Biomonitoring of heavy metals (Cd, Hg, and Pb) and metalloid (As) with the Portuguese common buzzard (Buteo buteo)

Abstract

The accumulation of heavy metals in the environment may have a wide range of health effects on animals and humans. Thus, in this study, the concentrations of arsenic (As), cadmium (Cd), lead (Pb), and mercury (Hg) in the blood and tissues (liver and kidney) of Portuguese common buzzards (Buteo buteo) were determined by inductively coupled plasma-mass spectrometer (ICP-MS) in order to monitor environmental pollution to these elements. In general, Hg and As were the elements which appeared in the highest and lowest concentrations, respectively. A highest percentage of non-detected concentration was found for blood Cd (94.6 %) but, in turn, it was the only metal that was detected in all kidney samples. The kidney was the analyzed sample which showed the highest concentrations of each element evaluated. Statistically, significant differences among blood, liver, and kidney samples were observed for As and Cd (P < 0.05). Cd concentrations in kidney and liver varied significantly with age: Adults showed higher hepatic and renal Cd concentrations than juveniles. Blood Pb concentration seems to show an association with the hunting season. Although raptors are at the top of the food chain and are thus potentially exposed to any biomagnification processes that may occur in a food web, the individuals evaluated in this study generally had low levels of heavy metals in blood and tissues. However, chronic exposure to these metals was verified. The results presented here lend weight to arguments in favor of continuous biomonitoring of metals and metalloids, since heavy metals may accumulate to levels that will pose a risk to both human health and the environment.

Full text

Biomonitoring of heavy metals (Cd, Hg, and Pb) and metalloid
(As) with the Portuguese common buzzard (Buteo buteo)
Manuela Carneiro &Bruno Colaço &Ricardo Brandão &
Carla Ferreira &Nuno Santos &Vanessa Soeiro &Aura Colaço &
Maria João Pires &Paula A. Oliveira &Santiago Lavín
Received: 21 November 2013 /Accepted: 30 June 2014
#Springer International Publishing Switzerland 2014
Abstract The accumulation of heavy metals in the
environment may have a wide range of health effects
on animals and humans. Thus, in this study, the concen-
trations of arsenic (As), cadmium (Cd), lead (Pb), and
mercury (Hg) in the blood and tissues (liver and kidney)
of Portuguese common buzzards (Buteo buteo)were
determined by inductively coupled plasma-mass spec-
trometer (ICP-MS) in order to monitor environmental
pollution to these elements. In general, Hg and As were
the elements which appeared in the highest and lowest
concentrations, respectively. A highest percentage of
non-detected concentration was found for blood Cd
(94.6 %) but, in turn, it was the only metal that was
detected in all kidney samples. The kidney was the
analyzed sample which showed the highest concentra-
tions of each element evaluated. Statistically, significant
differences among blood, liver, and kidney samples
were observed for As and Cd (P<0.05). Cd concentra-
tions in kidney and liver varied significantly with age:
Adults showed higher hepatic and renal Cd concentra-
tions than juveniles. Blood Pb concentration seems to
show an association with the hunting season. Although
raptors are at the top of the food chain and are thus
potentially exposed to any biomagnification processes
Environ Monit Assess
DOI 10.1007/s10661-014-3906-3
M. Carneiro :A. Colaço
Animal and Veterinary Research Centre (CECAV), University
of Trás-os-Montes and Alto Douro, 5000-801 Vila Real,
Portugal
B. Colaço
Department of Zootechnics, ECAV, University of
Trás-os-Montes e Alto Douro, Quinta dos Prados,
5000-801 Vila Real, Portugal
B. Colaço :M. J. Pires :P. A . O l i v e i r a
Centre for the Research and Technology of
Agro-Environmental and Biological Sciences, University of
Trás-os-Montes e Alto Douro, Quinta dos Prados,
5000-801 Vila Real, Portugal
R. Brandão
Ecology, Monitoring and Recovery Center of Wild Animals
(CERVAS), 6290-909 Gouveia, Portugal
C. Ferreira
Research and Recovery Center of Wild Animals of the Ria
Formosa Natural Park (RIAS), 8700 Olhão, Portugal
N. Santos
Wildlife Rehabilitation Center of the Peneda-Gerês National
Park, 4704-538 Braga, Portugal
V. S o e i ro
Wildlife Rehabilitation Center of the Gaia Biological Park,
4430-757 Avintes, Portugal
A. Colaço :M. J. Pires :P. A. Oliveira (*)
Department of Veterinary Sciences, ECAV, University of
Trás-os-Montes e Alto Douro, Quinta dos Prados,
5000-801 Vila Real, Portugal
e-mail: pamo@utad.pt
S. Lavín
Servei d’Ecopatologia de Fauna Salvatge (SEFaS),
Department of Medicine and Animal Surgery, Autonomous
University of Barcelona, 08193 Bellaterra, Spain

that may occur in a food web, the individuals evaluated
in this study generally had low levels of heavy
metals in blood and tissues. However, chronic
exposure to these metals was verified. The results
presented here lend weight to arguments in favor
of continuous biomonitoring of metals and metal-
loids, since heavy metals may accumulate to levels
that will pose a risk to both human health and the
environment.
