LEAD POISONING IN THE GLOBALLY THREATENED MARBLED TEAL AND WHITE-HEADED DUCK IN SPAIN

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1 LEAD POISONING IN THE GLOBALLY THREATENED MARBLED TEAL AND WHITE-HEADED DUCK IN SPAIN RAFAEL MATEO,* ANDY J. GREEN, CLINTON W. JESKE, VICENTE URIOS,W nd CATI GERIQUE# Lortory of Toxiology, Fulty of Veterinry, Autonomous University of Brelon, Bellterr 08193, Spin Deprtment of Applied Biology, Estió n Bioló gi de Doñ n, CSIC, Avenid de Mrí Luis s/n, Sevill 41013, Spin Ntionl Wetlnds Reserh Center, 700 Cjundome Boulevrd, Lfyette, Louisin 70506, USA WDeprtment of Environmentl Siene nd Nturl Resoures, University of Alinte, Alinte 03080, Spin #Center for Nture Protetion nd Study, Deprtment de Medi Amient, Generlitt Vlenin, Avenid de los Pinres 106, El Sler 46012, Spin Astrt Mrled tel (Mrmronett ngustirostris) nd white-heded duk (Oxyur leuoephl) re the two Europen duks thretened with glol extintion. We investigted led (P) poisoning in stifftils (Oxyur spp., n = 83) nd mrled tel (n = 80) shot or found ded or moriund in Spnish wetlnds vi gizzrd exmintion nd liver, one, nd lood P nlysis. Ingested P shot ws present in 32% of shot stifftils nd 70 nd 43% of ded or moriund stifftils nd mrled tel, respetively. Ledshot ingestion ws more frequent in Vleni (estern Spin), where P-shot densities were higher nd grit srer. Seletion of lrger grit similr in size to P shot my explin the higher rte of P-shot ingestion oserved in stifftils. Ingested shot ws found more frequently in juvenile stifftils thn in dults. Led one onentrtions were higher in duklings <9 d old thn in fully grown tel nd were lso higher in dult thn in juvenile tel. Our results show the need for n of P shot for wterfowl hunting in Spin nd the lenup of spent shot t mjor wetlnds. Keywords Hevy metl ontmintion Led shot Wterfowl Grit Wetlnds INTRODUCTION Led poisoning s result of ingestion of spent P shot in wetlnds hs een mjor use of wterfowl mortlity [1]. Led-shot densities in wetlnds long the Mediterrnen ost of Europe, where ll hunters still use P shot, re mong the highest reorded nywhere in the world [2]. The prevlene of P-shot ingestion in the widespred mllrd (Ans pltyrhynhos) rnges in Mediterrnen wetlnds from 20 to 45% [2 6]. The prevlene registered for mllrd or the relted lk duk (Ans ruripes) in Britin or North Ameri, where P shot hs een nned for over dede, ws <10% [7 9]. However, in some southern sttes of the United Sttes, the exposure in mllrd nd the relted mottled duk (Ans fulvigul) equled the vlues oserved in Mediterrnen Europe [8,10]. Two of the speies most ffeted y P poisoning in Mediterrnen wetlnds re the northern pintil (Ans ut) nd the ommon pohrd (Aythy ferin), whih hve prevlenes of P-shot ingestion of 50 to 70% [2 4,11] nd hve undergone popultion delines in Europe over the pst 25 yers [12]. These re still reltively widespred nd undnt speies of wterfowl. However, it hs previously een suggested tht P poisoning my e signifint use of mortlity for the two duk speies present in Europe tht meet the World Conservtion Union riteri for glolly thretened sttus [13], the mrled tel (Mrmronett ngustirostris) nd the whiteheded duk (Oxyur leuoephl) [14]. Here we present the first study of P poisoning in the mrled tel nd white-heded duk. We fous on Spin, the most importnt Europen rnge ountry for oth speies, whih re * To whom orrespondene my e ddressed (rfel.mteo@u.es). resident there. The mrled tel hs undergone long-term deline in Spin [15] nd elsewhere in its rnge, hving remining world popultion of 15,000 to 25,000 individuls [13]. Sine 1990, totl numers in Spin hve flututed etween pproximtely 50 nd 700 ([16], A.J. Green, unpulished dt). The mrled tel is dling duk in eologil (ut not txonomi) terms [16]. The white-heded duk is the only stifftil (trie Oxyurini, group of diving duks) ntive to the Plerti. It hs frgmented distriution nd delining world popultion of 10,000 to 20,000 irds [13]. In Spin, numers hve reovered from 22 irds in 1977 to 1,453 in 1999 in response to onservtion mesures, espeilly protetion from hunting [17]. The Spnish popultion of the white-heded duk is ffeted y the spred of the North Amerin ruddy duk, Oxyur jmiensis jmiensis, introdued to northern Europe [14]. Ruddy duks produe fertile hyrids with the white-heded duks; oth ruddy duks nd hyrids re shot y governmentl onservtion orgniztions to ounter geneti integrtion [14]. The white-heded duk, ruddy duk, nd their hyrids flok together nd hve very similr forging eology [18] nd grit seletion (this study) nd n e expeted to respond to P exposure in similr wy. We therefore inlude the ruddy duk nd their hyrids shot or found ded in Spin in this study s useful surrogte for studying P exposure in the glolly thretened white-heded duk. We ssess the frequeny of P-shot ingestion in oth stifftils nd tel in different prts of Spin nd the proportion of the popultions ffeted y toxi levels of P in tissues. We ssess the differenes etween ge lsses nd sexes in the prevlene of P exposure y shot ingestion. We ompre the omposition of grit ingested y eh txon in different regions 2860

