Skeletal Pathology in White Storks (Ciconia Ciconia) Associated With

Toxicologic Pathology, 33:441–448, 2005 Copyright QC by the Society of Toxicologic Pathology ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1080/01926230590953097

Skeletal Pathology in White Storks (Ciconia ciconia) Associated With Heavy Metal Contamination in Southwestern Spain

1 2 3 2 3 4 JUDIT E. G. SMITS, GARY R. BORTOLOTTI, RAQUEL BAOS, JULIO BLAS, FERNANDO HIRALDO, AND QIANLE XIE

1 Department of Veterinary Pathology, 2 Department of Biology, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada 3 Estacio´ n Biolo´ gica de Donˇ ana, C.S.I.C., 41013 Sevilla, Spain, and 4 Trent University, Peterborough, ON, Canada

ABSTRACT In 1998, a mine tailings dyke in southwestern Spain broke, ﬂooding the Agrio-Guadiamar river system with acid tailings up to the borders of one of the largest breeding colony of white storks in the western Palearctic, Dehesa de Abajo. Over the following years, a high proportion of nestlings developed leg defects, prompting this study. Ten ﬂedglings with leg deformities from the spill area were compared with 11 normal storks of the same year class from another region far from the spill. However, metals were analyzed as a continuum rather than by site, as reference birds also contained high levels of metals. Gross pathology of the legs was supported by histopathology, which showed that bone remodeling activity was greater in the deformed storks, which also had more irregular subperiosteal bone, and tended to have higher residual islets of cartilage in their metaphyses, which, in turn were related to metal contaminant residues. Both Ca and P in bone were affected independently by metals. Deformed birds had lower serum bone alkaline phosphatase. Bone malformations, measured by leg asymmetry, was only partially explained by bone metals, indicating that a combination of factors was involved with the abnormal development in these young storks. Keywords. Skeletal pathology; white storks; toxic metals; bone alkaline phosphatase; deformities; environmental contamination.

INTRODUCTION pri nci In April 1998, an environmental disaster struck ple southwestern Spain. The tailings dyke from an iron pyrite co mine in Aznalco´ llar (Sevilla) broke open, flooding the mp on valleys of the Agrio and Guadiamar rivers up to the border ent of the Don˜ ana National Park, a critical breeding and ; wintering site for many species of threatened and PC endangered animals (Grimalt et al., 2, 1999). The slurry of toxic acidic mud and water covered an sec area 40 km long and 0.5 km wide, was approximately 2 × on 6 3 d 10 m in volume and contained potentially toxic elements pri such as Pb, Zn, As, Cu, Cd, and other sulphide-related ele- nci ments (Grimalt et al., 1999). Of particular concern was the ple co proximity of the inundation to Dehesa de Abajo, the loca- mp tion of one of Western Europe’s largest breeding colony of on white storks (Ciconia ciconia). The tailings flooded the ent river valley and marshes as close as 1 km from the colony. ; PC The chemical nature of the tailings, plus hydrological, 3, geomor- phological details, as well as effects on soil, thi groundwater, plants, invertebrates, fish and birds in 1998, rd were detailed in an issue of Science of the Total pri Environment (1999) devoted to examining this accident. nci ple In the years since the spill, researchers have documented co different problems in the nestling storks. Genotoxic damage mp has been tracked in the colony for several years (Pastor et on al., ent .

