Trace elements in blood collected from birds feeding in
the area around Doñana National Park affected by the
toxic spill from the Aznalcóllar mine
V. Benito a , V. Devesaa , O. Muñoz a , M.A. Suñer a , R. Montoro a,* , R.
Baos b , F. Hiraldo b , M. Ferrer b , M. Fernández c , M.J. González c
a
Instituto de Agroquı́mica y Tecnologı́a de Alimentos, IATA, (CSIC), Apdo Correos 73, 46100 Burjassot, Valencia, Spain
b
Estación Biológica de Doñana, Pabellon del Perú s/n (CSIC), Se illa, Spain
c
Departamento de I. A. y Quı́mica Ambiental, IQOG, CSIC, Juan de la Cier a, 3, 28006-Madrid, Spain
Abstract
A long-term monitoring plan was established to study if bird populations around Doñana National Park were
affected by the toxic spill from the Aznalcóllar mine. The concentrations of Zn, Pb, As, Cu, Sb, Co, Tl and Cd in the
blood of 11 bird species feeding in the area were determined. The parameters which most affect the accumulation of
trace elements in the birds studied are, firstly, species and, secondly, trophic position, sex, days of exposure and
weight. In some individuals, Zn and Cu occurred at higher levels than the reference values for contaminated areas.
Concentrations of Pb and Cd in a considerable number of individuals were higher than those found in birds from
uncontaminated areas. The present data, together with the lack of data on blood metal concentration prior to the
spill, do not offer any conclusive evidence of the influence of the spill on avian blood metal concentrations.
Keywords: Acid mine waste; Heavy metals; Arsenic; Doñana National Park; Blood; Birds
*
Corresponding author. Tel.: +34-963900022; fax: +34-963636301.
E-mail address: rmontoro@iata.csic.es ŽR. Montoro.
8
1. Introduction
On the 25 April 1998, a dam retaining 5 Hm3
of pyritic sludge gave way and released heavy
metals to the Guardiamar River. The most abundant metals, considered highly toxic for wildlife,
were Zn, Pb, As, Cu, Sb, Co, Tl and Cd ŽWood,
1974.. Although great efforts have been made to
remove the sludge, soils have been contaminated
with variable amounts of these elements ŽSimon
et al., authors in this issue.. The contaminated
surface represents a relatively small percentage of
the protected area of Doñana wetlands, but some
of the affected locations are important feeding
and breeding grounds for nesting and wintering
waterfowl, thus a significant part of these populations could be affected by the contamination.
Land birds may not be affected to the same
extent as aquatic birds, since their feeding area
generally does not extend into the contaminated
part, although species that cover large distances,
such as some raptors, may be affected.
The Doñana National Park is a Biosphere Reserve ŽComisión de Expertos, 1992. and the whole
area is considered to be of special interest for
species of waterfowl ŽGarcı́a et al., 1989; Máñez,
1991; Garcı́a-Novo, 1995.. Doñana is a breeding
ground and wintering area for species such as the
Spanish imperial eagle Ž Aquila adalberti., considered in the European Union to be one of the
birds in greatest danger of extinction ŽFerrer,
1993.. In addition, some sedentary birds, and birds
that winter in Doñana are hunted in neighbouring areas, and this may provide a link by which
contamination could reach human populations.
The Scientific Research Council ŽCSIC. was
instructed by the Spanish Government to monitor
the effect that contamination by heavy metals and
arsenic may have on wildlife in the ecosystems of
Doñana. For the reasons stated above, and because aquatic birds are considered good indicators of contamination in wetlands since they accumulate metals and As in various organs, they
were included among the various groups of animals studied. In the short term, the aim of monitoring was to determine whether contamination
by metals affected summer
avian mortality
ŽHernández et al., authors in this issue.. A long-
term monitoring plan sampling living animals was
also established to identify the sublethal effects
that this contamination episode may have on individual birds and future bird populations.
To achieve these aims, concentration of metals
in blood were measured since blood reflects the
current status of a toxic substance in the body
and is increasingly used in wildlife sampling programs ŽHenny and Meeker, 1981.. It is known
that the concentration of Cd and Pb in blood is a
good short-term and long-term indicator of contamination ŽGarcı́a-Fernández et al., 1995, 1997..