2 Led poisoning in thretened wterfowl from Spin Environ. Toxiol. Chem. 20, Fig. 1. Lotion of utonomous ommunities nd wetlnds where stifftils nd mrled tel were olleted in Spin. Wetlnds 1 through 11, mrked with irles, re within the rnge of the white-heded duk nd mrled tel. Numers of irds (L = white-heded duk, J = ruddy duk, H = hyrid, M = fully grown mrled tel, m = tel hik) olleted t eh wetlnd re s follows. Andluí: 1 = Vet l Plm, Doñ n, Sevill, H (6), J (1), M (6); 2 = Luio del Cngrejo Grnde, Doñ n, Sevill, J (1); 3 = Lgun de Medin, Cádiz, J (1); 4 = Lguns de Puerto Rel, Cá diz, J (1); 5 = Lgun de Trelo, Doñ n, Cá diz, H (4), J (2); 6 = Alufer de Adr, Almerí, H (2), J (1); 7 = Punt Entins-Sinr nd nery, Almerí, J (3), H (2). Vleni: 8 = Snt Pol, Alinte M (4); 9 = El Hondo, Alinte, J (8), H (12), L (28), M (48), m (20); 10 = Alufer de Vleni, L (1), M (1), m (1). Cstill-l Mnh: 11 = Dehes de Monrel, Toledo, H (1). Argón: 12 = Lgun de Sriñ er, Hues, J (1). Pís Vso: 13, Ullirri-Gmo, Álv, J (7). Cntri: 14, Mrism de Sntoñ, J (1). nd onsider how this reltes to the rtes of P-shot ingestion. We lso investigte the levels of P found in newly hthed duklings (not sujeted diretly to ingested P shot), n issue not previously studied in ny wterfowl speies. Colletion of speimens MATERIALS AND METHODS We studied totl of 83 stifftils (29 white-heded duks, 26 ruddy duks, nd 28 hyrids) olleted from 14 Spnish wetlnds (Fig. 1) t different times of the yer [18]. Most ruddy duks nd hyrids were shot during ontrol progrm orgnized y the Spnish regionl nd ntionl dministrtions. These irds were trked down nd hunted individully y mrksmen, nd we do not ntiipte is towrd shooting irds more exposed to P. The white-heded duks were either found ded or dying (usully during epizooti outreks of slmonellosis, otulism, or other diseses) or shot (either inidentlly during the ruddy duk ontrol progrm or shot illeglly nd then onfisted). We studied totl of 80 mrled tel, inluding 59 fully grown irds nd 21 duklings (19 of the youngest ge lss I of 1 8 d old, 2 of lss II of d old [19]). All exept seven fully grown irds nd one dukling me from El Hondo nd Snt Pol long the Mediterrnen ost in the utonomous ommunity of Vleni in estern Spin (Fig. 1). All ut three fully grown irds were found ded or dying, nd 45 of them were olleted during die-off t El Hondo from Septemer to Novemer 1997 tht ws pprently used y orgnophosphte poisoning nd otulism (C. Gerique, unpulished dt). Most duklings died shortly fter eing resued from onrete-lined dringe nl in El Hondo, in whih hundreds of duklings were trpped eh yer [20] until rmps were instlled in In order to study regionl differenes in the prevlene of P exposure y shot ingestion, we ompred those irds from the utonomous ommunity of Vleni with those oming from other prts of Spin. Compred with other res, Vlenin wetlnds hve prtiulrly high densities of P shot [6], nd most of our irds me from El Hondo, where densities of up to 163 shot/m 2 re reorded [21]. El Hondo is the most importnt Europen wetlnd for oth mrled tel nd whiteheded duks [15]. Tretment of irds vried onsiderly in this heterogeneous smple, involving mny orgniztions nd people. Blood smples ( ml) were tken from eight sik whiteheded duks upon rrivl t the Center for Nture Protetion nd Study in Vleni. Blood smples were olleted in metlfree tues with sodium heprin nd frozen to 20 C efore nlysis. Most ded irds were frozen shortly fter olletion nd were sent to the Doñ n Ntionl Prk or Doñ n Biologil Sttion for neropsy. Fully grown irds were sexed nd ged (dults or juveniles) sed on plumge hrteristis, gond inspetion, nd presene or sene of the urs of Friius [19,22]. Smples of liver nd one (humerus nd/or femur) were removed nd stored in metl-free plsti vils. Anlysis of P-shot presene nd P onentrtions Gizzrds were exmined externlly for the presene of shot entry holes to estlish whether shot found within hd een shot in or ingested. Eh gizzrd ws ut with sissors nd its ontents wshed with wter nd then dried. Food items were removed y flottion. The preipitte ws dried nd pled in plsti Petri dishes. After P shot were removed nd weighed, grit ws sieved (sieve sizes 4, 3, 2, 1.5, 1, nd 0.5 mm) nd