Address correspondence to: Dr. Judit E. G. Smits, Department of Veterinary Pathology, Western College of Veterinary Medicine, Univer- sity of Saskatchewan, Saskatoon, SK S7N 5B4 Canada; e-mail: judit. [email protected] Abbreviations: AP, alkaline phosphatase; ANOVA, analysis of variance; BAP, bone alkaline phosphatase; DdA, Dehesa de Abajo; HPF, high power field; LSI, liver somatic index; MT, tarsometatarsal bone; OP, organophosphate; PCA, principle component analysis; PC1, first 441 20 d previously in this or any other stork colony in Spain. The 04 peak in deformities occurred in 2001, with nearly 10% of ). the nestlings being affected to some degree (R. Baos, Si unpublished). As detailed below, legs were consistently nc affected just distal to the tarsal joint, with thickening, local e malformation, and lateral or medial devia- tions of the th lower leg. In some nestlings, the bill, especially the maxilla, e was curved dorsally or laterally. spi Our objective was to conduct a comprehensive examina- ll, tion characterizing the nature and extent of physiological a and anatomical aberrations in young storks in relation to va metal contaminant loads in tissues. To this end, deformed ria juveniles from Dehesa de Abajo (DdA) were compared bl with another small group of storks of the same age collected e from different breeding sites in Caćeres province, an area bu over 200 Km away from the spill . Pathological plus other t morphologi- cal and physiological endpoints were su compared between the groups of birds. bst MATERIALS AND METHODS an tia Animals. In 2001, 10 nestling storks with obviously de- l formed tarsometatarsal (MT) bones were collected from the pr DdA colony adjacent to the spill site just prior to reach- op ing fledging age (birds ranged from 40–55 days old), and ort were housed in the Acebuche wildlife recuperation center io (Don˜ ana National Park, Huelva) for 3 months. Eleven n storks, also young of the year and apparently healthy of (except they had fallen from their nests or suffered some th other misadven- ture) were collected from a wildlife e recovery center “Sierra de Fuentes” in the neighbouring ne area of Extremadura. Birds were housed in groups of 5 or 6 stl colony mates, in 4 open pens. Pens (4w × 6d × 3h m) in were enclosed with solid mesh gs we re dis co ve re d to ha ve de for m ed le gs an d bil ls th at ha d no t be en do cu m en te 186 SMITS ET AL. TOXICOLOGIC PATHOLOGY walls, open wire fronts, and roofs with partial cover. Birds had access to small ponds within their enclosures, and were maintained on a standard diet of various species of marine fish purchased from a fish-processing factory, which produces fish for human consumption. Any fish that were too small or otherwise unsuitable for the human food market, made up the diet of the storks. Their diet was not analyzed for metals, but was assumed to meet human food quality criteria. Once the birds arrived at the wildlife recovery centre, their legs were radiographed. The storks remained in the rehabilitation centre for 3 months before it was logistically possible to conduct the necropsy examinations. All work was conducted under the proper Animal Care Protocols. Study areas. The colony referred to as DdA, was located in a natural area (Puebla del R´ıo, province of Sevilla, 6◦ 10t 16tt W: 37◦ 12t 33tt N), while the Caćeres birds came from a variety of habitats in the Extremadura region of southwestern Spain. The linear distance between the origins of these 2 groups of birds was greater than 200 km. The DdA colony was far from urban environments, close to a permanent water source (within 2 km of a marsh in Don˜ ana National Park which is a very productive ecosystem), with nests in wild olive trees. Details of the origins of the birds that came from the Caćeres rehabilitation center were not available. Pathology. Storks were humanely euthanized by overdos- ing with Halothane inhalation anaesthetic. Their heads were placed into a plastic bag containing halothane soaked cotton swabs, until respiratory and cardiac arrest within 30 to 45 sec- onds. Body mass was determined immediately. A complete necropsy examination was conducted. Because of the leg deformities in DdA birds, length of both right and left MT bones (i.e., area of leg spanning the tarsometatarsal bone) were measured to the nearest mm and the difference between the 2 was used to describe the degree of leg asymmetry. All joints, mus- culoskeletal system and viscera were examined, and the mass of the liver, bursa of Fabricius, and spleen were recorded. Or- gan mass was expressed as “somatic indices” (SI) (e.g., (liver SI = (liver mass/(body mass − liver mass)) ×100). Samples of liver and kidney were wrapped in xylene rinsed foil and