As far as As and Co are concerned, there is no
evidence if blood concentration of these elements
represents a short- or a long-term exposure, although in humans arsenic levels in blood have a
short biological half-life and reflect recent exposure ŽCornelis et al., 1995; Nicolas and Descotes,
1996.. Moreover, there is also a great difference
between organisms in the retention of arsenic in
erythrocytes ŽEisler, 1994.. Large amounts of Zn
are initially stored in the human liver, but it also
tends to accumulate in red blood cells and bones,
whilst copper is initially bound to serum albumin
and later more firmly bound to alpha-ceruloplasmin, the liver and bone marrow being the main
copper storage organs. Trivalent forms of antimony generally concentrate in red blood cells,
while pentavalent compounds are found in plasma.
ŽHammond and Beliles, 1980..
The toxic effects of the various heavy metals
and arsenic on birds have been documented. Lead
can cause bird mortality ŽRamo et al., 1992; Mateo et al., 1998., have sublethal effects ŽOchiai et
al., 1992., or a negative effect on reproduction
ŽBurger, 1995., depending on the dose. High doses
of Cd can cause death, and sublethal effects have
been described in birds at lower concentrations
ŽBokori et al., 1995.. Arsenic, especially in its
inorganic forms, can bring about the death of an
individual, produce sublethal effects and affect
reproduction ŽEisler, 1994.. There are no known
studies on the effects of Sb and Tl on birds, but
sublethal effects have been described in both
adult rats and fetuses ŽAlkhawajah et al., 1996;
Barroso Moguel et al., 1996; Albiser et al., 1997;
Poon et al., 1998.. Zn, Cu and Co are essential
elements for birds, but they can be toxic in high
concentrations, and various sublethal effects have
been described ŽRamey and Sterner, 1995;
Martı́nez and Dı́az, 1996; Ewing et al., 1998..
Moreover, when studying the effects of an
episode of local contamination on various species,
one must bear in mind that there are numerous
sources of individual variation. Among those most
frequently quoted are: physiological characteristics related to rates of absorption and assimilation
ŽPeterle, 1991.; trophic position and weight ŽPain
and Amiard-Triquet, 1993; Garcı́a-Fernández et
al., 1997.; sex ŽFinley et al., 1976., and age
ŽGarcı́a-Fernández et al., 1996, 1997.. Moreover,
the distance of breeding grounds from the spill
and the length of time that birds have been
exposed to its effects may affect the degree of
avian contamination.
The aim of this paper is to present the first
data on metal concentrations in the blood of
birds found in Doñana during the breeding season, and to determine whether these levels correspond to areas contaminated by heavy metals and
arsenic or to uncontaminated areas. Finally, Generalised Linear Models ŽNelder and Wedderburn,
1972; McCullagh and Nelder, 1983. were used to
determine the importance that the various sources
of variation mentioned above may have in explaining the contamination observed in individual
specimens.
2. Materials and methods
2.1. Sampling procedure
The birds initially included in the sampling
mainly fed in the wetlands, since it was unlikely
that other birds would be affected by this contamination episode. Sufficient species were selected to represent the various trophic levels
found in the wetland bird community of Doñana.
Breeding had already started, and thus the collection of samples to some extent was conditioned.
For many of the species selected significant numbers of samples were not obtained, and results for
these species are therefore not included in this
paper. The species for which the concentration of
metals in blood was analysed are given in Table 1,
which includes species, number of individuals of
each species sampled, position in the food chain,
weight, days of exposure to the spill, sex, age,
sampling point and social and ecological interest.
The weights assigned to each one of the studied
species and introduced in the analysis as continuous variable were taken directly from the bibliography ŽCramp and Simmons, 1977, 1980, 1983,
1985., selecting the weight of individuals belonging to the analysed species, sampled in the same
season Žspring—summer. in places next to Doñana.
Days of exposure were calculated as the difference between the dates of capture and toxic spill.
The locations of the sampling stations are shown
in Fig. 1.
A molecular technique based on the structural
difference of sex chromosomes was used to determine the sex of the specimens captured. Unlike mammals, male birds have two identical sex
chromosomes ŽZZ., whereas the females are heterogametic ŽZW. ŽLessels and Mateman, 1996.;
consequently, the DNA sequences of the W chromosome belong exclusively to females. Griffiths
et al. Ž1996. described a gene ŽCDH-W. which is
found in the W chromosome in females, with the
exception of ostriches and related species; this
knowledge was applied and the specimens were
sexed by means of Polymerase Chain Reaction
ŽPCR..