eh size lss of prtiles ws weighed [5]. Led shot ws exluded from nlyses of grit omposition. Livers were nlyzed t the Autonomous University of Brelon (Brelon, Spin), ones t the Ntionl Wetlnds Reserh Center, nd lood smples t the Sgunto 99 Lortory of Veterinry Anlysis (Vleni, Spin). Livers were homogenized nd portion ws dried t 60 C until its mss ws onstnt. A 500-mg liquot of dry liver ws mirowve digested in 70% nitri id for 45 min t 180 psi in mirowve digester MDS2000 (CEM, Mtthews, NC, USA). Led onentrtions were mesured y Zeemn grphite furne-tomi sorption spetrosopy on 4100 ZL spetrometer (Perkin- Elmer, Norwlk, CT, USA). Our results from three replites of ertified referene smple of ovine liver (BCR 185, Promohem, Wesel, Germny) with men ± stndrd devition of 501 ± 38 ng/g dry weight were 540 ± 14 ng/g. Bones were lened of inserted musles, oven dried to onstnt mss, nd weighed to the nerest 0.1 mg. Eh one ws then mirowve digested nd P onentrtions were mesured y indutively oupled plsm-tomi emission spetrosopy on n Optim 3000 spetrometer (Perkin-Elmer). Blood P onentrtion ws determined in duplites of eh smple y grphite furne-tomi sorption spetrosopy on GBC GF 2000 Controller spetrometer (GBC, Dndenong, Vitori, Austrli) fter dilution of 50 iil of lood in 50 iil of Triton X-100 (1%) nd 150 iil of deionized wter. Blood P onentrtion of eh smple ws otined from the verge of duplites. Results from duplite nlyses, fortified smples, nd referene smples of humn lood (Lyphohek, Bio-Rd Lortories, Rihmond, CA, USA) with three levels of P (0.081, 0.285, nd iig/ml) were within qulity ontrol guidelines. Minimum detetion limits of P were 0.1 iig/g dry

3 2862 Environ. Toxiol. Chem. 20, 2001 R. Mteo et l. Tle 1. Proportions of fully grown duks from wetlnds of Vleni nd other prts of Spin ontining ingested P shot in the gizzrd nd those ove threshold levels of P exposure in liver nd one (humerus nd/or femur); perentges re presented in prentheses P shot in gizzrd Liver P Bone P >5 iig/g dry weight >10 iig/g dry weight Vleni Others Totl Vleni Others Totl Vleni Others Totl Mrmronett ngustirostris 21/50 0/6 21/56 10/25 0/5 10/30 3/41 0/6 3/47 (42) d (0) (38) (40) (0) (33) (7) (0) (6) Oxyur leuoephl e 13/25 0/1 13/26 9/17 0/1 9/18 1/1 0/1 1/2 (52) (0) (50) (53) (0) (50) (100) (0) (50) Oxyur jmiensis f + hyrids g 9/20 4/33 13/53 4/16 1/12 5/28 1/5 0/12 1/17 (45) (12) (25) (25) (8) (18) (20) (0) (6) Totl Oxyur spp. 22/45 4/34 26/79 13/33 1/13 14/46 2/6 0/13 2/19 (49) (12) (33) (39) (8) (30) (33) (0) (11) [5]. [23]. Birds exposed/n. d Perentge. e White-heded duk. f Ruddy duk. g Oxyur leuoephl X jmiensis. weight in liver of fully grown irds, 0.3 iig/g dry weight in liver of duklings, 0.05 iig/g dry weight in ones, nd 0.01 iig/ml in lood smples. Birds were onsidered exposed to norml P levels with liver onentrtion >5 iig/g dry weight [5], one onentrtion >10 iig/g dry weight, nd lood onentrtion >0.2iig/ml [23]. Those irds found ded with liver P onentrtions >20 iig/g dry weight n e onsidered to hve died y P poisoning nd those with lood P onentrtions >1 iig/ml s severely poisoned [23]. Sttistil nlyses Prevlenes of P-shot ingestion nd P onentrtions ove the hosen threshold levels were ompred mong speies, lotions, nd olletion method (shot or found ded) with Fisher ext tests. Generlized liner models (GLMs) were onstruted using GLIM softwre [24] to investigte the ftors explining vrition in presene or sene of P shot (s inry vrile) nd the onentrtion of P in liver nd one tissue in fully grown irds. Led-shot prevlene ws nlyzed using inomil error nd logit link funtion, while tissue onentrtions were nlyzed using norml error nd identity link funtions (log-trnsforming the dependent vrile to remove heterosedstiity). Preditors used in the models were region where the irds were olleted (ftor of two levels, Vleni or other res), olletion method used (ftor, shot or found ded), sex (ftor), nd ge (ftor, dult or juvenile). Seprte models were onstruted for stifftils nd mrled tel (exluding the three irds shot nd the ftor olletion method from the ltter models). Smple size vried onsiderly etween models euse not ll tissues were nlyzed in ll irds nd not ll irds ould e sexed or ged. We present the finl models in whih olletion method (for stifftils) nd region were inluded together with sex or ge, when these hd signifint effets. We lso present the prtil effets of the nonsignifint vriles when these were dded to the finl models. In stifftils, the humerus ws the only one nlyzed. In mrled tel, the humerus nd/or the femur were nlyzed. For the GLMs, we nlyzed humerus onentrtions or femur onentrtions when the former were not