Keywords Buteo buteo .Common buzzard .Heavy
metals .Metalloid .Raptors .Portugal
Introduction
Trace elements are present in the environment through
the geological cycle and various anthropogenic activi-
ties, with the latter the most relevant (Licata et al. 2010;
Naccari et al. 2009). These elements can easily enter the
food chain, and at high doses they can be acutely lethal,
while at lower doses they may have a wide range of
health effects—such as mutagenicity, carcinogenici-
ty, teratogenicity, inmunosuppression, poor body
condition, and impaired reproduction in humans and
animals (Florea and Busselberg 2006; Scheuhammer
1987). All of which make them a serious threat to the
stability of ecosystems and living organisms (Battaglia
et al. 2005; López-Alonso et al. 2007; Naccari et al.
2009).
Levels of trace elements and their effects on organ-
isms are influenced by numerous factors related to hab-
itat, physiology, and life history (Peakall and Burger
2003). Exposure to nonessential elements generally is
below levels thought to be acutely toxic (Henny et al.
1995; Stout and Trust 2002; Wayland et al. 2001b).
Although acute toxicity is unlikely to occur, chronic
exposure to nonessential elements may interact with
other environmental stressors namely parasitic infec-
tions or other pathogens and this could compromise
bird’s survival and reproduction (Wayland et al.
2008). It should be appreciated that potentially sub-
lethal effects caused by chronic exposure to envi-
ronmental contaminants are largely unknown in wild
birds (Shlosberg et al. 2011).
Biomonitoring of trace elements in the environment
has enabled the identification of many sources of pol-
lutants. Typically, the bioavailability of environmental
pollutants is assessed by measuring chemical residues in
tissues or fluids taken from animals living in specific
habitats (López-Alonso et al. 2007). The direct measure-
ment of contaminants in blood and internal tissues is the
best indicator of the degree and type of exposure to them
(García-Fernández et al. 1997), presenting blood the
advantage of being easily accessible, sampling can be
relatively harmless, and it is in contact with all tissues
where chemicals are deposited and stored (Esteban and
Castano 2009). Some species have biological habits that
increase the likelihood of exposure to contaminants and
can produce relevant information that would be missed
if only the areas water or soil were analyzed (Battaglia
et al. 2005). However, assessing an ecosystem’shealth
adequately by means of biomonitoring requires the
selection of species that are representative. Territorial
birds of prey, ones that are non-migratory and have
long life spans, are likely to reflect chemical contam-
ination within their extended home ranges (Pérez-
López et al. 2008). These localised, upper-trophic
level species are also believed to be especially vul-
nerable to metals and play a very important role as
environmental-contamination indicators (Stout and
Trust 2002; Wayland et al. 1999).
The common buzzard (Buteo buteo), a diurnal bird of
prey belonging to the order Accipitriformes and to the
family Accipitridae, was the sentinel species selected for
this study for several reasons: it is abundant within
Portuguese territory and the Portuguese population of
these birds is essentially resident, though in autumn and
winter a comparatively small number of common buz-
zards from northern Europe do reach the Iberian Penin-
sula. What is more, these birds are very territorial, are
present in different habitats (forests, agricultural zones,
mountain regions, and sub-urban areas), they feed on a
wide range of prey, and are very opportunistic hunters
(Catry et al. 2010).
The present study was carried out in order to evaluate
the degree and type of exposure to trace elements that
the Portuguese common buzzards may be exposed and
to monitor environmental pollution. For this purpose,
we determine the arsenic (As), cadmium (Cd), lead (Pb),
and mercury (Hg) concentrations in whole blood,
liver, and kidney samples taken from common buz-
zards from different areas of Portugal. Also, differ-
ences between their areas of origin and the influence
of age and gender on toxic-metal concentrations
were studied. The influence of the season the sam-
ples were taken in, on blood-metal concentrations,
was also investigated.