When drawing up the sampling program, we
attempted to sample a particular species on various dates and at various distances from the area
directly affected by the spill. The latter was impractical, since many of the species live in colonies
and only a single colony was accessible during the
sample collection period, thus it was not possible
to consider this variable when sources of variation
were studied.
The greater flamingo Ž Phoenicopterus ruber . was
included in the sampling
although its closest
breeding colony is 135 km from the site of the
spill
because monitoring of marked birds revealed that they frequently visit Doñana to feed
during the breeding period and the winter season.
The specimens were captured in the nest Žpullets. or in their feeding grounds Žyoung birds;
adults., using various trapping methods. For each
specimen, 2 ml of blood was taken from the radial
Fig. 1. Map of the area studied and the sampling stations.
1.
2.
3.
4.
5.
Dehesa de Abajo
Matasgordas
Caño de la Escupidera.
Veta Hornitos.
La FAO.
6. La Algaida.
7. Caño de Guadiamar.
8. Marilópez.
9. Veta La Palma
10. Las Pajareras.
11.
12.
13.
14.
El Pinar de San Agustı́n.
El Puntal.
Odiel, Ayamonte. Huelva.
Fuente de la Piedra. Málaga.
Table 1
Characteristics of waterfowl species in Doñana National Park studied in this work a
Individuals
sampled
Podiceps cristatus Žgreat
crested grebe
Ardea cinerea Žgrey heron.
20
Ciconia ciconia Žwhite stork.
30
Platalea leucorodia Žspoonbill.
30
Plegadis falcinellus Žglossy
ibis.
Phoenicopterus ruber Žgreater
10
7
20
flamingo.
Anas platyrhynchos Žmallard.
8
Anas strepera Žgadwall.
Aythya ferina Žpochard.
Mili us migrans Žblack kite.
5
10
25
Larus cachinnans Žyellowlegged gull.
11
a
Trophic level
Weight
Žg.
Fish predator
920
b
Days of
c
exposure
Age
1505
43 — 113 f
3571
16
Fish and crayfish
predator
Invertebrate
predator
Invertebrate
predator
Omnivorous
1960
39
30 — 87 f
S& E
e
Interest
7 Žpull.
3 Ž7.
Protected
3 M; 14 F; 3 N.K.
17 Žpull.; 3 ŽN.K..
4 Ž4.; 10 Ž14.; 12 Ž2.
Protected
16 M; 14 F
30 Žpull.
1 Ž30.
Protected
15 M; 12 F; 3 N.K.
28 Žpull.; 2 Žadult.
4 Ž1.; 10 Ž15.; 13 Ž14.
Protected
557
25
4 M; 6 F
10 Žpull.
5 Ž10.
Protected
3579
111
14 M; 6 F
20 Žpull.
20 Ž14.
Protected
1216
65 — 107 f
f
659
849
807
74 — 86
82 — 88 f
45 — 78 f
971.5
46 — 108 f
2 M; 1 F; 6 N.K.
3 M; 2F
10 N. K.
13 M; 7 F; 4 N.K.
2 M; 9 N.K.
M, male; F, female; N.K., not known; Pull, pullet.
The weights quoted are typical weights referred by Cramp and Simmons Ž1977, 1980, 1983, 1985 ..
c
Days of exposure were calculated as the difference between capture date and toxic spill date.
d
Sampling point: see Fig. 1 to locate the sampling point on the map.
e
S & E Interest: Social and Ecological Interest.
f
Days exposure interval in which the captures were carried out.
b
Sampling point
Žindividuals
sampled at
d
each location .
7 N.K.
106
Fish and crayfish
predator
Fish and crayfish
predator
Omnivorous
Omnivorous
Bird and mammal
predator
Carrion consumer
Sex
3 Žpull.; 5 Žadult.;
4 Ž2.; 7 Ž1.; 8 Ž2.;
1 ŽN.K..
9 Ž4.
4 Žpull.; 1 ŽN.K..
4Ž1.; 8 Ž4.
10 Žpull.
4 Ž1.; 8 Ž9.
23 Žpull.; 2 Žadult. 1 Ž2.; 2 Ž9.; 6 Ž3.; 11 Ž2.;
N.K. Ž9.
