Toxicologic Pathology, 33:441–448, 2005
C by the Society of Toxicologic Pathology
Copyright Q
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
JUDIT E. G. SMITS,1 GARY R. BORTOLOTTI,2 RAQUEL BAOS,3 JULIO BLAS,2 FERNANDO HIRALDO,3
1
AND
QIANLE XIE4
Department of Veterinary Pathology, 2 Department of Biology, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada
3
Estación Biológica de Doňana, 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
In April 1998, an environmental disaster struck southwestern Spain. The tailings dyke from an iron pyrite mine in
Aznalcó llar (Sevilla) broke open, flooding the valleys of the
Agrio and Guadiamar rivers up to the border of the Doñ ana
National Park, a critical breeding and wintering site for many
species of threatened and endangered animals (Grimalt et al.,
1999). The slurry of toxic acidic mud and water covered an
area 40 km long and 0.5 km wide, was approximately 2 ×
106 m3 in volume and contained potentially toxic elements
such as Pb, Zn, As, Cu, Cd, and other sulphide-related elements (Grimalt et al., 1999). Of particular concern was the
proximity of the inundation to Dehesa de Abajo, the location of one of Western Europe’s largest breeding colony of
white storks (Ciconia ciconia). The tailings flooded the river
valley and marshes as close as 1 km from the colony. The
chemical nature of the tailings, plus hydrological, geomorphological details, as well as effects on soil, groundwater,
plants, invertebrates, fish and birds in 1998, were detailed in
an issue of Science of the Total Environment (1999) devoted
to examining this accident.
In the years since the spill, researchers have documented
different problems in the nestling storks. Genotoxic damage
has been tracked in the colony for several years (Pastor et al.,
Address correspondence to: Dr. Judit E. G. Smits, Department of
Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4 Canada; e-mail: judit.
smits@usask.ca
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 principle component;
PC2, second principle component; PC3, third principle component.
441
2004). Since the spill, a variable but substantial proportion
of the nestlings were discovered to have deformed legs and
bills that had not been documented previously in this or any
other stork colony in Spain. The peak in deformities occurred
in 2001, with nearly 10% of the nestlings being affected to
some degree (R. Baos, unpublished). As detailed below, legs
were consistently affected just distal to the tarsal joint, with
thickening, local malformation, and lateral or medial deviations of the lower leg. In some nestlings, the bill, especially
the maxilla, was curved dorsally or laterally.
Our objective was to conduct a comprehensive examination characterizing the nature and extent of physiological and
anatomical aberrations in young storks in relation to metal
contaminant loads in tissues. To this end, deformed juveniles
from Dehesa de Abajo (DdA) were compared with another
small group of storks of the same age collected from different breeding sites in Cáceres province, an area over 200 Km
away from the spill . Pathological plus other morphological and physiological endpoints were compared between the
groups of birds.
MATERIALS AND METHODS
Animals. In 2001, 10 nestling storks with obviously deformed tarsometatarsal (MT) bones were collected from the
DdA colony adjacent to the spill site just prior to reaching fledging age (birds ranged from 40–55 days old), and
were housed in the Acebuche wildlife recuperation center
(Doñ ana National Park, Huelva) for 3 months. Eleven storks,
also young of the year and apparently healthy (except they
had fallen from their nests or suffered some other misadventure) were collected from a wildlife recovery center “Sierra
de Fuentes” in the neighbouring area of Extremadura. Birds
were housed in groups of 5 or 6 colony mates, in 4 open
pens. Pens (4w × 6d × 3h m) were enclosed with solid mesh
442
SMITS ET AL.
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 Rı́o, province of Sevilla, 6◦ 10t 16tt
W: 37◦ 12t 33tt N), while the Cáceres 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 Doñ 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 Cáceres
rehabilitation center were not available.
Pathology. Storks were humanely euthanized by overdosing 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 seconds. 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, musculoskeletal system and viscera were examined, and the mass
of the liver, bursa of Fabricius, and spleen were recorded. Organ 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 evaluation of the bone sections was carried out by the pathologist unaware of the animals’ identification or origin. The
H&E-stained bone sections were examined microscopically
for evidence of pathology. After consultation with a colleague/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× magnification), 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
TOXICOLOGIC PATHOLOGY
bone was determined based upon the presence of an open,
haphazard pattern of newly formed bone with remaining collagen 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 distilled, 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 deionized water to 100 ml, and stored at 4◦ C until analysis. For the
bone samples, the sections were crushed with an agate pestle, ground and homogenized with corundum grinder, 0.1 g
of the powder was used for digestion following the procedure 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 Expansion, 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
Vol. 33, No. 4, 2005
LEG DEFORMITIES IN STORKS EXPOSED TO METALS
443
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 water
standard SLRS-4, a dogfish liver standard DOLT-2 and a dogfish muscle standard DORM-2 (National Research Council
of Canada, Ottawa, Canada).