Environ Monit Assess

Material and methods
Sample collection
All samples collected from common buzzards came
from five Portuguese wildlife rehabilitation centers
(Centro de Ecologia,Recuperação e Vigilância de
Animais Selvagens (CERVAS); Centro de Recuperação
de Animais Selvagens do Parque Nacional da Peneda
do Gerês;Centro de Recuperação de Animais Selvagens
do Parque Biológico de Gaia;Centro de Recuperação e
Investigação de Animais Selvagens da Ria Formosa
(RIAS); and Centro de Estudos e Recuperação de
Animais Selvagens de Castelo Branco (CERAS)) be-
tween November 2007 and January 2012.
Collected animals were either found dead or brought
to the centers alive but injured or debilitated due to
several potential reasons. These reasons were as fol-
lows: collision with a vehicle (n=18), collision with
power lines (n=17), lead shot (n=22), fall from nest
(n=14), malnutrition (n=3), and injury of unknown
origin (n=51). The following data were registered for
each bird: date of arrival at the center, area of origin,
reason for being brought in, gender (male, female, or
unknown), and age (juvenile, adult, or unknown). The
birds’origin was divided into two different Portuguese
regions: littoral (urban and industrial areas) and up-
country (rural and natural areas).
From a total of 125 common buzzards, blood (n=93),
liver (n=56), and kidney (n=36) samples were collect-
ed. The number of samples collected across different
years, seasons, regions, and for different gender and age
classes were listed in Table 1. Blood samples were
collected via the brachial vein at the moment of arrival
to the rehabilitation center and immediately transferred
into collection tubes without the use of an anticoagulant.
Liver and kidney samples were collected from animals
that were found dead, died of natural causes, or were
sacrificed when their state of health indicated a potential
recovery was unlikely. Liver and kidney samples were
placed individually in plastic bags. In this study, only
samples from animals that died in the rehabilitation
center within the first month after admission were in-
cluded. All samples were stored at −20ºC until analysis.
Analytical procedure
Sample analysis was performed in the laboratories of the
Scientific-Technical Services of the University of
Barcelona (SCT-UB), Spain. Liver and kidney sub-
samples [250–350 mg wet weight (w.w.)] were digested
in Teflon reactors with 3 ml of 65 % concentrated nitric
acid (HNO
3
) and 2 ml of 30 % hydrogen peroxide
(H
2
O
2
) at 90ºC in an oven and left overnight. According
to the volume of blood contained in the tubes, different
amounts of HNO
3
and H
2
O
2
were used until the diges-
tion of blood samples was complete. After digestion,
each liver and kidney sample was brought up to a
volume of 40 ml with milli-Q water and according to
the blood volume to be analyzed, blood samples were
brought up to a volume of 20, 30, or 40 ml with milli-Q
water. All samples were transferred to the measuring
vessel and then analyzed for As, Cd, Hg, and Pb in an
inductively coupled plasma-mass spectrometer (ICP-
MS) (Perkin Elmer Model Elan 6000, Perkin Elmer,
Waltham, USA). All material used in the digestion
process was thoroughly acid-rinsed. A second set of
identical liver and kidney samples (1–2 g) was oven-
dried at 60ºC until reaching a constant weight in order to
calculate the percentage of humidity in each sample,
which enabled the transformation of wet-weight results
into dry-weight (d.w.) values (Ribeiro et al. 2009).
Tabl e 1 Data relating to common buzzards (Buteo buteo)ofthis
study
Blood
(n=93)
Liver
(n=56)
Kidney
(n=36)
Year 2007 5 0 0
2008 8 6 0
2009 24 19 5
2010 33 24 24
2011 22 7 7
2012 1 0 0
Season Spring 15 11 6
Summer27157
Autumn 34 17 15
Winter 17 13 8
Origin Littoral 47 28 24
Up-country 46 28 12
Age Adult 30 19 16
Juvenile 44 24 9
Unknown191311
SexFemale21158
Male 35 26 19
Unknown27159
Environ Monit Assess

An analytical quality-control program was applied
throughout the study, according to López-Alonso et al.
(2007). Blank absorbance values were monitored
throughout the survey and were subtracted from the
readings before the results were calculated. The limits
of detection (LOD) in the acid digest (set at three times
the standard deviation of the reagent blanks) were in all
cases <0.5 μg/l, and the limits of quantification (LOQ),
expressed as a concentration in the blood and tissue,
were calculated on the basis of the mean sample weight
and volume analyzed. Analytical recoveries were deter-
mined from the certified standard reference materials
(Whole-blood Seronorm, Trace Element, Whole Blood
2 ref. 201605, and Whole Blood 3 ref. 102405 from
SERO AS, Norway and Bovine Liver-1577b from Na-
tional Institute of Standards and Technology, Gaithers-
burg, USA) analyzed together with the samples. The
range of recovery rates (in view of the concentrations in
the reference material) ranged between 93.67 % for
hepatic Pb and 138.43 % for blood As.