10 Žpull.; 1 ŽN.K..
9 Ž7.; N.K. Ž3.
Consumption
Consumption
Consumption
Protected
Protected
V. Benito et al. / The Science of the Total Eni ironment 242 (1999) 309—323
Species
313
vein. The specimens collected were marked with
plastic rings that could be identified individually
at a distance to allow monitoring of individuals in
the future.
2.2. Treatment of samples
Blood was kept frozen from collection to
preparation. Blood Ž1 ml. was taken and added to
9 ml of 0.5% Žw/v. Triton X-100 ŽMerck Farma y
Quı́mica, S.A., Valencia, Spain.. The solution was
mechanically shaken for 30 s and then shaken
vigorously in an ultrasound bath for 30 min. The
mixture was centrifuged at 2000 rev. min—1 for 5
min. Aliquots of 1 ml were taken and the precipitate was discarded. The aliquots obtained were
stored at —20°C prior to elemental analysis. An
MSE Minor centrifuge ŽPacisa, S.A., Madrid,
Spain . , a Vortex M S2 M inishaker Ž IKA
Labortechnik, Merck Farma y Quı́mica, S.A.,
Barcelona, Spain. and a Selecta Ultrasons P bath
ŽJ.P. Selecta, S.A., Barcelona, Spain. were used.
2.3. Determination of As, Cd, Co, Cu, Pb, Sb and Tl
The determination of As, Cd, Co, Cu, Pb, Sb
and Tl in whole blood samples was accomplished
by graphite furnace Zeeman-effect atomic absorption spectroscopy. A Perkin-Elmer ŽPE. longitudinal AC Zeeman ŽAAnalyst 600. atomic absorption spectrometer, equipped with a transversely heated graphite atomiser and a built-in
fully computer-controlled AS-800 autosampler
ŽPerkin Elmer Hispania, S.A., Madrid, Spain.,
and PE pyrolitic graphite coated tubes with an
inserted L’vov platform were used. Calibration
was performed with the method of Additions
Standard Curve. Deionised water Ž18 M
cm.
obtained with a Milli-Q water system ŽMillipore
Inc., Millipore Ibérica, Madrid, Spain. was used
for the preparation of reagents and standards. All
chemicals including standards and solutions were
of pro analysi quality or higher: nitric acid Ž p =
1.38 g ml—1 ., 1000 mg l—1 standards of As, Cd,
Co, Cu, Pb, Sb, Tl, palladium powder, magnesium
nitrate hexahydrate and ammonium dihydrogen
phosphate as matrix modifiers ŽMerck Farma y
Quı́mica, S.A., Valencia, Spain. were used. All
glassware, Eppendorf vials, and disposable sampling cups were treated with 10% nitric acid for 1
week, and then rinsed three times with Milli-Q
water, before use. Between uses, glassware was
placed in 10% nitric acid for 24 h. Method detection limits were: As Ž0.006 mg l—1 ., Cd Ž0.0001 mg
l—1 ., Co Ž0.001 mg l—1 ., Cu Ž0.013 mg l—1 ., Pb Ž
0.002 mg l—1 ., Sb Ž0.002 mg l—1 . and Tl Ž0.002 mg
l—1 .. The precision as measured by triplicate analysis, expressed as the relative standard deviation
ŽR.S.D.. was as follows: As 4%, Cd 5%, Co 5%,
Cu 2%, Pb 4%, Sb 3% and Tl 3%. The range of
recovery evaluated by spiking blood samples with
each of the elements was 85—115%.
Accuracy for Pb and Cd was established by
analysing a certified reference sample of bovine
blood ŽCRM-195. from the Institute for Reference Materials and Measurements ŽIRMM.. The
values found ŽPb: 413 ± 17; Cd: 5.40 ± 0.69 pg
l—1 . overlapped with the interval found for the
certified values ŽPb: 416 ± 9; Cd: 5.37 ± 0.24 pg
l—1 .. For As, Cu and Co, a CRM sample or
TORT-2 Žlobster hepatopancreas, Canada National Research Council, CNRC. was used. The
values obtained for these elements in our CRM
analyses were consistent with the certified values.