Statistical analyses. Although we present the metal
residues in liver and kidney as well as bone to allow comparison with findings by other researchers, we limit all statistical analyses to metal levels in the bones. The rationale
for the latter is that all the birds had been on the same standard fish diet at the rehabilitation centre for 3 months, allowing differences that might have originally existed in the
liver and kidney to disappear. Bone metal concentrations
were assumed to be unchanged from the time the fledglings
were taken from their original colonies. Bone elements are
quite stable in full-sized animals compared with rapidly
growing nestlings (Scheuhammer, 1987 and references
therein).
The full biological impact of metals was unlikely to be
revealed by repeated testing of individual elements, as there
are numerous interactions and correlations among the metals
themselves. Therefore, we used principal component anal-
ysis (PCA) to derive fewer and independent variables. In 1
analysis we took this approach for 9elements in the bone
that we interpret as contaminants (Pb, Co, Cr, Ti, Zn, Sn,
V, Ba, Sr). For a second analysis we used 5 normal, physiologically important elements in the bone (Ca, P, Mg, Na, S).
For these PCAs, we chose elements that were consistently
detectable and that were, or could be transformed to be, normally distributed. The PCA of contaminants resulted in three
components accounting for 63.7% of the total variability:
PC1 explained 29.7%, and PC2 explained 21.7%, and PC3
explained 12.3% of the variance in the data set. Examination
of the relative loading of the variables on the 3 components,
and subsequent correlation analyses, yielded the following
interpretation. PC1 was largely an axis of V, Ba, Sr, and Cr
all varying positively. PC2 was Zn and Ti (positively), and
Co, Sn, and Cr (negatively). PC3 was largely influenced by
a positive relationship with Pb and Co. For the PCA of the
normal bone elements 2 components explained 91.0% of the
variance: (PC1 70.6%, PC2 20.4%). Ca, P, Mg, and Na were
all highly, and positively associated with PC1, whereas PC2
was primarily influenced by S.
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TOXICOLOGIC PATHOLOGY
SMITS ET AL.
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 Cáceres.
Tissue
Colony
Kidney DdA
Liver
Bone
Al ppm
2657
1110–7256
Caceres
3960
1959–11612
DdA
2109
942–33882
Caceres
4463
1107–11592
DdA
0
0–6.2
Caceres
0
0–76.5
Ti ppm
V ppb
Cr ppb
Co ppb
28426
494
1354
251
22542–33456 235–1420
2–2043
171–534
25528
383
1456
276
22417–33238 299–692
2–4054
166–512
27157
191
1528
101
24205–34954 81–316 1021–2137 65–158
26480
165
866
143
21353–30415 100–234 339–2767 71–185
185
704
811
127
123–273
130–1936
0–1473
0–277
196
243
743
190
109–229
162–1348 21–1233 121–327
We used ANOVA where nonsignificant interactions and
variables were removed iteratively, to obtain the most parsimonious model that explained variation in the dependent variable. Two-tailed tests were performed using SPSS (Norušis,
1993), and we considered results significant at the 0.05 level.
For the analysis of leg asymmetry, it was not valid to simply
compare birds on the basis of location since all deformed
birds came from the same location. However, it was clear that
individuals varied in the degree of leg deformity and so we
derived the variable, absolute leg asymmetry, i.e., difference
in length between the right and left metatarsi. In the ANOVAs
we thus used this variable, and location as a factor.