Data analysis
Statistical analyses were performed with IBM SPSS
Statistics for Windows, V.19.0.
A statistical significance level of P<0.05 was used
for null hypothesis rejection.
Each sample below the LOQ was assigned a value of
one-half the LOQ and included in the data set for statis-
tical treatment, a technique which minimizes nominal
type I error rates (Clarke 1998).
Normal-distribution assumption was checked
using the Kolmogorov–Smirnov test. When normal
distribution assumption was violated, the data sets
were log-transformed before analysis and checked
with the Kolmogorov–Smirnov test. However, most
of the variables did not follow normal distribution
even after transformation, so a non-parametric ap-
proach to the data analysis was necessary. The
Mann–Whitney Utest was used to test the statistical
significance for area of origin, age, and gender in
the blood and tissues concentrations. Birds with
unknown age and gender were not included in the
statistical analysis when testing the significance for
these variables. Comparisons across the different
seasons in terms of blood concentrations and differences
between metal concentrations in the blood, liver, and
kidney samples were tested using the Kruskal–Wallis test
followed by the Dunn’s post-hoc test. A non-parametric
Spearman`s test was applied to test the correlation be-
tween blood and tissues and between tissue concentra-
tions for each analyzed metal.
Results
Heavy metals and metalloid concentrations found in the
blood, liver, and kidney of common buzzards are listed
in Table 2.
Concerning As concentrations, blood As was not
detected in 30.1 % of total samples, in the liver and
kidney samples it was not detected in 37.5 % and
19.4 %, respectively (Table 2). Mean As concentrations
were significantly statistically different between ani-
mals’blood, liver, and kidney (P<0.01): the lowest
mean concentration was found in blood (0.014±
0.014 μg/g w.w) and the highest in the kidney (0.041±
0.026 μg/g w.w.) (Table 2and Fig. 1). As concentrations
in kidney samples varied significantly with age and
gender: Adults showed higher (P<0.05) renal-As con-
centrations than juveniles and females showed renal-As
concentrations that were nearly twice as high as average
concentrations in males (P<0.05). No significant influ-
ence from any factor was detected in blood- and hepatic-
As concentrations (Table 3).
When considering Cd results, this was not detected in
94.6 % of total blood samples, and for that reason, the
influence of age, gender, origin, and season on concen-
trations of this metal in blood was not studied. In con-
trast, Cd was detected in 96.4 % of liver samples and in
all the kidney samples (Table 2). Mean Cd concentra-
tions were significantly (nearly four times) higher
(P<0.05) in the kidney (0.373±0.381 μg/g w.w.) than
in the liver (0.089±0.097 μg/g w.w.) (Fig. 1). Cd con-
centrations in tissues varied significantly with age:
Adults showed higher hepatic- (twice as high, P<0.01)
and renal- (three times as high, P<0.05) Cd concentra-
tions than juveniles. No significant influence of origin
and gender in hepatic and renal levels were detected for
Cd accumulation (Table 3).
In this study, Pb was detected in most of the samples:
97.8 % (blood), 87.5 % (liver), and 94.4 % (kidney)
(Table 2). Mean Pb concentrations were very similar
between blood (0.142±0.628 μg/g w.w.) and liver
(0.152± 0.194 μg/g w.w.). In the kidney, the highest
mean Pb concentration (0.245±0.364 μg/g w.w.) was
found, but this difference was not statistically significant
(Fig. 1). Blood Pb was significantly affected by age
Environ Monit Assess

(P<0.01), adults had higher concentrations than juve-
niles, and by season (P<0.01), blood samples collected
in autumn had higher Pb concentrations than those
collected in spring and summer. Hepatic and renal Pb
concentrations were not significantly affected by age,
gender, and origin (Table 3).
Turning to Hg results, this metal was also detected in
most of the samples: 88.2 % (blood), 94.6 % (liver), and
97.2 % (kidney) (Table 2). Blood Hg concentrations
were significantly lower than hepatic and renal Hg
(P<0.001). Hg accumulation was mainly in the kidney,
although there is no significant difference between he-
patic and renal concentrations (Fig. 1). Blood Hg con-
centrations were significantly higher (P<0.01) in adults
than in juveniles. The season the samples were taken in
also had significant effects on blood Hg concentrations
(P<0.001): Higher levels were found in autumn and
winter than in spring and summer, but significant dif-
ferences were only verified between autumn and spring
and autumn and summer. Hepatic and renal Hg was not
significantly affected by any of the variation factors
considered in this study (Table 3).