frozen in sample bags at −20◦ C. Tarsometatarsal bones were immersed in 10% neutral buffered formalin. From the proximal epiphysis, extending 2 to 3 cm distally along the shaft, to encompass the area of the deformations, the MT bones were sectioned midsagitally. The bones were then placed in decalcification solution (20% formic acid in water) until they were demineralized sufficiently for proper histological preparation. To avoid information bias, the eval- uation of the bone sections was carried out by the pathol- ogist unaware of the animals’ identification or origin. The H&E-stained bone sections were examined microscopically for evidence of pathology. After consultation with a col- league/osteopathologist, the bones were assessed according to the following features. The osteal-periosteal surface was described as smooth or irregular (Figure 1a and b). After examination of 6 high power fields (HPF: 200× magnifica- tion), the occurrence of irregularly shaped islets of cartilage in the diaphyseal or cortical bone around the growth plate was scored as “mild” if there were 0–1 islets per HPF, moderate if there were 2–4 per HPF, and marked, if there were greater than or equal to 5 per HPF. The remodeling activity in the Vol. 33, No. 4, 2005 LEG DEFORMITIES IN STORKS EXPOSED TO METALS 187 bone was determined based upon the presence of an open, haphazard pattern of newly formed bone with remaining col- lagen and occasional round osteoblasts visible (Figure 2a). Remodeling was most easily determined using a polarizing filter to distinguish it from the well organized, lamellar, parallel pattern of older bone (Figure 2b). It was categorized as mild if <10% of each field showed remodeling, moderate if 10–50% showed newly forming bone, and marked if >50% showed remodeling activity. When remodeling activity changed along the bone section, the most commonly noted pattern was recorded for that case. Clinical pathology. Prior to euthanasia, a blood sample was taken from each bird. Because of the important role of Ca and P in bone development, levels of both minerals were assessed in serum using an automated instrument (Hitachi 912 automatic analyzer, Montreal, Canada). In veterinary medicine, serum levels reported as total alkaline phosphatase (AP) are the sum of AP isoenzymes from the liver, intestine, and bone. Because of the specific role of bone AP, which is produced at the highest rate by osteoblasts during bone growth and development (Hoffman and Solter, 1999), we measured the total, hepatic and bone AP, to determine if there was a detectable change in bone AP activity associated with the deformed long bones. Serum levels of bone AP were determined using an adaptation of the mammalian assay by Hoffman et al. (1994). Mineral and metal analysis. Liver and kidney samples from these storks were placed into a drying oven in covered porcelain dishes for several days until all moisture was gone from the tissues. A 2 cm slice of each MT bone was sectioned 3 cm distal to the proximal epiphysis, individually bagged and submitted to the analytical laboratory at the Department of Geological Sciences, University of Saskatchewan. The dried liver and kidney samples were then ground and homogenized using a corundum grinder. Aliquots of the powdered tissues (0.1 g) were placed into Teflon vials with 2 ml double dis- tilled, ultrapure HNO3 and a few drops of H2 O2 . Vials were left open in a clean fume hood until the reaction subsided. Vials were then capped and placed on a hot plate at 100◦ C until samples were completely digested and a clear solution was formed. The vial was then opened so the liquid could evaporate ◦ at 70 C until it reached ambient dryness, 1 ml HNO3 was added to the residue and the vials capped for 1 hour until all residues were dissolved, the solution was diluted with deion- ized water to 100 ml, and stored at 4◦ C until analysis. For the bone samples, the sections were crushed with an agate pes- tle, ground and homogenized with corundum grinder, 0.1 g of the powder was used for digestion following the proce- dure described above. Analysis for metals was performed using a Platform collision cell inductively coupled plasma mass spectrometer (ICP-MS, Micromass, Manchester, UK). A low flow Meinhard concentric nebulizer (Glass Expan- sion, Hawthorne, Australia) was employed. A water-cooled (4◦ C) Scott type double-pass spray chamber was also used to generate homogeneous sample aerosols. Calibration was achieved using a series of multi-element standards (1, 5, 10, 20 ppb), and a calibration curve was established. Rhodium was used as an internal standard to correct for sample matrix effects and instrument drift. Three standard reference 188 SMITS ET AL. TOXICOLOGIC PATHOLOGY