mesured. Preliminry nlyses showed tht there were no signifint differenes etween these two mesures in fully grown tel. In order to study the reltionship etween P levels in tissues nd the mount of P ingested, we used the numer of P shot oserved in the gizzrd nd the estimted mss of P eroded. The ltter ws lulted y sutrting the oserved mss of the shot from the expeted mss of uneroded shot (onsidered to e 128 mg [2]). We dded these vriles to GLMs to identify their prtil orreltions with P tissue levels while ontrolling for region nd olletion method. The P onentrtions in tel tissues showed log-norml distriution (onfirmed with Kolmogorov Smirnov tests) nd omprisons etween tissue levels within nd mong ge lsses (duklings nd fully grown irds) were mde using logtrnsformed onentrtions nd t tests. Person orreltions mong log-trnsformed P tissue onentrtions were onduted for eh ge lss. Beuse of the log-norml distriution of P onentrtions, results re expressed with geometri mens nd 95% onfidene intervls (CI) rther thn with rithmeti mens. Differenes in the totl weight of grit in the gizzrd etween tx, sex, ge, lotion, olletion method, nd the presene of ingested shot were studied with nlyses of vrines. Beuse of the unit-sum onstrint of grit size frtions for eh individul duk, differenes in grit omposition due to the ove ftors were studied y mens of ompositionl nlysis of the frtions s perentges, using multiple nlysis of vrine of the log of the weight of eh frtion divided y the weight of grit <0.5 mm [22]. RESULTS A high proportion of mrled tel nd stifftils studied hd ingested P shot or norml P onentrtions in liver or one tissues, prtiulrly in Vleni (Tle 1). Eighty perent of the white-heded duks nd 20% of the mrled tel found ded hd lethl liver P onentrtions (Fig. 2), nd ommon neropsy findings in these irds were ingested P shot; weight loss; ple tissues; ile-stined gizzrd, liver, nd vent; nd white streks in the hert. Led levels in the lood of eight white-heded duks resued from El Hondo, seven of them not inluded in Tle 1, were ll inditive of P poisoning (rnge iig/ml) nd ll of them died within few dys of their dmittne to the Center for Nture Protetion nd Study in Vleni. These irds showed wekness, right green

4 Led poisoning in thretened wterfowl from Spin Environ. Toxiol. Chem. 20, Fig. 2. Distriution of the numer of ingested P shot nd P liver onentrtions in fully grown mrled tels nd stifftils (Oxyur spp.) from Spin, seprting irds shot (lk rs) nd those found ded or moriund (white rs). Perentges within two rnges of liver P onentrtion or with P shot in the gizzrd re shown for irds shot (regulr) nd found ded (itlis). Ext numer of irds with different numers of P shot in the gizzrd re lso shown. dirrhe, pleness of muous memrnes, nd low hemtorit (30 35%). The differenes oserved etween tx in prevlenes were strongly influened y the wy the irds were olleted (Fig. 2). Most of the fully grown mrled tel (95%, 56 of 59) nd 69% (20 of 29) of white-heded duks were found ded, wheres 98% (53 of 54) of ruddy duks nd hyrids were shot. Thus, the dt in Tle 1 for mrled tel nd white-heded duk re likely to exggerte the prevlene of P exposure in their popultions euse they re minly irds found ded, wheres the dt for ruddy duks nd hyrids, minly shot, re likely to reflet more urtely the prevlenes in the group of the stifftils. For exmple, 70% (14 of 20) of stifftils found ded in Vleni hd ingested P shot, ompred with only 32% (8 of 25) stifftils shot in this region (Fisher ext, p < 0.017), nd similr differenes were oserved for ll res omined (Fig. 2). Liver P onentrtions suggest tht P-shot ingestion is more likely to e diret use of mortlity for stifftils thn for mrled tel (Fig. 2). Among irds found ded in Vleni, 43% of mrled tel (20 of 47) hd ingested P shot, ompred with 70% of stifftils (14 of 20) (Fisher ext, p = 0.06). Similrly, 40% of these mrled tel (10 of 25) hd P liver onentrtions over 5 iig/g, ompred with 80% of stifftils (8 of 10) (Fisher ext, p = 0.06). The GLMs showed tht the proportion of stifftils ontining ingested P shot ws signifintly higher in Vleni, higher in irds found ded thn those shot (Fig. 2), nd lower in dults thn in juveniles (Tle 2). For mrled tel, the prevlene of ingested P shot ws lso signifintly higher in Vleni, ut there were no ge or sex effets (Tle 2). The P onentrtions in liver were signifintly higher in stifftils found ded thn in shot irds (Fig. 2), ut the regionl nd ge effets were not signifint (Tle 3). In mrled tel, no preditor vriles hd signifint effet on liver onentrtions (Tle 3), lthough they were mrginlly higher in Vleni (p < 0.08). The GLMs for P one onentrtions reveled rther