryn-kiewicz et al., 1980), we are not aware of anymeasurements of fluoride in lichens in thearea receiving tephra. Apparently, therewas no important volatile phase of fluoridein relation to PCCVE. This conclusion issupported by the absence of reports ofeffects in humans or animals. Plants,however, can also absorb fluorides liberatedvia leaching from tephra and can harborsignificant amounts in particles attached tothe plant surface. The only measurementsknown to us are from Chile, where fluoridelevels in forage and livestock were signifi-cantly elevated (Araya, pers. comm.). How-ever, we do not know if those measurementswere based only on plant matter. Shupe etal. (1984) cautioned that vegetation analysismust be done on unwashed plant samples,which would be particularly relevant inareas exposed to much fine dust and rain-splattered soil on low-growing plants.

Studies on fluorosis in Argentina following the2011 eruption

There have been no reports in Argentinaregarding fluorosis related to PCCVE;rather, livestock losses were attributed tofactors other than known toxic elements

(Wilson et al., 2012), and high fluoride levelsin forage from Chile remain unreported.Nevertheless, the overt clinical signs foundin red deer (damaged enamel, biophysicaldamage expressed as breakage and rapidwear, pitting, mottling, very variable stagesof development, loss of teeth functionality)and the high bone fluoride levels are clearlyrelated to fluoride intoxication.

Instructively, early tephra analyses fol-lowing the Hudson volcano eruption(1991, Chile) revealed high fluoride levels,yet fluorosis was not identified. Instead,the thousands of sheep deaths wereattributed to physical—not chemical—properties of tephra (Rubin et al., 1994).However, considering that the study wasconducted only 1 mo after the eruption,not even mild chronic fluorosis could yethave been observed, as chronic fluorosisusually develops gradually and insidiously,and overt signs do not appear until sometime after initial exposure. Severe dentalfluorosis, as reported here, and skeletalfluorosis require longer-term exposure(Shupe et al., 1979; Livesey and Payne,2011). Extensive studies among severalwild and domestic ungulates showed thatdental changes correlated with fluoridelevels in vegetation and water (Shupe etal., 1984), which would predict that areasaffected by PCCVE would also revealelevated fluoride levels in the vegetation-particle complex.

Future perspectives

We next asked how these observedpathologies relate to future developmentsin the affected ecosystem. Tephra depos-ited already from PCCVE—an estimated100 million tons over some 36 millionha—will continue to be slowly redepositedfurther downslope and downwind, facili-tated by dry summers, strong eolic condi-tions, and trampling, burrowing, androoting by animals. Animals walking orrunning behind the first few individualsare immersed in clouds of tephra. Deerand other animals will therefore continueto be exposed to abrasive tephra for years.




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It is, therefore, also likely that animals willcontinue to accumulate fluoride and sufferthe associated long-term problems ofacquiring osteofluorosis. Tephra from thenearby Lonquimay volcano eruption of1988 caused elevated forage fluoridelevels for 2 yr of monitoring after theeruption, and high forage levels alonewere concluded to be dangerous foranimals at least 2 yr after cessation of theeruption (Araya et al., 1993). The occur-rence of osteofluorotic alterations hasbeen diagnosed in red deer with fluorideconcentrations greater than 4,000 ppm indry bone (Schultz et al., 1998). The factthat adult deer in this study had reached5,175 ppm in merely 15.5 mo of exposureto tephra suggests that deer will reachstages of osteofluorosis.

What is less clear is the potential foradditional impacts from fluoride, as we donot yet know the route of exposure causingthe observed dental pathology in deer.The extraction of fluoride from tephradepends chiefly on mineral composition ofthe water solution used, pH, particle size,and time allowed for the extraction. Thelow fluoride levels in surface water re-ported early agree with studies showingthat oligotrophic water has little potentialto dissolve fluoride from tephra, particu-larly at neutral pH; regional lakes have apH of 6.8 and are considered ultraoligo-trophic (Markert et al., 1997). Instead,possibly the ingestion of tephra and thetype of digestive system of deer (processesof rumination, fermentation, and diges-tion) result in more liberation of fluoridethan indicated by low levels measured inultraoligotrophic creeks and lakes. Fur-thermore, low and admissible water-solu-ble fluoride levels in PCCVE tephrareported initially, based on single 30-minwater-based extractions, may confoundtrue bioavailability. Taves (1980) showedthat multiple water-based extractions re-sulted in 50% more fluoride recoveredover single extractions, indicating thatsome fluoride is released slowly. More-over, considerably more fluoride was

obtained by acid-based extraction overseveral days: Values then averaged142 ppm versus 4 ppm from waterextracts, a 33-fold increase (Taves, 1980).Similarly, while tephra contained 300 ppmfluoride, water leachates contained only1.8 ppm, whereas acid leaching releasedanother 1.3 ppm. However, fluoride con-sistently occurred in highest concentra-tions in alkaline leachates (Smith et al.,1983). Importantly, tephra from differentvolcanoes had up to four times moreleachability, making each case particular(Smith et al., 1983). Remarkably, Taves(1980) cautioned that it is unknownwhether water-soluble fluoride is the onlybiologically important form. Furthermore,a large proportion of total tephra fluoridewas suggested to be soluble in digestivesystems of grazing animals that ingesttephra via grooming (Walton, 1988), vialarge amounts of ingested soil, and viaparticles adhering to low-growing forage(particularly characteristic of rangelands),which results in passive absorption (Gre-gory and Neall, 1996; Cronin et al., 2000,2003). Other avenues of fluoride ingestiondepend on water and forage concentra-tions determined by precipitation patternsand soil conditions, and thus vary amonglocations (Shupe et al., 1984).