The existence of a statistical relationship between
metal concentrations in blood and in the different tissues
was studied by means of simple correlation coefficients
(Table 4). The relationship between Cd contents in
blood and tissues was not studied, since it was barely
detected in blood. The highest correlation was ob-
served between hepatic and renal Hg concentrations
(R=0.946, P<0.001).
Tabl e 2 Arsenic (As), cadmium (Cd), lead (Pb), and mercury (Hg) concentrations in the blood, liver, and kidney of common buzzards from
Portugal analyzed in the present study
Blood (n=93) Liver (n=56) Kidney (n=36)
μg/dl μg/g w.w. μg/g d.w. μg/g w.w. μ/g d.w. μg/g w.w
As
Mean±S.D 1.489± 1.457 0.014± 0.014 0.104± 0.136 0.029± 0.039 0.180± 0.133 0.041± 0.026
Median 1.391 0.013 0.074 0.022 0.149 0.036
MinimumND NDNDNDNDND
Maximum 8.508 0.082 0.978 0.281 0.588 0.112
n<LOQ/% 28/30.1 % 21/37.5 % 7/19.4 %
Cd
Mean±S.D 0.201± 0.567 0.002± 0.005 0.322± 0.361 0.089± 0.097 1.553± 1.706 0.373± 0.381
Median 0.102 0.001 0.184 0.050 0.865 0.216
Minimum ND ND ND ND 0.033 0.009
Maximum 4.447 0.043 1.801 0.450 8.344 1.697
n<LOQ/% 88/94.6 % 2/3.6 % 0
Pb
Mean±S.D 14.711±65.156 0.142± 0.628 0.541± 0.687 0.152± 0.194 0.945± 1.356 0.245± 0.364
Median 5.864 0.056 0.284 0.079 0.443 0.102
MinimumND NDNDNDNDND
Maximum 631.473 6.089 3.468 0.949 5.331 1.386
n<LOQ/% 2/2.2 % 7/12.5 % 2/5.6 %
Hg
Mean±S.D 20.940± 26.728 0.202± 0.258 1.387± 1.242 0.389± 0.346 2.086± 1.689 0.503± 0.310
Median 12.603 0.121 1.168 0.319 1.850 0.448
MinimumND NDNDNDNDND
Maximum 164.895 1.590 5.314 1.479 5.945 1.482
n<LOQ/% 11/11.8 % 3/5.4 % 1/2.8 %
S.D. standard deviation, nnumber of samples, LOQ limit of quantification, ND non-detected
Environ Monit Assess

Discussion
Previous data on the concentrations of nonessential
elements in the blood and tissues of common buzzard
are scarce. In fact, this study reports the first data on the
concentrations of trace elements (As, Cd, Hg, and Pb) in
blood and tissues of raptors residing in Portugal.
The common buzzards, being birds of prey, are at the
top of the food chain and consequently are potentially
exposed to any biomagnification processes that may
occur in a food web (Gochfeld and Burger 1987). In
the common buzzard, concentrations of toxic elements
reported in the literature were usually determined in the
liver and kidney (Battaglia et al. 2005; Castro et al.
2011; Naccari et al. 2009; Pérez-López et al. 2008).
Concentrations of heavy metals in the liver and kidney
can be considered suggestive of chronic exposure to
metals while concentrations in blood reflect recent
exposure and thus can be used as a real-time mon-
itor for all stages of the birds’life-cycles. Better
understanding of the relationship between heavy-metal
concentrations in tissues and blood would enable the
researchers to assess whether high concentrations in
blood warrant concern regarding their toxic effects,
without having to use lethal sampling techniques
(Burger and Gochfeld 1997;Castroetal.2011;
Naccari et al. 2009; Wayland et al. 2001a).
There have been a few studies on the transfer of As
through terrestrial food chains to predatory birds and on
the presence of such metalloid in the raptor tissues, but it
is known that vertebrate top predators experiencing
higher As burdens (Erry et al. 1999; Lebedeva 1997;
Lucia et al. 2012b). In fact, published data on arsenic
levels in common buzzards is sparse (Naccari et al.
2009; Pérez-López et al. 2008), and this study reports
the first data on blood As concentrations in this species,
so it is difficult to compare our results with other studies.