different ptterns. In stifftils, onentrtions were signifintly higher in Vleni nd in femles (Tle 4). The dt suggest tht, for given exposure to ingested P shot, femle stifftils deposit more P in one thn mles. Even when ontrolling for the numer of ingested P shot (i.e., dding this preditor to the model of Tle 4), femles still hd higher P levels in one thn mles (F 1,14 = 4.84, p < 0.05). In mrled tel, one onentrtions were higher in dults thn in juveniles (Tle 4). Although not inluded in the models presented in Tles 3 nd 4, the mount of P shot found in the gizzrd is strong preditor of the P onentrtion in liver nd one tissues, onfirming ingested shot s the primry soure of ontmintion. While ontrolling for region (plus olletion method for stifftils) in GLMs, oth numer of ingested shot in the gizzrd nd eroded mss of P showed strong prtil orre- Tle 2. Generlized liner models of P-shot prevlene in the gizzrds of fully grown mrled tel (Mrmronett ngustirostris) nd stifftils (Oxyur spp.) Mrmronett ngustirostris Oxyur spp. Prmeter estimte Stndrd Prmeter error 2 2 estimte Stndrd error 2 2 Constnt Region d Vleni * * Colletion method d Shot ** Sex d Mle (+) 0.00 ( ) 1.17 Age d Adult ( ) ** Residul devine * *** Residul df The dependent vrile ws one (when shot were present) or zero, using inomil error nd logit link funtion. Colletion method ws exluded from the tel model. Prtil 2 2 vlues nd the sign of the prmeter estimte (in prentheses) re shown for independent vriles exluded from the finl models. d Region other, olletion method found ded, sex femle, nd ge juvenile were lised. * p < ** p < *** p <

5 2864 Environ. Toxiol. Chem. 20, 2001 R. Mteo et l. Tle 3. Generlized liner models of P liver onentrtion (expressed s ln[onn. in ng/g dry weight + 2]) of fully grown mrled tel (Mrmronett ngustirostris) nd stifftils (Oxyur spp.) Mrmronett ngustirostris Oxyur spp. Prmeter Stndrd Prmeter Stndrd estimte error F rtio estimte error F rtio Constnt Region d Vleni Colletion method d Shot Sex d *** Mle (+) 0.05 ( ) 0.04 Age d Adult ( ) 0.25 ( ) 1.30 Residul devine Residul df *** Using norml error nd identity link funtion. Colletion method ws exluded from the tel model. Prtil F vlues nd the sign of the prmeter estimte (in prentheses) re shown for independent vriles exluded from the finl models. d Region other, olletion method found ded, sex femle, nd ge juvenile were lised. *** p < ltions with the P onentrtions in liver of stifftils (r = 0.48, F 1,39 = 11.8, p = ; nd r = 0.52, F 1,39 = 14.4, p = , respetively) nd mrled tel (r = 0.47, F 1,27 = 7.6, p < 0.011; nd r = 0.49, F 1,39 = 8.4, p < 0.008, respetively). The equivlent prtil orreltions of P onentrtions in one tissue with the numer of ingested shot nd the eroded mss of P were even stronger in stifftils (r = 0.82, F 1,15 = 30.4, p < ; nd r = 0.88, F 1,15 = 49.2, p < , respetively) nd mrled tel (r = 0.56, F 1,41 = 18.4, p = ; nd r = 0.56, F 1,41 = 18.4, p = , respetively). Mjor differenes were oserved in tissue onentrtions etween duklings nd fully grown mrled tel (Tle 5). Sine the presene of ingested P shot in the gizzrd is mjor determinnt of P onentrtions, we exluded irds with in- gested shot from sttistil omprisons of tissue onentrtions mong nd within ge groups. Ingested P shot ws only reorded in one mrled tel hik, ird of intermedite ge (lss II). Liver P onentrtions were higher in fully grown irds thn in duklings, ut the reverse ws true for oth humerus nd femur (Tle 5). Humerus P onentrtions in mrled tel duklings, lthough djusted to log-norml distriution, showed high dispersion (Tle 5), nd two groups of duklings re pprent. In eight irds, P onentrtion ws 4 iig/g dry weight, nd in the other six irds, the vlues rnged from 8 to 26.6 iig/g dry weight, the ltter possily orresponding to duklings whose mothers were exposed to P shot prior to or during luth formtion. However, this imodl distriution ws not oserved in femur P onen- Tle 4. Generlized liner models of P onentrtion in humerus or femur (expressed s ln[onn. in iig/g dry weight ]) of fully grown mrled tel (Mrmronett ngustirostris) nd stifftils (Oxyur spp.) Mrmronett ngustirostris Oxyur spp. * p < *** p < Prmeter Stndrd Prmeter Stndrd estimte error F rtio estimte error F rtio Constnt Region d Vleni *** Colletion method d Shot Sex d Mle (+) *** Age d Adult * (+) 0.01 Residul devine * *** Residul df Using norml error nd identity link funtion. Colletion method ws exluded from the tel model. Prtil F vlues nd the sign of the prmeter estimte (in prentheses) re shown for independent vriles exluded from the finl models. d Region other, olletion method found ded, sex femle, nd ge juvenile were lised.