Ruminants indeed present a complexphysiochemical environment for process-ing any compound. Rumination involvesregurgitation of rumen content for repeat-ed mastication to diminish the size ofparticles. This process likely results inpulverization of tephra as evidenced byrapid tooth wear, and thereby increasesthe surface:volume ratio, with the result-ing effect of liberating more fluoride.Moreover, this mastication process occursin a chemical medium dominated bysaliva, which is highly alkaline (pH of8.2–8.5; Aschenbach et al., 2011). Rumi-nation is a major component of this type ofdigestion, as evidenced by 20–25 chewsbefore swallowing (Prinz and Lucas,1997), and the copious production ofsaliva to replace what is swallowed each




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time. Cattle have been measured toproduce 100–200 L/day or more of saliva(Silanikove and Tadmor, 1989). The great-ly increased fluoride solubility in alkalinemedia (e.g., Markert et al., 1997) likelymakes this a crucial step in the massbalance of fluoride in ruminants. Uponswallowing, the mixture enters the rumen,which essentially represents a water-basedextraction system with nearly neutral pH,but with presence of various solutes.Finally, the suspension passes to theabomasum for final digestion at a pH of1–2. Again, it is well documented thatfluoride solubility is greatly increased inacidic conditions as compared to neutralpH. Clearly, the initial analyses done afterPCCVE on surface water and on water-based leachates of tephra did not provideany indications of future fluoride intoxica-tion among ruminants. Considering theaforementioned, we posit that the suscep-tibility of ruminants resides in their foodprocessing: 1) intensive mastication andtephra size reduction (with concomitantexcessive tooth wear), 2) thorough mixingof tephra with alkaline saliva at repeatedrumination cycles, 3) water-soluble extrac-tion in rumen, and 4) extraction in theacidic abomasum.

The lesser impact on deer observedfrom areas with more browse species mayrelate to different forage (Fig. 3C, bot-tom). Although those areas received lesstephra (25–50 mm) than the other sam-pling site (50–100 mm; Wilson et al.,2012), tephra layers of even ,1 mmresulted in the death of several thousandsheep, mainly from fluorosis (Gregory andNeall, 1996; Cronin et al., 2003). Consid-ering that fluoride will continue accumu-lating in the body under excessive inges-tion, skeletal fluorosis could become afuture concern for the study population.Another important factor is recognitionthat due to particle size, tephra depositedfarthest from a volcano have highestfluoride levels (Taves, 1980; Rubin et al.,1994; Gregory and Neall, 1996; Croninet al., 2003). Hence, fluorosis might be

even more severe in deer or other wildliferesiding at distances farther from PCCVEthan our study population, thus demand-ing studies in additional areas.

CONCLUSIONS

These findings have major implicationsfor the region affected by the PCCVE. Theimpacts from recent tephra deposits docu-mented on all examined subadults corrob-orate the toxic fluoride levels reported inforage from nearby Chile, the absence ofsuch pathology in these deer monitoredsince 1991 prior to PCCVE (Flueck andSmith-Flueck, 2008), bone fluoride con-centrations, and diagnostic symptomatolo-gy (Shupe et al., 1984; Walton, 1988). Ifexcessive fluoride intake continues, sub-adult deer will be subjected to furtherimpact because crown formation of addi-tional emerging permanent teeth will lastanother 4–5 mo (Kierdorf et al., 1996b).The dry, windy conditions of the affectedregion further exacerbate impacts from theabrasive tephra on mastication. Add tothese fluorosis, and the deterioration ofthese animals’ teeth would then accelerateconsiderably more (Burns, 1969). Becausefluoride accumulates under chronic expo-sure and causes osteofluorosis, it may resultin skeletal weakness and bone damage.Importantly, the impact from fluoride isfurther exacerbated by the region beingiodine deficient, with endemic goiter ratesin 1965 of 50–80% before iodized salt wasintroduced (Salvaneschi and Garcıa, 2009).Epidemiologic and experimental data showthat iodine deficiency increases the inci-dence of dental fluorosis and severity ofdamages caused by excessive fluoride, andalso increases bone fluoride content (Xu etal., 1994; Zhao et al., 1998). Lastly, basedon clinical and biochemical symptoms, theregion, at least in Chile, is deficient inselenium, which also has well-known im-pacts on iodine metabolism (Flueck andSmith-Flueck, 2008, 2011). The describedimpact will reverberate through severalaspects of the ecology of the deer, including




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effects on population dynamics, morbidity,and predation susceptibility, as well asother components of the ecosystem, in-cluding scavenger and plant communities.

ACKNOWLEDGMENTS

This research was done on private landwithin a natural reserve of the ArgentineAdministracion de Parques Nacionales (permit070-2012). The authors are grateful to JuanJones, Konrad Bailey, and Pio Pigorini forfacilitating access and allowing us to work ontheir properties. We also thank Swazi NewZealand for protective field garments, RubenKodjaian for providing our office with cruciallogistics via the Hosterıa El Retorno, and BeatFuchs for his dedicated field assistance withinclouds of tephra. We appreciate the diverseconstructive comments from several review-ers. W.F. and J.S. contributed equally to thefield study, the analysis, and writing of themanuscript. This work was made possiblethrough the support of DeerLab, Bariloche,Argentina, and the Asociacion de Pesca y CazaNahuel Huapi and the Sociedad Rural de SanCarlos de Bariloche kindly covered the pub-lication costs.

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Submitted for publication 2 November 2012.Accepted 20 November 2012.




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severe dental fluorosis in juvenile deer linked to a recent volcanic

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