2.4. Determination of zinc
In the determination of Zn by flow injectionflame-atomic absorption spectrometry, a PE
Model 3300 atomic absorption spectrometer
equipped with a PE flow injection analysis system
for atomic spectroscopy ŽFIAS-400. and an autosampler ŽPE AS-90. were used. Precision was
established by analysis of an A-13 reference sample of animal blood obtained from the International Atomic Energy Agency ŽIAEA.. The recorded
values Ž13.6 ± 0.6 pg g—1 . overlapped with the
certified interval for this element Ž13 ± 1 pg g—1 ..
The evaluation of Zn recovery in spiked blood
samples was 95%. The method detection limit for
this element was 0.23 mg l—1 .
2.5. Statistical analysis
A Generalised Linear Model ŽGLM., ŽNelder
and Wedderburn, 1972; Dobson, 1983; McCullagh
and Nelder, 1983. was used to derive a mathematical description of individual variations in concentrations of metals and arsenic in blood. Models of
this kind are used when determining the individual effect of several variables on a given phenomenon ŽDonázar et al., 1993; Bustamante, 1997;
Forero et al., 1999; Tella et al., 1999.. Generalised Linear Models can be considered as a
particular case of multiple linear regression. Three
components must be defined for a GLM: a linear
predictor, an error function and a link function. A
linear predictor ŽLP. is defined as LP = a +b* x 1
+ c* x2 + . . . where a is a constant to be estimated; b, c, . . . are parameters to be estimated
from observed data; and x 1 , x 2 . . . are the explanatory variables. The error function depends
on the nature of the data. The concentration of
the various metals in blood was ln-transformed
and a normal error distribution was assumed for
the models. An identity link was used as link
function. In this case the model does not differ
from a multiple linear regression with a ln-transformed-dependent variable. The explanatory variables considered were introduced into the model
as factors Žspecies, sex, trophic position. or continuous variables Ždays of exposure to the spill and
weight.. Although age is relevant as a factor of
individual variation ŽGarcı́a-Fernández et al.,
1996, 1997., it was not included in the statistical
analysis since most of the individuals sampled
were pullets ŽTable 1.. We fitted each explanatory
variable to the observations using the GLM program ŽBaker, 1987. following a modification of a
traditional forward stepwise procedure. Each
variable was tested in turn for significance, and
only those significant at the 5% level were included in the model. Recent papers have criticised automatic stepwise procedures as they are
not necessarily able to select the most influential
variable from a subset ŽJames and McCulloch,
1990.. The modification of the stepwise modelling
procedure involved testing the alternative models
that were obtained when the second or the third
most significant variable was included Žprovided
that it was significant at the 5% level., instead of
the most significant one at each step. This Forward Stepwise Branching Modelling Procedure
ŽDonázar et al., 1993. eventually gave a set of
different models which mostly converged into a
single model or a set of models from which similar relationships could be inferred.
The expression of the model becomes:
[ metal ] = e a+ b
*
x+ c* x+ . . .
Ž1.
3. Results
Table 2 shows the total blood metal content of
the species sampled, and Table 3 gives the percentage of individuals of each species with higher
levels of metals than those reported in the literature for birds in uncontaminated areas ŽPb, As
and Cd. or contaminated areas ŽCu and Zn..
Data for birds in uncontaminated areas were not
found for the latter elements. Reference values
were not found for Co, Sb and Tl levels in the
blood of birds. For each element, in the case of
measurements below the limit of detection ŽLOD.,
the value introduced in the database was that of
LOD.
3.1. Zinc
The zinc contents found in the species studied
ŽTable 2. varied between 0.3 and 8.6 mg l—1 . The
species that presented the highest mean level of
this metal was the gadwall, followed by the yellow-legged gull. The mean value found in glossy
ibis was notably low. Twenty percent of the gadwall individuals analysed ŽTable 3. presented values even higher than those established for birds
in contaminated areas Ž7.5 mg kg—1 wet weight
Žww., Falandysz et al., 1988., this being the only
species in which an individual exceeded the reference values.
3.2. Lead
The levels of Pb ranged between 0.002 and
0.454 mg l—1 ŽTable 2.. The species with the
highest mean levels of lead in blood was the
mallard, followed by the gadwall. Some individual
specimens of mallard had levels close to 0.5 mg
kg—1 , a value indicative of lead poisoning in swans
316
Table 2
Metal and arsenic concentrations in blood of wild birds feeding in the area around Doñana National Park a
No.
specimens
Ciconia ciconia
N = 30
Žwhite stork.