6 Led poisoning in thretened wterfowl from Spin Environ. Toxiol. Chem. 20, Tle 5. Led onentrtions (iig/g dry wt) in tissues of mrled tel (Mrmronett ngustirostris) without ingested P shot Liver Humerus Femur n GM 95% CI n GM 95% CI n GM 95% CI Duklings Fully grown Geometri men (i.e., men of the log-trnsformed distriution). Signifintly different from onentrtions in the sme tissue of fully grown irds (Student s t tests, p < 0.01). Signifintly different from humerus nd femur onentrtion in duklings (pired t tests, p < 0.001). No signifint differenes were found etween different tissue onentrtions in fully grown tel or etween humerus nd femur onentrtions in duklings. trtions. In duklings, higher P onentrtions were found in ones thn in liver (humerus vs liver, n = 14, geometri mens = 4.32 vs 0.22; femur vs liver, n = 12, geometri mens = 2.71 vs 0.19; pired t tests, p < 0.001), ut there were no suh differenes in fully grown irds (Tle 5). Correltions mong P onentrtions in liver, humerus, nd femur were signifint for fully grown tel (liver with humerus: n = 21, r = 0.581, p = 0.005; liver with femur: n = 16, r = 0.503, p = 0.046; humerus with femur: n = 34, r = 0.678, p < 0.001) ut not for the duklings (p > 0.05). Liver nd humerus P onentrtions were lso orrelted in stifftils (n = 19, r = 0.633, p = 0.003). Grit size omposition ws different etween fully grown mrled tel nd stifftils (Wilk s X = 0.49, F 5,116 = 24.12, p < ), stifftils seleting grit more similr in size to P shot (Fig. 3). Grit size omposition ws different etween Vleni nd other regions (mrled tel: Wilk s X = 0.77, F 5,49 = 2.87, p = ; stifftils: Wilk s X = 0.83, F 5,61 = 2.56, p = 0.036), with smller proportions of orse grit in Vleni, espeilly in the tel (Fig. 3). The totl mss of grit ws higher in stifftils thn in tel (F 1,126 = 4.744, p = 0.031) nd lower for oth tx in Vleni (F 1,126 = 9.81, p = ). Men mss ± stndrd error of grit in stifftils ws 0.79 ± 0.13 g in Vleni (n = 40) nd 1.23 ± 0.12 g in other res (n = 34). Mss of grit in mrled tel ws 0.44 ± 0.04 g in Vleni (n = 50) nd 0.93 ± 0.19 g in other res (n = 6). No signifint differenes were oserved in the totl mount or size omposition of grit etween olletion methods, the presene or sene of ingested shot, sex, or ge or etween Fig. 3. Mss of the different grit size tegories from the gizzrds of fully grown mrled tels nd stifftils (Oxyur spp.) from Vleni (lk rs) nd other regions in Spin (white rs). Signifint differenes in the mss of frtions etween res re mrked with sterisks (nlysis of vrine, p < 0.05). different stifftil tx. Mrled tel duklings hd no mesurle grit. DISCUSSION Previous studies of P poisoning in the Mediterrnen prt of Europe showed it to e mjor prolem for reltively widespred, undnt speies of wterfowl wintering in the region ut reeding further north [2 6]. Ours is the first study to fous on wterfowl speies resident in the Mediterrnen region nd shows, sed on linil signs, neropsy findings, nd liver P onentrtions, tht P poisoning is use of mortlity [23] in two glolly thretened speies in Spin, espeilly in El Hondo nd other wetlnds in the Vlenin ommunity where oth mrled tel nd white-heded duk hve eome inresingly onentrted over the pst dede [15]. Other wterfowl speies of onservtion onern re likely to e similrly ffeted y P poisoning in Mediterrnen wetlnds. During the ourse of our study, we nlyzed one ferruginous duk (Aythy nyro), speies lose to extintion in Spin nd onsidered ner thretened t glol level [13]. This ird, found ded t El Hondo on Ferury 29, 1997, hd 30 ingested P shot. Mrled tel nd stifftils olleted in Vleni hd signifintly more ingested P shot thn those olleted elsewhere, s ws expeted given the higher densities of shot in Vlenin wetlnds (rnge of mens: shot/m 2 ) ompred with the Doñ n wetlnds in Andluí (mximum men: 14 shot/m 2 ), one of our other study res [6]. The prevlenes of P-shot ingestion in other wterfowl speies in Vlenin wetlnds re mong the highest reorded in Spin [6], nd epizootis of P poisoning in greter flmingos (Phoeniopterus ruer roseus) hve ourred nnully over the lst dede in El Hondo [21]. Bone P onentrtion ws higher in stifftils from Vleni, refleting the higher rte of shot ingestion in tht wetlnd. Low lium intke my lso inrese P deposition in ones [25], nd grit, n importnt soure of lium, is sre in these wetlnds [6]. Our results suggest tht stifftils were more ffeted y P poisoning thn mrled tel. The lower prevlene of ingested shot nd P levels in liver in stifftils tht were shot ws expeted sine mny irds found ded hd pprently died of P poisoning. However, P exposure in shot irds ws still ommonple (e.g., 32% of stifftils shot in Vleni hd ingested shot). The prevlene of ingested P shot in stifftils shot my even underestimte the exposure of white-heded duks to P sine most irds olleted y shooting were ruddy duks or hyrids tht were killed s soon s possile fter they were first oserved t the site [14]. They re thus likely to hve een newly rrived or newly fledged irds hving hd less time exposed to the high P-shot densities t sites suh s El Hondo thn the verge white-heded duk. The time of residene in n re n ffet the risk of P-shot ingestion in wterfowl [26,27]. Among Austrlin wterfowl, two stifftils, the lue-illed duk (Oxyur ustrlis) nd the musk duk (Biziur lot), pper to e the speies most ffeted y P exposure, with 24 nd 50% of individuls, respetively, showing elevted levels in one [28]. Few dt exist on the prevlene of shot ingestion in ruddy duks in their nturl rnge in North Ameri, ut these suggest they re s suseptile to P exposure s other diving duks [10], whih tend to e more ffeted thn dling duks euse they n esily reh P shot present t the ottom of wetlnds [8]. Given tht lmost ll our fully grown mrled tel were