Figures 1–2

FIGURE 1.—Comparison of (A) smooth, or (B) irregular osteal-periosteal surface of the tarsometatarsi in storks with varying levels of metals in their long bones (H&E bar = 200 µm). 2.—Remodelling activity in the proximal tarsometatarsi was described as (A) “immature” if there was primarily open, haphazard bone formation, or (B) “mature” if there was primarily parallel, laminar bone formation. (H&E, bar = 50 µm).

materials were used for the data quality control; a river ysis (PCA) to derive fewer and independent variables. In 1 water standard SLRS-4, a dogfish liver standard DOLT-2 analysis we took this approach for 9elements in the bone and a dogfish muscle standard DORM-2 (National that we interpret as contaminants (Pb, Co, Cr, Ti, Zn, Sn, Research Council of Canada, Ottawa, Canada). V, Ba, Sr). For a second analysis we used 5 normal, physio- Statistical analyses. Although we present the metal logically important elements in the bone (Ca, P, Mg, Na, S). residues in liver and kidney as well as bone to allow com- For these PCAs, we chose elements that were consistently parison with findings by other researchers, we limit all sta- detectable and that were, or could be transformed to be, tistical analyses to metal levels in the bones. The rationale normally distributed. The PCA of contaminants resulted in for the latter is that all the birds had been on the same stan- three components accounting for 63.7% of the total dard fish diet at the rehabilitation centre for 3 months, al- variability: PC1 explained 29.7%, and PC2 explained lowing differences that might have originally existed in the 21.7%, and PC3 explained 12.3% of the variance in the data liver and kidney to disappear. Bone metal concentrations set. Examination of the relative loading of the variables on were assumed to be unchanged from the time the fledglings the 3 components, and subsequent correlation analyses, were taken from their original colonies. Bone elements are yielded the following interpretation. PC1 was largely an quite stable in full-sized animals compared with rapidly axis of V, Ba, Sr, and Cr all varying positively. PC2 was growing nestlings (Scheuhammer, 1987 and references Zn and Ti (positively), and Co, Sn, and Cr (negatively). therein). PC3 was largely influenced by a positive relationship with The full biological impact of metals was unlikely to be Pb and Co. For the PCA of the normal bone elements 2 revealed by repeated testing of individual elements, as there components explained 91.0% of the variance: (PC1 70.6%, are numerous interactions and correlations among the PC2 20.4%). Ca, P, Mg, and Na were all highly, and metals themselves. Therefore, we used principal positively associated with PC1, whereas PC2 was primarily component anal- influenced by S. Vol. 33, No. 4, 2005 LEG DEFORMITIES IN STORKS EXPOSED TO METALS 189

TABLE 1.—Metal contaminants (median, minimum and maximum concentrations) in kidney, liver and bone of white storks exposed to a mine tailing spill in southwestern Spain (Dehesa de Abajo—DdA) and from a reference area in Ca´ceres.

Tissue Colony Al ppm Ti ppm V ppb Cr ppb Co ppb Cu ppm Zn ppm As ppb Se ppm Sn ppb Sr ppm Pb ppb Kidney DdA 2657 28426 494 1354 251 17 111 1558 8.6 736 0.588 310 1110–7256 Caceres 3960 22542–33456 235–1420 2–2043 171–534 12–26 89–139 635–2950 5.0–10.7 348–1144 0.408–0.784 203–604 1959–11612 25528 383 1456 276 23 108 1664 6.6 774 0.472 587 Liver DdA 2109 22417–33238 299–692 2–4054 166–512 12–26 71–126 666–3406 4.1–13.4 292–1231 0.363–0.798 83–1403 942–33882 27157 191 1528 101 36 292 3236 7.4 555 0.191 93 Caceres 4463 24205–34954 81–316 1021–2137 65–158 21–51 223–339 1266–6072 5.6–9.0 244–1597 0.140–0.309 27–236 1107–11592 26480 165 866 143 30 317 3375 5.3 781 0.147 264 Bone DdA 0 21353–30415 100–234 339–2767 71–185 19–88 176–445 1621–4697 3.1–8.7 286–2171 0.094–0.271 23–1031 0–6.2 185 704 811 127 0.003 55 0 0 239 229.2 882 Caceres 0 123–273 130–1936 0–1473 0–277 0–1.2 0–94 0–104 0–6.0 81–439 159.2–305.1 0–2779 0–76.5 196 243 743 190 0 39 0 287 134.3 2008 109–229 162–1348 21–1233 121–327 0–0.8 0–185 0–338 142–341 68.5–236.2 0–12178