RESULTS
Metal contamination by location. Table 1 shows clearly
that neither birds from the reference location nor from the
Cu ppm
Zn ppm
As ppb
Se ppm
Sn ppb
Sr ppm
Pb ppb
17
111
1558
8.6
736
0.588
310
12–26 89–139 635–2950 5.0–10.7 348–1144 0.408–0.784 203–604
23
108
1664
6.6
774
0.472
587
12–26 71–126 666–3406 4.1–13.4 292–1231 0.363–0.798 83–1403
36
292
3236
7.4
555
0.191
93
21–51 223–339 1266–6072 5.6–9.0 244–1597 0.140–0.309 27–236
30
317
3375
5.3
781
0.147
264
19–88 176–445 1621–4697 3.1–8.7 286–2171 0.094–0.271 23–1031
0.003
55
0
0
239
229.2
882
0–1.2
0–94
0–104
81–439 159.2–305.1 0–2779
0–6.0
0
39
0
287
134.3
2008
0–0.8
0–185
0–338
142–341 68.5–236.2 0–12178
spill site were free of metal contamination. In fact, for some
of the metals, concentrations were higher in the “reference”
birds. For the PCA analysis of bone metals, location was not
significant for PC1 (F1,19 = 1.672, p = 0.211), marginally
nonsignificant for PC2 (F1,19 = 3.837, p = 0.065) and significant for PC3 (F1,19 = 5.436, p = 0.031) in which the difference was driven by the higher Pb in bones of the Cáceres
birds.
Gross pathology. Radiographic evidence of leg deformities are shown in Figure 3. The deformed, healed bones were
locally thicker with a triangular, rather than round, cross section. In all affected birds, the cortical bone just distal to the
tibiotarsal joint was abnormally thickened with callus formation and varying severity of valgus deviation of the distal
portion of the limbs. There was no radiographic evidence of
decreased cortical thickness or density, or of any generalized
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
445
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 Cáceres
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 Cáceres, 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).
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 deformities, as measured on a continuous scale of leg asymmetry, were associated with a number of other physiologically relevant variables. When the histological appearance
of the osteal/periosteal surface was smooth, leg asymmetry
was significantly less than if the birds had irregular bone
surfaces (F1,17 = 6.819, p = 0.018) (Figure 5). Considering the bone remodeling activity, the leg asymmetry increased significantly with increasing evidence of remodeling (F2,14 = 4.981, p = 0.023) (Figure 6). The presence and
density of cartilaginous islets in the proximal tarsometarsal
sections of bone was independent of the symmetry of the leg
bones (F1,15 = 0.787, p = 0.473).
In the ANOVAs, body mass and sex were never found to
be significantly related to leg asymmetry and so were always
dropped from models. Using asymmetry as the dependent
variable, both effect of location and levels of bone alkaline
phosphatase (BAP) levels in plasma (F1,17 = 4.483, p < 0.05)
FIGURE 5.—Subperiosteal bone surface was more irregular in birds with more
asymmetrical legs.
FIGURE 4.—Level of remodeling activity in tarsometatarsi related to asymmetry in bone length.
FIGURE 6.—The density of islets of cartilage in diaphyseal bone is related to
contaminant metal burdens in the bone.
were significantly different between DdA and Caceres birds.
For birds in both locales, increasingly asymmetrical legs had
decreasing amounts of the bone enzyme BAP (Figure 7).
We could not detect an association between asymmetry and
either PC1 (p > 0.13) or PC2 (p > 0.71) of the normal bone
minerals. Of the organs measured, only liver showed any
relationship with limb pathology. As liver somatic index (liver
size relative to body mass) increased, leg asymmetry also
increased (F1,17 = 8.805, p = 0.009), while again location
explained considerable variance (F1,17 = 14.088, p = 0.002).
The degree of leg asymmetry was also examined with respect to the 3 PCAs based on metal contaminants. Although
location was considered as well, it was dropped from the final
model, and PC3 remained significant (F1,18 = 10.385, p =
0.005). However, the relationship was unexpected, as bones
with low PC3 scores (i.e., driven by low Pb levels) were most
asymmetrical.
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SMITS ET AL.
FIGURE 7.—Serum levels of bone alkaline phosphatase (BAP) are higher in
storks with more symmetrical bones.
Body and organ mass. Independent ANOVAs were conducted for the dependent variables of liver, bursa and spleen
somatic indices, as well as body mass. The initial models
used sex, location, and the 3 PCA contaminant-independent
variables. For body mass, sex (F1,17 = 11.717, p = 0.003) and
PC3(F1,17 = 5.406, p = 0.033) were significant, where males
were heavier (as is known for this species), as were birds with
higher PC3 levels. For the liver somatic index, PC3 was again
a significant covariate (F1,18 = 5.102, p = 0.037). Neither the
splenic nor bursal indices were significantly associated with
the PCA for metals.