7 2866 Environ. Toxiol. Chem. 20, 2001 R. Mteo et l. found ded (i.e., the sene of less ised smple olleted y shooting), it is hrder to ssess the true prevlene of P exposure in this speies. However, this prevlene must e reltively high given tht the proportion of the mrled tel popultion tht died in the epizooti of orgnophosphte poisoning nd otulism t El Hondo in 1997 tht provided most of our smple ws t lest 25%, nd possily over 50%. Thus, even in the unlikely event tht ll individuls exposed to P shot died in the epizooti, the overll prevlene of ingested shot in the El Hondo popultion would still hve een t lest 11% t tht point in time (we found prevlene of 45% in the 44 individuls olleted in the 1997 epizooti, ut t lest 150 tel died from n estimted totl of 600). The mrled tel in Spin hs prtiulrly high nnul mortlity rte [29], nd P poisoning is likely to e one ftor responsile, exerting its thretened sttus. Overll, stifftils seleted more nd lrger grit sizes thn mrled tel, result onsistent with the oser ved differene in P-shot exposure. In onfusion, wterfowl selet P shot for lrge grit prtiles [30]. The mounts of grit seleted pproximtely 1 to 2 mm in dimeter y oth tx were similr to those seleted y other wterfowl speies showing high prevlenes of P-shot ingestion of 28 to 74% in the Ero delt, nother Spnish Mediterrnen wetlnd [22]. The regionl differenes we found in grit seletion my e produt of differenes in grit vilility. In oth tx, irds olleted in El Hondo hd smller proportion of orse grit, where the prtiles of grit >0.5 mm onstitute only 0.86% of the totl weight of sediment, while in other Spnish wetlnds, they onstitute 1.5 to 13.4% [6]. Some regionl vrition in stifftil diet hs een reorded in Spin [18], nd hnges in diet ould ffet grit requirements nd seletion [30]. The mrled tel olleted in Vleni were feeding minly on Sirpus litorlis seeds, while those olleted elsewhere were feeding minly on Ruppi mritim seeds (A.J. Green, unpulished dt). These seeds re similr in size ut my vr y in hrdness, R. mritim possily requiring lrger grit to rek them up. Adult stifftils hd lower prevlene of ingested shot thn juveniles, perhps due to the forging inexperiene of juvenile irds tht inreses their likelihood of ingesting shot [26,31]. Alterntively, the higher P-shot ingestion in juvenile stifftils my reflet the ft tht higher proportion of dults were ruddy duks (49%, ompred with 30% of juveniles). Some ruddy duks were olleted from northern Spnish wetlnds (Fig. 1), whih perhps hve prtiulrly low shot densities. Others shot on Mediterrnen wetlnds hd proly rrived from further north in Europe shortly efore eing shot [14], thus hving less time to ingest P shot thn the white-heded duks or hyrids ntive to these wetlnds with high P-shot densities. This ould explin why we found differenes in Pshot prevlene etween ge lsses in the sene of ny differenes in grit size seletion. Other studies hve found no ge-relted differenes in the prevlene of shot ingestion [3,7,32] or hve oserved higher prevlenes of P-shot ingestion [8] or elevted lood P [33] in dults. Mjor differenes within nd etween tx were oserved in the preditors of vrition in P-shot ingestion nd P onentrtions in liver nd one. From sttistil perspetive, it is diffiult to ssess whih of the differenes etween nlyses re relile sine the dt set for eh nlysis ws somewht different (sine not ll tissues were nlyzed in ll irds nd not ll irds ould e ged or sexed) nd the mesurement error for eh dependent vrile is likely to differ somewht. For exmple, the only signifint effet of sex ws oserved in stifftil ones, yet less thn third (n = 19) of ll stifftils were inluded in this dt set, of whih only two were femles, oth of whih showed extremely high levels. Differenes in P-shot ingestion or tissue onentrtions relted to sex hve een desried in severl studies of different speies, ut this is fr from eing generl pttern [1,3,7,10,26,32,34,35]. Experimentl studies suggest tht the higher one P onentrtion in femles ould e relted to lium requirements nd to the tive one metolism during egg lying [25,35,36]. However, White nd Stendell [34] did not find differenes in one P onentrtions in field study with mllrd, northern pintil, nd lk duk, nd sexul differenes in sorption rtes my not exist outside the reeding seson [37] or my even e higher in mles [38]. No dt re ville on P onentrtions in eggs of mrled tel from El Hondo, ut trnsfer of P from exposed hens to their eggs nd the high deposition of P following the lium pthwy for one formtion in hiks [25,39] my explin the high P one levels in tel duklings. However, in fully grown tel with ones tht hve rehed full size, juveniles hd lower one P onentrtions thn dults, possily due to the dilution of mternl P in the lrger ones of