Plegadis falcinellus
N = 10
Žglossy ibis.
Mili us migrans
N = 25
Žblack kite.
Aythya ferina
N = 10
Žpochard.
Anas platyrhynchos
N =9
Žmallard.
Ardea cinerea
N = 20
Žgrey heron.
Phoenicopterus ruber N = 20
Žgreater flamingo.
Anas strepera
N =5
Žgadwall.
Platalea leucorodia
N = 30
Žspoonbill.
Podiceps cristatus
N =7
Žgreat crested grebe.
Larus cachinnans
N = 10
Žyellow-legged gull.
Total Zn
Žmg l—1 .
Total Pb
Žmg l— 1 .
Total As
Žmg l— 1 .
Total Cu
Žmg l— 1 .
Total Sb
Žmg l— 1 .
Total Co
Žmg l— 1 .
Total Tl
Žmg l— 1 .
Total Cd
Žmg l— 1 .
1.9b
0.8—2.8c
0.9b
0.7—1.3c
3.3b
2.3—4.5c
3.7b
2.5—6.0c
3.3b
1.3—4.0c
2.2b
1.5—3.3c
1.7b
0.3—2.6c
5.9b
3.5—8.6c
3.2b
1.4—5.5c
2.2b
1.3—2.9c
4.4b
3.1—5.2c
0.071b
0.002—0.320c
0.061b
0.020—0.233c
0.054b
0.002—0.179c
0.073b
0.025—0.274c
0.208b
0.045—0.454c
0.015b
0.002—0.089c
0.076b
0.035—0.120c
0.120b
0.069—0.174c
0.008b
0.002—0.034c
0.002b
0.002c
0.020b
0.009—0.032c
0.019b
0.006—0.121c
0.008b
0.006—0.017c
0.008b
0.006—0.035c
0.006b
0.006c
0.011b
0.006—0.042c
0.006b
0.006c
0.006b
0.006c
0.017b
0.006—0.029c
0.019b
0.006—0.181c
0.006b
0.006c
0.006b
0.006c
0.586b
0.180—1.530c
0.133b
0.067—0.241c
0.211b
0.120—0.303c
0.203b
0.151—0.396c
0.258b
0.142—0.361c
0.352b
0.204—0.650c
0.334b
0.187—0.531c
0.526b
0.346—0.753c
0.307b
0.190—0.569c
0.395b
0.327—0.488c
0.429b
0.271—0.535c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.007b
0.001—0.016c
0.029b
0.019—0.041c
0.004b
0.001—0.013c
0.011b
0.005—0.016c
0.006b
0.001—0.015c
0.019b
0.008—0.025c
0.054b
0.039—0.070c
0.027b
0.005—0.047c
0.048b
0.001—0.110c
0.001b
0.001—0.002c
0.040b
0.015—0.070c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.002b
0.002c
0.0015b
0.0001—0.0090c
0.0110b
0.0060—0.0150c
0.0068b
0.0010—0.0140c
0.0009b
0.0001—0.0030c
0.0048b
0.0001—0.0190c
0.0002b
0.0001— 0.0010c
0.0006b
0.0001—0.0010c
0.0006b
0.0001—0.0290c
0.0003b
0.0001—0.0010c
0.0001b
0.0001c
0.0002b
0.0001—0.0005c
a
In those cases in which the value was not detectable, the detection limit was taken ŽZn 0.23 mg l—1 ; Pb 0.002 mg l— 1 ; As 0.006 mg l— 1 ; Cu 0.013 mg l— 1 ; Sb 0.002
mg l—1 ; Tl 0.002 mg l— 1 ; Co 0.001 mg l— 1 ; Cd 0.0001 mg l— 1 ..
b
Mean of n values.
c
Range of n values.
V. Benito et al. / The Science of the Total Eni ironment 242 (1999) 309—323
Species
Table 3
Percentage of specimens of each species of bird feeding in the area around Doñana National Park that presented higher levels of metals and arsenic in blood than
values found in the literature for contaminated and uncontaminated areas
No. of
individuals
Ciconia ciconia
Žwhite stork.
Plegadis falcinellus
Žglossy ibis.
Mili us migrans
Žblack kite.
Aythya ferina
Žpochard.
Anas platyrhynchos
Žmallard.
Ardea cinerea
Žgrey heron.