We used ANOVA where nonsignificant interactions and spill site were free of metal contamination. In fact, for some variables were removed iteratively, to obtain the most parsi- of the metals, concentrations were higher in the “reference” monious model that explained variation in the dependent birds. For the PCA analysis of bone metals, location was variable. Two-tailed tests were performed using SPSS not significant for PC1 (F1,19 = 1.672, p = 0.211), (Norusˇis, marginally nonsignificant for PC2 (F1,19 = 3.837, p = 1993), and we considered results significant at the 0.05 0.065) and significant for PC3 (F1,19 = 5.436, p = 0.031) level. in which the difference was driven by the higher Pb in For the analysis of leg asymmetry, it was not valid to bones of the Caćeres birds. simply Gross pathology. Radiographic evidence of leg deformi- compare birds on the basis of location since all deformed ties are shown in Figure 3. The deformed, healed bones birds came from the same location. However, it was clear were locally thicker with a triangular, rather than round, that cross section. In all affected birds, the cortical bone just individuals varied in the degree of leg deformity and so we distal to the tibiotarsal joint was abnormally thickened with derived the variable, absolute leg asymmetry, i.e., difference callus formation and varying severity of valgus deviation in length between the right and left metatarsi. In the of the distal portion of the limbs. There was no radiographic ANOVAs evidence of decreased cortical thickness or density, or of we thus used this variable, and location as a factor. any generalized RESULTS Metal contamination by location. Table 1 shows clearly that neither birds from the reference location nor from the 190 SMITS ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 3.—Radiographs of (a) abnormal tarsometatarsal bones in a stork from DdA compared with (b) normal limbs from a reference area stork. Vol. 33, No. 4, 2005 LEG DEFORMITIES IN STORKS EXPOSED TO METALS 191 increase in lucency of the cortices over the length of the bones. No fracture lines were visible. Histopathology. A Chi-square test revealed remodeling bone score to be dependent on location, as birds in Ca ćeres had minimal to moderate bone remodeling whereas those from DdA had moderate to active remodeling activity (Pearson Chi-Square Test, p = 0.015). The nature of the surface of the osteum/periosteum was similarly dependent on location (Fisher’s Exact Test, p = 0.001). There was no statistically significant effect of location on scores for the cartilage islets in the bone, although this may be due to the small sample size, as the trend was for 70% of birds in Ca ćeres, vs only 33% in DdA, to have low amounts of residual cartilage in their metaphyses. When contaminant levels, as described using PCA, were examined in relation to histological scores, there was no difference among scores for bone remodeling or surface of the osteum/periosteum; however, PC2 values increased as the occurrence of residual islets of cartilage increased (F2,16 = 5.412, p = 0.016) (Figure 4). FIGURE 5.—Subperiosteal bone surface was more irregular in birds with more asymmetrical legs. Leg asymmetry, associated pathology, and clinical biochemistry. Legs of DdA birds were more asymmetrical than the reference birds (F1,17 = 10.123, p < 0.005). Leg de- were significantly different between DdA and Caceres formities, as measured on a continuous scale of leg asym- birds. For birds in both locales, increasingly asymmetrical metry, were associated with a number of other physiolog- legs had decreasing amounts of the bone enzyme BAP ically relevant variables. When the histological appearance (Figure 7). We could not detect an association between of the osteal/periosteal surface was smooth, leg asymmetry asymmetry and either PC1 (p > 0.13) or PC2 (p > 0.71) of was significantly less than if the birds had irregular bone the normal bone minerals. Of the organs measured, only surfaces (F1,17 = 6.819, p = 0.018) (Figure 5). Consider- liver showed any relationship with limb pathology. As liver ing the bone remodeling activity, the leg asymmetry in- somatic index (liver size relative to body mass) increased, creased significantly with increasing evidence of remodel- leg asymmetry also increased (F1,17 = 8.805, p = 0.009), ing (F2,14 = 4.981, p = 0.023) (Figure 6). The presence and while again location explained considerable variance (F1,17 density of cartilaginous islets in the proximal tarsometarsal = 14.088, p = 0.002). sections of bone was independent of the symmetry of the The degree of leg asymmetry was also examined with re- leg bones (F1,15 = 0.787, p = 0.473). spect to the 3 PCAs based on metal contaminants. Although In the ANOVAs, body mass and sex were never found to location was considered as well, it was dropped from the be significantly related to leg asymmetry and so were final model, and PC3 remained significant (F1,18 = 10.385, always dropped from models. Using asymmetry as the p = dependent variable, both effect of location and levels of 0.005). However, the relationship was unexpected, as bones bone alkaline phosphatase (BAP) levels in plasma (F1,17 = with low PC3 scores (i.e., driven by low Pb levels) were 4.483, p < 0.05) most asymmetrical.