Normal bone elements. In the multivariate analysis of normal bone constituents, we considered length of right metatarsus as a potential covariate. There was no significant effect
of the three PCA contaminant variables on PC1 of the normal bone minerals. However, for PC2 of bone minerals, there
were significant effects of metatarsal length (F1,16 = 8.752,
p = 0.009), and PC1 of metal contaminants (F1,16 = 5.099,
p = 0.038). Because Ca and P are the 2 major elements comprising bone, they were examined individually in relation to
the PCA of contaminant metals. In ANOVAs we used bone Ca
(and subsequently P) as the dependent variable, and considered sex and location as factors. We considered body mass,
P (or subsequently Ca) in bone and the 3 components of
the PCA of contaminants as potential covariates. In the final
significant model, Ca was explained by body mass (F1,16 =
9.748, p = 0.007), P in bone (F1,16 = 369.785, p < 0.001), and
PC1 metals (F1,16 = 6.188, p = 0.024). Similar analysis of
bone P levels showed significant effects of body mass (F1,15 =
11.701, p = 0.004, Ca in bone (F1,15 = 441.029, p < 0.001),
location (F1,15 = 6.347, p = 0.024) and PC2 metals (F1,15 =
4.722, p = 0.046). In addition, PC1 proved, predictably, to
be significantly correlated with Ca:P ratio (rs = 0.644, p =
0.002), but PC2 and PC3 were nonsignificant.
DISCUSSION
In this study we examined physiological and anatomical
aberrations in juvenile storks in relation to metal residues
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 explore 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 the bones would have gone through
substantial changes during the nestling period, readily masking or correcting early defects in mineralization. Metals,
once bound in bone matrix, are considered non-mobilizable
(Klaassen, 2001). The metals were not different between the
birds from DdA and Cáceres, so it is possible that the rate
or timing of exposure to those metals would have played an
important role in expression of toxicity in young, quickly
developing birds. Maybe very young storks are fed on more
contaminated, smaller, filter-feeders so they get higher doses
of mud-associated contaminants early in life. Field observations indicate that one important prey item for DdA nestlings
is the red swamp crayfish Procambarus clarkii (Negro et al.,
2000), which can accumulate metals in their tissues. Crayfish
captured in the spill area in 2000 had metal levels exceeding
that allowed for human consumption (Sánchez-López et al.,
2003).
The limb deformities were originally suspected to be due
to toxicity by metals associated with the spill, that could directly disturb bone formation. Cadmium and Pb as well as Al
are known to interfere with bone turnover and Ca metabolism
(Goyer et al., 1994; Berglund et al., 2000). As it turned out, the
levels of toxic metals were often lower in the affected birds
than those from the reference site distant from the tailings accident. The Pb in tissues from the DdA storks was confirmed
as coming from the Aznalcollar mine spill through isotopic
identification (Meharg et al., 2002). Of the metals used for
PCA analyses, only Pb, Co, and Zn were identified in the mud
slurry. The remaining metals included in the analyses (V, Ba,
Sr, Cr, Ti) were identified as coming from the natural river
sediments unaffected by the spill (Alastuey et al., 1999).
By using multivariate analyses, relationships between
toxic metal concentrations and essential bone components
became evident. Using a PCA allowed us to recognize physiological costs associated with exposure to a complex mixture
of metals in an acid matrix. Sulfur levels in the bones had a
significant relationship with contaminant metals possibly implying 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
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LEG DEFORMITIES IN STORKS EXPOSED TO METALS
associated with metal concentrations, and which was different 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 attempt 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 facilitate 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 contributors 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 extensive biotransformation in all forms of life, with very species
specific routes and rates of metabolism (Ecobichon, 2001).
Organophosphorous (OP) esters stimulate the cytochrome
447
P450 isoenzyme system. The normal role of P450 enzymes is
to enhance eventual excretion of metabolic breakdown products. 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 metalothionine 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, blocking 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. Garcı́a, J. M. Terrero, H.
Lefranc, and R. Rodrı́guez for their able field assistance.
J.L.Tella and the Ayuntamiento de Puebla del Rı́o provided
logistic support and access to the DdA stork colony. We thank
Consejerı́a de Medio Ambiente—Junta de Andalucı́a for providing 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 Extremadura) provided the reference birds. The wildlife/stork
monitoring program (16/98) was financed by CSIC. This
work was supported by fellowships from the Spanish Ministerio de Educació 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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