However, other species of birds sampled in natural areas
(Burger and Gochfeld 1997) and near potential sources
of metals (Baos et al. 2006a,b; Blanco et al. 2003)
showed similar and higher blood As concentrations,
respectively. With respect to As concentrations in the
liver and kidney, our results are much lower than those
observed for the common buzzard in Galicia, Spain
(Pérez-López et al. 2008) and in Sicily, Italy (Naccari
et al. 2009) where the exposure to As was considered of
no toxicological concern. However, we obtained similar
or slightly lower concentrations than those obtained by
Erry et al. (1999)inkestrels(Falco tinnunculus)from
Britain, which have similar feeding habits to common
buzzards. Erry et al. (1999) also measured hepatic and
renal As concentrations in barn owls (Tyto alba)that
despite having similar feeding habits with kestrels and
common buzzards, presented lower As concentrations
Fig. 1 Bar chart showing the
toxic metal concentrations
(expressed as arithmetic means,
μg/g wet weight) in the blood,
liver, and kidney in the 125
common buzzards considered in
this study. *P<0.05, **P<0.01,
***P<0.001
Environ Monit Assess

than those obtained in these two species. Taking into
account this previous information, the study of common
buzzards in Portugal showed a low level of exposure to
this metalloid. However, the significantly higher As
levels found in kidney samples of adult birds suggest
that As is being bioconcentrated over time. This mean
that common buzzards face chronic exposure to this
metalloid (Eisler 1998). Despite the As burdens detected
in common buzzards were not likely to have been asso-
ciated with adverse health effects (this element could not
be considered as a threat for the survival of the studied
birds), it could be involved in sublethal effects (Lucia
et al. 2012a,b).
Cd has been described as one of the most dangerous
trace elements from environmental and toxicological
standpoints, both for humans and animals, not only for
its high toxicity but also for its persistence (Battaglia
et al. 2005; García-Fernández et al. 1996;Licataetal.
2010). Cd distribution and bioaccumulation patterns
observed in our study are consistent with the previous
studies in the common buzzard (Battaglia et al. 2005;
Castro et al. 2011; García-Fernández et al. 1996;Jager
et al. 1996; Naccari et al. 2009). The kidney was the
organ with the highest concentration. In contrast, Cd in
Tabl e 3 Heavy metals in adults and juveniles birds, females and male birds, from littoral and up-country birds and in blood samples
collected in the different seasons expressed as mean value (M.V.) ±standard deviation (S.D.) μg/dl in blood and μg/gd.w.intissues
Sample As Cd Pb Hg
M.V±S.D. M.V ±S.D. M.V. ±S.D. M.V.±S.D.
Blood (μg/dl) Age Adult 1.578± 1.178 −9.865± 8.302
a
20.853±13.770
b
Juvenile 1.451±1.647 −5.704±7.534
a
14.856±20.964
b
Gender Female 1.698± 1.188 −5.645± 8.000 14.688± 14.085
Male 1.662±1.734 −6.291±5.692 22.642± 26.565
Origin Littoral 1.389±1.088 −20.852 ± 91.300 27.605 ±34.075
Up-country 1.593±1.769 −8.436±8.860 14.131±13.409
Season Spring 2.267±2.220 −5.413±6.493
c
11.871±15.363
d
Summer 1.297±1.249 −6.310±9.278
c
8.191±9.571
d
Autumn 1.438±1.271 −9.608± 7.353
c
30.268±28.510
d
Winter 1.205± 1.265 −9.899± 8.560 29.429±38.931
Liver (μg/g d.w.) Age Adult 0.104± 0.055 0.460± 0.454
e
0.443±0.433 1.481±1.330
Juvenile 0.118± 0.192 0.209± 0.278
e
0.441±0.618 1.130±1.132
Gender Female 0.096± 0.048 0.199± 0.196 0.323± 0.348 1.364±1.341
Male 0.083±0.058 0.342±0.405 0.508± 0.555 1.415±1.385
Origin Littoral 0.077±0.044 0.273±0.359 0.484± 0.519 1.618±1.489
Up-country 0.132± 0.184 0.371± 0.364 0.596± 0.829 1.155±0.903
Kidney (μg/g d.w.) Age Adult 0.217± 0.139
f
2.165±2.162
g
0.828±1.326 1.895±1.725
Juvenile 0.139± 0.068
f
0.698±0.975
g
0.822±1.558 1.816±1.331
Gender Female 0.245± 0.106
h
2.056±1.793 0.706±0.762 2.407± 1.705
Male 0.166±0.131
h
1.496±1.990 0.892±1.533 1.745± 1.720
Origin Littoral 0.150±0.092 1.620±1.972 0.874± 1.207 2.455±1.871
Up-country 0.241± 0.179 1.421± 1.048 1.087± 1.665 1.348±0.928
a
(Z=−2.940, P<0.003),
b
(Z=−3.164, P<0.002),
c
(H=11.639, P<0.008),
d
(H=24.190, P<0.001),
e
(Z=−2.641, P<0.008),
f
(Z=−2.040, P<0.043),
g
(Z=−1.981, P<0.048),
h
(Z=−2.447, P<0.013)
Tabl e 4 Correlations coefficients (R) between blood and tissues
and between tissues for each element in common buzzard
Blood*Liver
(n=24)
Blood*Kidney
(n=15)
Liver*Kidney
(n=36)
As R=0.572** R=0.530* R=0.886***
Cd −− R=0.831***
Pb R=0.859*** R=0.875*** R=0.811***
Hg R=0.857*** R=0.832*** R=0.946***
*
P<0.05,
**
P<0.01,
***
P<0.001
Environ Monit Assess

the blood was not detected in 94.6 % of blood samples,
which reflect its low concentrations in blood and/or may