Phoenicopterus ruber
Žgreater flamingo.
Anas strepera
Žgadwall.
Platalea leucorodia
Žspoonbill.
Podiceps cristatus
Žgreat crested grebe.
Larus cachinnans
Žyellow-legged gull.
N = 30
0a
N = 10
Reference value
Zn
As
Cu
33a
17a
3a
33a
0a
20a
0a
0a
100a
N = 25
0a
32a
8a
0a
92a
N = 10
0a
30a
0a
0a
20a
N =9
0a
78a
11a
0a
78a
N = 20
0a
5a
0a
0a
0a
N = 20
0a
70a
0a
0a
0a
N =5
20a
100a
40a
0a
0a
N = 30
0a
0a
20a
0a
0a
N =7
0a
0a
0a
0a
0a
N = 10
0a
0a
0a
0a
0a
Ž7.5 mg kg—1 ph.b
Pb
Ž0.062 mg l— 1 .c
Ž0.020 mg l— 1 .d
Ž1.15 mg kg— 1 ph.e
Cd
Ž0.001 mg l— 1 .f
V. Benito et al. / The Science of the Total Eni ironment 242 (1999) 309—323
Species
% Žno. of specimens with value higher than reference value found in the literature/no. of specimens analysed.
Reference value of Zn for birds in contaminated areas ŽFalandysz et al., 1988..
c
Reference value of Pb for birds in uncontaminated areas ŽDieter et al., 1976..
d
Reference value of As for birds in uncontaminated areas ŽBurger and Gochfeld, 1997..
e
Reference value of Cu for birds in contaminated areas ŽVan Eeden and Schoonbee, 1996..
f
Reference value of Cd for birds in uncontaminated areas ŽGarcı́a-Fernández et al., 1995..
a
b
317
ŽBlus et al., 1991.. The values found in ducks in
uncontaminated areas by other authors are
around 0.062 mg l—1 ŽDieter et al., 1976.. No
individuals of spoonbill, yellow-legged gull or great
crested grebe exceeded the reference value ŽTable 3..
Eeden and Schoonbee, 1996.. Only a few examples of white stork had levels above or around
this value ŽTable 3..
3.5. Antimony and thallium
None of the species analysed presented values
which exceeded the detection limits Ž0.002 mg l—1
for both metals..
3.3. Arsenic
Levels of As ranged between 0.006 and 0.181
mg l—1 ŽTable 2.. The highest mean contents
were found in the white stork, spoonbill and
gadwall, in all cases they were less than the reference value for uncontaminated areas Ž0.02 mg
l—1 , Burger and Gochfeld, 1997.. Nevertheless,
some individuals of five species studied presented
levels that exceeded the reference value. There
was great variability in arsenic levels between
specimens of the same species. The high number
of species with levels below the detection limit
was also noteworthy Žpochard, grey heron, greater
flamingo, great crested grebe and yellow-legged
gull..
3.4. Copper
Levels of Cu content found was between 0.067
and 1.530 mg l—1 ŽTable 2.. The species with the
highest copper blood levels were the white stork,
gadwall and yellow-legged gull. These mean values are lower than those found in the literature
for contaminated areas Ž1.15 mg kg—1 ww; Van
3.6. Cobalt
The Co range varied between 0.001 and 0.110
mg l—1 ŽTable 2., with particularly high levels
found in the greater flamingo, spoonbill and yellow-legged gull. The species with the lowest mean
level was the great crested grebe.
3.7. Cadmium
Cd concentrations were between 0.0001 and
0.029 mg l—1 ŽTable 2.. The species that had the
highest mean concentrations were the glossy ibis,
black kite and mallard, in descending order. When
the individual values for each of the species were
compared with the reference values found in the
literature for uncontaminated areas Ž0.001 mg
l—1 ; Garcı́a-Fernández et al., 1995., only five of
the eleven species included in the study had individuals that exceeded the reference value. It must
be emphasised, however, that in three of these
five species the percentage of individuals with
Table 4
Variables Žfactors and continuous. that are significant at 5% in the Generalised Linear Models
Element
Model 1a
Model 2b
Accounted
deviance Ž%.
Factors
Continuous
variable
Accounted
deviance Ž%.