FIGURE 4.—Level of remodeling activity in tarsometatarsi related to asym- FIGURE 6.—The density of islets of cartilage in diaphyseal bone is related to metry in bone length. contaminant metal burdens in the bone. 192 SMITS ET AL. TOXICOLOGIC PATHOLOGY

in bones. Fledglings were collected from the DdA colony adjacent to the inundated area because of having deformed tarsometarsal bones. Through pathology, biochemistry, and tissue residue analyses, we describe the toxicology and ex- plore the nature of the physiological disturbances that have resulted in bone problems. Leg asymmetry was an important feature related to bone contaminant levels. The leg deformities were most likely the result of fractures which occurred at an early stage during the nestling period, and, because nestlings do not bear weight on their legs until about 45 to 50 days of age, the fractures could heal through apositional bone production. The consistent location of the leg pathology at the proximal end of the tarsometatarsus indicates that physical stress occurs at this site. Radiographs taken after fledging when the birds would have reached maximum skeletal size, did not show deficient cortical bone compared with the legs of the reference birds, although FIGURE 7.—Serum levels of bone alkaline phosphatase (BAP) are higher in the bones would have gone through substantial changes storks with more symmetrical bones. during the nestling period, readily mask- ing or correcting early defects in mineralization. Metals, once bound in bone matrix, are considered non-mobilizable (Klaassen, 2001). Body and organ mass. Independent ANOVAs were con- The metals were not different between the birds from DdA ducted for the dependent variables of liver, bursa and spleen and Caćeres, so it is possible that the rate or timing of somatic indices, as well as body mass. The initial models exposure to those metals would have played an important used sex, location, and the 3 PCA contaminant-independent role in expression of toxicity in young, quickly variables. For body mass, sex (F1,17 = 11.717, p = 0.003) developing birds. Maybe very young storks are fed on more and PC3(F1,17 = 5.406, p = 0.033) were significant, where contaminated, smaller, filter-feeders so they get higher males were heavier (as is known for this species), as were doses of mud-associated contaminants early in life. Field birds with higher PC3 levels. For the liver somatic index, observa- tions indicate that one important prey item for PC3 was again a significant covariate (F1,18 = 5.102, p = DdA nestlings is the red swamp crayfish Procambarus 0.037). Neither the splenic nor bursal indices were clarkii (Negro et al., significantly associated with the PCA for metals. 2000), which can accumulate metals in their tissues. Normal bone elements. In the multivariate analysis of Crayfish captured in the spill area in 2000 had metal levels normal bone constituents, we considered length of right exceeding that allowed for human consumption (Sańchez- metatar- sus as a potential covariate. There was no Lo´ pez et al., significant effect of the three PCA contaminant variables on 2003). PC1 of the normal bone minerals. However, for PC2 of The limb deformities were originally suspected to be due bone minerals, there were significant effects of metatarsal to toxicity by metals associated with the spill, that could di- rectly disturb bone formation. Cadmium and Pb as well as length (F1,16 = 8.752, p = 0.009), and PC1 of metal Al contaminants (F1,16 = 5.099, p = 0.038). Because Ca and P are the 2 major elements com- prising bone, they were are known to interfere with bone turnover and Ca examined individually in relation to the PCA of contaminant metabolism metals. In ANOVAs we used bone Ca (and subsequently P) (Goyer et al., 1994; Berglund et al., 2000). As it turned out, as the dependent variable, and considered sex and location the as factors. We considered body mass, P (or subsequently levels of toxic metals were often lower in the affected birds Ca) in bone and the 3 components of the PCA of than those from the reference site distant from the tailings contaminants as potential covariates. In the final significant accident. The Pb in tissues from the DdA storks was model, Ca was explained by body mass (F1,16 = 9.748, p = 0.007), P in bone (F = 369.785, p < 0.001), confirmed 1,16 as coming from the Aznalcollar mine spill through isotopic and PC1 metals (F1,16 = 6.188, p = 0.024). Similar analysis of bone P levels showed significant effects of body identification (Meharg et al., 2002). Of the metals used for mass (F = PCA analyses, only Pb, Co, and Zn were identified in the 1,15 mud 11.701, p = 0.004, Ca in bone (F1,15 = 441.029, p < 0.001), location (F = 6.347, p = 0.024) and PC2 metals slurry. The remaining metals included in the analyses (V, 1,15 Ba, (F1,15 = 4.722, p = 0.046). In addition, PC1 proved, predictably, to Sr, Cr, Ti) were identified as coming from the natural river sediments unaffected by the spill (Alastuey et al., 1999). be significantly correlated with Ca:P ratio (rs = 0.644, p = 0.002), but PC2 and PC3 were nonsignificant. By using multivariate analyses, relationships between toxic metal concentrations and essential bone components DISCUSSION became evident. Using a PCA allowed us to recognize physi- In this study we examined physiological and anatomical ological costs associated with exposure to a complex aberrations in juvenile storks in relation to metal residues mixture of metals in an acid matrix. Sulfur levels in the bones had a Vol. 33, No. 4, 2005 LEG DEFORMITIES IN STORKS EXPOSED TO METALS 193 significant relationship with contaminant metals possibly im- plying that this essential mineral was being compromised in birds with higher metal contamination. Calcium and optimal Ca:P ratios, which are critical for normal bone development and remodeling, were linked to the combination of toxic metals in the bone. Because blood Ca is tightly regulated in the body through hormonal and renal controls, and the Ca:P ratio was abnormal, it stands to reason that the P, which was 194 SMITS ET AL. TOXICOLOGIC PATHOLOGY