be associated with the detection limit of our analytical
method. In the blood samples where Cd was detected,
the mean concentration was higher than that obtained by
García-Fernández et al. (1996) in samples of whole
blood from wild birds. Comparison of the Cd concen-
trations in tissues obtained in this study with those
obtained in other recent studies with the same species
reveals that our results differ depending on the geo-
graphical area: similar with the results obtained in Italy
by Alleva et al. (2006), Battaglia et al. (2005), and
Naccari et al. (2009), lower than those observed in
Galicia, Spain by Pérez-López et al. (2008) and Castro
et al. (2011), and higher than the results obtained by
García-Fernández et al. (1995) in Murcia, Spain. Ac-
cording to Scheuhammer (1987), the hepatic and renal-
Cd concentrations obtained in our study are indicative of
a prolonged exposure to low and background amounts
of this metal.
Pb is a highly toxic heavy metal that acts as a non-
specific poison, affecting all body systems (Hernández
and Margalida 2009). Blood-Pb concentrations obtained
in this study were slightly higher than those obtained by
García-Fernández et al. (1995) and García-Fernández
et al. (1997) in common buzzards from south-eastern
Spain. According to Franson (1996), 90.3 % of the
common buzzards analyzed in our study had blood-Pb
concentrations compatible with an absence of abnormal
Pb exposure, 8.6 % had Pb levels indicative of subclin-
ical exposure, and only 1.1 % had a potentially lethal
blood-Pb concentration. Taking hepatic-Pb concentra-
tions into consideration, several researchers determined
higher concentrations in the same species than those we
quantified (Alleva et al. 2006; Battaglia et al. 2005;
Jager et al. 1996; Licata et al. 2010; Naccari et al.
2009; Pain et al. 1995; Pérez-López et al. 2008). In
contrast, García-Fernández et al. (1995) and García-
Fernández et al. (1997) quantified lower hepatic- and
renal-Pb concentrations. According to the ranges
established by Pain et al. (1995), none of the studied
animals exceeded the calculated dry weight threshold
for massive exposure and most of them had very low
hepatic and renal Pb concentrations of <2 μg/g, with
many <1 μg/g d.w., indicating a safe environmental
exposure. These results suggest that common buzzards
in Portugal are exposed to relatively low levels of Pb.
However, some studies provide evidence that low-level
Pb exposure, although not causing the clinical
symptoms of classical Pb poisoning, may nevertheless
have subtle detrimental effects on normal behavior and
cognitive function (Burger and Gochfeld 2000;
Scheuhammer 1987), while Pb poisoning has been rec-
ognized as one of the most significant causes of mortal-
ity in raptors (Pain et al. 2005).
There are very few studies on Hg concentrations in
birds of prey and, as far as we know, this is the first
report on Hg blood concentrations in the common buz-
zard and, in Portugal, the first in raptors. Tartu et al.
(2013) and Goutte et al. (2014) evaluated the effect of
Hg in seabirds predators, and they conclude that Hg
exposure could affect the ability of modulate their re-
productive effort. As threshold-effects levels for Hg
have yet to be established for bird blood, it is unclear
whether Hg levels were high enough to pose a risk to
any of these birds. Considering the hepatic and renal Hg
concentrations, other authors have quantified higher Hg
concentrations in the common buzzard (Alleva et al.
2006;Castroetal.2011). Hg concentrations observed
in our birds suggested that a source of Hg does exist.
According to the previous information and to
Scheuhammer (1987), the common buzzards studied
are chronically exposed to normal background levels.
It is perhaps because Portugal is a small country (with
a maximum extension in length of 561 and 218 Km in
width) that we did not observe significant differences
between areas of origin in the concentrations of various
elements analyzed.
Age was the only factor explaining Cd accumulation
in both the liver and kidney, as observed in other studies
with wild birds (García-Fernández et al. 1996;Naccari
et al. 2009; Ribeiro et al. 2009). With continued expo-
sure, even at low levels, this nonessential element is
accumulated throughout the life span of birds, due to
its extremely long biological half-life once bound to
metallothionein in tissues and its slow elimination from
these tissues (Scheuhammer 1987;Furness1996). Age
also had an influence on blood Pb and Hg concentra-
tions, but this influence was not verified in hepatic and
renal concentrations. Differences in blood Hg levels
between age classes seem to be related with prey-size
selection and stage of juvenile feather moult (Evers et al.