juveniles ompred with duklings [35] nd lso to the longterm exposure of dults to P shot. Given their young ge (<9 d), P in tel duklings proly me from the eggs or from ingested food or sediment ut is not likely to hve ome from ingested P shot or grit euse none ws oserved in their gizzrds. From the imodl distriution oserved in humerus P onentrtion, we estimte tht 42% of duklings my hve een young of P-exposed femles. The reltionship mong the duklings nlyzed nd the P levels in their mothers re unknown, so this perentge my not urtely reflet exposure levels for reeding femle tel. Trnsfer of P from femles to hiks hs een demonstrted in doves [39], lthough umultion of P in eggs is vrile [35]. In fully grown irds, we found signifint orreltions etween P onentrtions in different tissues nd signifint prtil orreltions of these tissue onentrtions with P-shot ingestion. As expeted, stronger orreltions were oserved etween humerus nd femur thn etween either one nd liver in fully grown tel. However, the numer of P shot ingested nd the mss of shot eroded showed stronger prtil orreltions with one thn with liver P onentrtions for oth tel nd stifftils. These results were unexpeted nd my e prtly due to the ft tht smple sizes were different for the nlyses of liver nd one onentrtions. Furthermore, these prtil r vlues were hevily influened y the other vriles inluded in the GLM model. Thus, when sex ws dded to the one model for stifftils, the prtil r vlues were redued to 0.58 for the eroded mss of P nd 0.36 for the numer of ingested shot. When ge ws dded to the mrled tel one model, the r vlues were redued to for oth. Diret reltionships etween P-shot ingestion nd one P onentrtions hve only een found in other studies when ompring popultions [34] nd re usully wek [40] euse the one P onentrtion tends to reflet long-term exposure rther thn the ute high-level exposure refleted in liver onentrtions [23]. The finding tht dult mrled tel umulte more P in one thn juveniles ould reflet this long-term exposure to P, s oserved in other studies [37,38]. Our strong prtil orreltions etween P-shot ingestion nd one P re lso

8 Led poisoning in thretened wterfowl from Spin Environ. Toxiol. Chem. 20, proly relted to the rpid hunting of ruddy duks nd hyrids upon their pperne in Spin nd to the presene of so mny juvenile tel (.65%) fledged in the weeks just efore the epizooti in El Hondo. For these resons, one P onentrtions were reltively low ompred with other studies [38,40] or similr to those oserved in immture irds of out 1 iig/g dry weight [37] nd therefore my etter reflet reent exposure to P shot. Conservtion mesures re urgently required to redue the mjor impt tht P exposure is hving on wterfowl in Spin, prtiulrly the glolly thretened mrled tel nd whiteheded duk. Aout 5.5% of the shot stifftils showed liver P onentrtions omptile with severe linil poisoning (>20 iig/g dry wt) [23], nd monthly mortlity rte of 7.4% ould e estimted from the numer of ingested P shot in the gizzrd, s lulted y Bellrose [1] for mllrds. Although our results suggest tht P poisoning is more frequent in the white-heded duk, the dditive effet of this prolem my e more signifint for the mrled tel. While the Spnish (ut not the glol) white-heded duk popultion hs inresed in the lst 20 yers[17], the Spnish mrled tel reeding popultion is smller, is reltively more dependent on the highly ontminted El Hondo, nd is undergoing long-term deline [15]. It is importnt tht the use of P shot in hunting tivity in Spin e nned s soon s possile nd tht the most highly ontminted prts of El Hondo nd other importnt wetlnds e lened of P shot to prevent die-offs of wterfowl of ll speies. Aknowledgement Funding support ws provided y the uthors institutes nd the Humne Soiety. J. MCoy, J.C. Dolz, M. Gimenez, M. Má ñ ez, J.D. Nvrro, P. Pereir, H. Queedeux, N. Selv, C. Surez, nd C. Urdiles ssisted in preprtion of speimens nd nlysis of smples. N.W. Beyer nd D.J. Pin provided helpful omments on the mnusript. REFERENCES 1. 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9 2868 Environ. Toxiol. Chem. 20, 2001 R. Mteo et l. 34. White DH, Stendell RC Wterfowl exposure to led nd steel shot on seleted hunting res. J Wildl Mng 41: Frnson JC, Petersen MR, Creekmore LH, Flint PL, Smith MR Blood led onentrtions of spetled eiders ner the Kshunuk River, Yukon Delt Ntionl Wildlife Refuge, Alsk. Eotoxiology 7: Finley MT, Dieter MP Influene of lying on led umultion in one of mllrd duks. J Toxiol Environ Helth 4: Stendell RC, Smith RI, Burnhm KP, Christensen RE Exposure of wterfowl to led: A ntionwide survey of residues in wingones of seven speies, Sientifi Report Wild- life 223. U.S. Deprtment of the Interior, Fish nd Wildlife Servie, Wshington, DC. 38. Merhnt ME, Shukl SS, Akers HA Led onentrtions in wing ones of the mottled duk. Environ Toxiol Chem 10: Kendll RJ, Snlon PF Effets of hroni led ingestion on reprodutive hrteristis of ringed turtle doves Streptopeli risori nd on tissue led onentrtions of dults nd their progeny. Environ Pollut A 26: Fisher FM, Hll SL Hevy metl onentrtions of duk tissues in reltion to ingestion of spent shot. In Feierend JS, Russell AB, eds, P Poisoning in Wild Wterfowl A Workshop. Ntionl Wildlife Federtion, Wshington, DC, USA, pp