associated with metal concentrations, and which was differ- esters stimulate the cytochrome ent between the 2 populations, was not being properly regulated in the DdA birds. Bone alkaline phosphatase is normally produced during active growth and remodeling in bone. It should be present in serum at relatively higher levels in animals that are growing quickly (Hoffmann and Salter, 1999). In other studies from a larger group of nestling storks, those which had slightly deformed limbs had significantly higher BAP than either normal or severely deformed chicks (Smits, unpublished), perhaps indicating that they were mounting a successful at- tempt to remodel the damaged bones. However, in the current study with full sized birds, decreased BAP levels were associated with more severe leg asymmetry, although histologically those limbs appeared to be at an active stage of remodelling. The lower BAP activity indicated defective or static development in bone that had never properly matured. According to recent studies in passerines, increased serum BAP is seen during the final maturation and mineralization of bone (Tilgar et al., 2004). Increased liver mass is a nonspecific response associated with induction of enzymes involved in biotransformation of environmental contaminants to enhance their polarity and fa- cilitate their excretion (Klaassen, 2001). The relatively larger livers seen in the storks with more severely affected legs, although a nonspecific response, is consistent with an increased detoxification effort by animals dealing with contaminant exposure. Because many metals were higher in the physically normal birds from the reference area, other sources and types of xenobiotics must be acknowledged as potential contrib- utors to the hepatic differences in these birds. There is no indication that other factors such as trauma, or other physical or genetic factors may have been responsible for the observed limb defects. In small mammals (Mus spretus) collected in the spill area, biochemical responses indicate the simultaneous occurrence of organic pollutants as well as the contaminant metals (Bonilla- Valverde et al., 2004). In DdA, storks generally forage in nearby marshes and rice paddies, feeding their nestlings crayfish, fish, insects, and other invertebrates (Negro et al., 2000). Both the DdA population (Jovani and Tella, unpublished) and other populations of storks are known to forage in garbage dumps (Tortosa and Caballero, 2002), which likely explains the chicken offal that has been identified in the regurgitation of nestling storks along with the more normal dietary items. The toxic mud resulting from the breached tailings dyke was removed within several months (Hudson-Edwards et al., 2003). Besides the residual mine contaminants, the DdA stork population is also close (within 3 km) to a vast delta area of rice production, which, during the breeding season of the storks, receives frequent applications of agrochemicals to support crop production and control pests. It was not possible to obtain reliable information about the compounds being used. However, from speaking with local agronomists, or by finding recently emptied pesticide containers, it was evident that both carbamate (carbaryl) and organophosphate compounds (malathion, fenitrothion, and triclorfon), along with several types of fungicides were being used in the delta region. Cholinesterase-inhibiting compounds undergo exten- sive biotransformation