2005). Knowing that blood Hg is strongly influenced by
dietary uptake, these age-related differences could be
due to adults and young eat different foods or eat dif-
ferent proportions of the same foods (Burger and
Gochfeld 1997). Apart from the dietary intake, the Hg
concentration in blood reflects physiological influences,
Environ Monit Assess

such as mobilization and storage in feathers and eggs
(Dauwe et al. 2000,2003; Honda et al. 1986). The
amount of Hg eliminated into eggs is usually small
compared to the amount transferred into feathers during
the moult (Furness 1993). Feather moult and growth is
the main Hg excretion pathway (Braune and Gaskin
1987; Honda et al. 1985; Monteiro and Furness 2001).
The ability to rapidly transfer blood Hg into growing
feathers partly accounts for the significant difference in
blood Hg levels between adults and juveniles prior to
fledging (Evers et al. 2005). Although the Pb excretion
into growing feathers occurs to a lesser extent compared
with Hg (Dmowski 1999; Furness 1993), the differences
between adults and juveniles in blood Pb concentrations
could also be related to the stage of juvenile feather
moult and growth. Once blood Pb concentrations reflect
immediate dietary intake (Furness 1993), the differences
for blood Pb concentrations between adults and juve-
niles could also be explained by the considerations of
feeding behavior (Burger and Gochfeld 1997).
Only Pb- and Hg-blood concentrations were influ-
enced by season. Pb poisoning in raptors is likely to be
more significant in autumn and winter, since the propor-
tion of carrion taken by certain species may be higher in
these seasons. In addition, since waterfowl and other
game species are generally hunted during autumn and
winter, killed, crippled, and poisoned individuals pro-
vide a readily available Pb-contaminated food source
(Pain and Amiard-Triquet 1993). Common buzzards
often act as scavengers and, in this way, are more likely
to be exposed to the lead shot prevalent in small game
species (Battaglia et al. 2005). This fact could help to
understand the generally higher blood Pb concentration
quantified in autumn and winter. In the case of Hg,
possible explanations for the significant differences
found between the different seasons are migration, diet
(Eisler 1987), and moult. It is during the moulting that
Hg is incorporated in the keratin structure of the
feathers, thus reducing the Hg levels in blood (Braune
and Gaskin 1987; Honda et al. 1986; Monteiro and
Furness 2001).
Regarding correlations, we found that Pb- and Hg-
blood concentrations were statistically related to their
corresponding concentrations in the liver or kidney—
which suggests that blood concentrations of these metals
may be a useful indicator of the degree of recent expo-
sure. García-Fernández et al. (1996) showed that blood-
Cd concentration may also be a useful indicator of the
degree of exposure to this metal. In this study, due to the
large number of samples in which it was not detected, it
was not possible to show whether blood could be a
useful indicator of the degree of exposure to Cd. Kidney
samples could be used to assess chronic exposure to As,
Cd, Pb, and Hg, a working hypothesis substantiated by
the significant correlations between liver and kidney
concentrations of these trace elements.
Conclusion
In general, Hg was the element studied present in the
highest concentrations in the three types of samples, and
the kidney was the sample with the highest concentra-
tions of each element. If possible, in future studies, it
would be important to exclude birds that had migrated
from northern Europe and to measure Hg concentrations
in feathers, in order to further examine the causes of the
higher blood-Hg concentrations in winter and autumn.
The generally higher blood-Pb concentrations quanti-
fied in autumn and winter are possibly due to birds’
higher consumption of individuals crippled and poi-
soned through the hunting of small game species, which
indicates that future measures regarding hunting practices
are necessary in order to avoid high Pb exposure and/or
Pb poisoning in wild birds. Although raptors are at the top
of the food chain, and thus potentially exposed to any
biomagnification processes that may occur in a food web,
the individuals studied in this study generally had low
levels of heavy metals in blood and tissues, compared
with other authors. However, there are unknown sources
of exposure to the trace elements studied, so further
studies are needed to determine their origin.
Acknowledgments This study was supported by the fellowship
SFRH/BD/62115/2009 provided by the Fundação para a Ciência
e Tecnologia. The authors wish to thank all personnel at the Serveis
Científics i Tècnics of the University of Barcelona, Spain and
especially all personnel from the Servei d’Ecopatologia de Fauna
Salvatge of the Autonomous University of Barcelona, Spain.
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