in all forms of life, with very species specific routes and rates of metabolism (Ecobichon, 2001). Organophosphorous (OP) Vol. 33, No. 4, 2005 LEG DEFORMITIES IN STORKS EXPOSED TO METALS 195 P450 isoenzyme system. The normal role of P450 enzymes is to enhance eventual excretion of metabolic breakdown prod- ucts. One theory that could be explored is that in cases of low grade, subchronic exposure to OPs, other phosphoric acids may be metabolized and excreted at an increased rate because of the overall upregulation of this enzyme system that is relatively indiscriminant, thus causing a disturbance of normal P regulation. Lower BAP was seen in the deformed birds, which was indicative of retarded or arrested bone maturation (Figure 2a, b). PC3 associated most with Pb levels, was making a negative contribution to bone maturation also. The metals appear to be at least in part responsible for increasing hepatic detoxification efforts (and thus higher liver SI) assuming that metaloth- ionine synthesis (the major protein produced to protect the body against toxic metals) is one contributing factor, along with induced biotransformation activity within the liver, plus hepatic storage of metals. One could speculate on a potential role of Se, which was higher in DdA birds, that could offer some protection against the metal residues. Selenium has the capacity to form stable associations with some metals, block- ing them from forming ligands with the sulfhydryl groups of essential enzyme systems (Sugiura et al., 1976). We have shown with this study, that the relationship between bone malformations and metal contamination occurs in a complex manner which cannot be explained by one or two metals. The juvenile storks with leg deformities were affected in part by toxic elements, but the metals alone do not explain the pathology that developed. Physiological costs of exposure to toxic metals have been exacerbated by unknown factors that may be related to the intense agricultural activities occurring in proximity to the affected colony. Rice production is one conspicuous source of xenobiotic inputs that is not occurring in the reference area. We know that the DdA population had higher genotoxic damage than did storks sampled from distant locations (Pastor et al., 2004). Although we could not prove a direct link with any particular metal, something about the contaminant burdens was affecting bone development. Clearly, a combination of factors is involved with the abnormal development seen in these young storks that requires further investigation.

ACKNOWLEDGMENTS The authors are indebted to G. Garc´ıa, J. M. Terrero, H. Lefranc, and R. Rodr´ıguez for their able ﬁeld assistance. J.L.Tella and the Ayuntamiento de Puebla del R´ıo provided logistic support and access to the DdA stork colony. We thank Consejer´ıa de Medio Ambiente—Junta de Andaluc´ıa for pro- viding permits, and the Acebuche wildlife recovery centre for tending the birds. Sierra de Fuentes wildlife recovery center (Consejeria de Agricultura y Medio Ambiente—Junta de Ex- tremadura) provided the reference birds. The wildlife/stork monitoring program (16/98) was ﬁnanced by CSIC. This work was supported by fellowships from the Spanish Min- isterio de Educacio´ n Cultura y Deporte (to RB and JB), and grants from EGMASA and the European Community (JB). The work was also supported by Canadian NSERC grants (to GB and JS).

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