Factors
Continuous
variable
Zn
Pb
71.98
57.48
Species
Species
—
—
52.77
24.93
—
—
Cd
79.53
Species
—
69.19
Trophic
Sex
Trophic
Trophic
Cu
Co
As
49.08
74.93
15.90
Species
Species
Species
—
—
—
40.98
50.74
9.27
Trophic
Trophic
Trophic
a
b
Days
Weight
Weight
—
Days
Model 1: all the variables studied have been included Žspecies; trophic level, sex, days of exposure and weight..
Model 2: the variable ‘species’ has been omitted in all cases.
Table 5
Generalised Linear Models with normal distribution of errors and identity link for each of the metals analysed in blood of birds captured in Doñana National Park.
All the variables studied have been included Žspecies, trophic level, sex, days of exposure and weight.
Model 1
Zn
Constant
0.6275
Anas strepera
1.1040
gadwall
Larus cachinnans
0.8385
yellow-legged gull
Aythya ferina
0.6520
pochard
Mili us migrans
0.5577
black kite
Anas platyrhynchos
0.5093
mallard
Platalea leucorodia
0.5028
spoonbill
Ardea cinerea
0.1577
grey heron
Podiceps cristatus
0.1325
great crested grebe
Ciconia ciconia
0.0000
white stork
Phoenicopterus ruber —0.1135
greater flamingo
Plegadis falcinellus —0.7372
glossy ibis
Residual deviance
d.f.
308.73
157
Cd
Cu
Co
As
S.E.
Estimated
parameter
S.E.
Estimated
parameter
S.E.
Estimated
parameter
S.E.
Estimated
parameter
S.E.
Estimated
parameter
S.E.
0.0477
0.1263
—3.4520
1.2880
0.2058
0.5444
—6.7480
—0.6799
0.0961
0.2802
—0.6295
—0.0445
0.0761
0.2014
—5.2190
1.3790
0.1235
0.3268
—5.1710
0.5821
0.1501
0.3972
0.0955
—0.5408
0.4115
—0.9265
0.1922
—0.2362
0.1522
1.8820
0.2471
—0.6384
0.3002
0.0994
0.5612
0.4115
—0.3562
0.1922
—1.0003
0.1522
—
—
—0.6384
0.3002
0.0716
0.7775
0.3052
1.5990
0.1425
—0.9483
0.1129
—0.4957
0.1832
—0.4553
0.2227
0.0994
1.5730
0.4283
0.9432
0.2000
—0.7495
0.1584
—0.3585
0.2571
—0.1524
0.3125
0.0701
—2.0300
0.2910
—0.7505
0.1396
—0.5876
—0.1086
1.9820
0.1747
0.1149
0.2123
0.0778
—1.7060
0.3253
—0.8124
0.1598
—0.4551
0.1266
1.2000
0.1953
—0.6384
0.2374
0.1097
—3.4560
0.4731
—0.8531
0.2209
—0.3080
0.1750
—1.6890
0.2840
—0.6384
0.3451
0.0477
0.0000
0.2058
0.0000
0.0961
0.0000
0.0761
0.0000
0.1235
0.0000
0.1501
0.0755
0.8165
0.3253
—0.5412
0.1519
—0.5054
0.1203
2.2760
0.1953
—0.6384
0.2374
0.0955
0.3404
0.4115
2.2180
0.1922
—1.4780
0.1522
1.6600
0.2471
0.0173
0.3002
115.87
165
1418.90
158
317.83
161
126.29
165
V. Benito et al. / The Science of the Total Eni ironment 242 (1999) 309—323
Estimated
parameter
Pb
151.81
165
S.E.: Standard Error.
d.f.: degrees of freedom
319
320
V. Benito et al. / The Science of the Total Eni ironment 242 (1999) 309—323
levels above the reference values was very high,
and in the case of the glossy ibis it was 100%.
4. Discussion
Two model groups were fitted for As and each
of the metals except Sb and Tl, which were notdetected in most of the birds studied. In Model 1
all the variables were included Žspecies, trophic
level, weight, time elapsed since the spill and sex.,
while in Model 2 the species variable was omitted
in all cases. Various significant models were found
in both groups. In each case only the best models
were selected, i.e. those with the smallest residual
deviance ŽTable 4..
When we considered Model 1, models were
obtained that explained a high percentage of the
original deviance for all the metals except As
ŽTable 5.. Moreover, for all the metals the best
model included only one variable, species, and
none of the other factors or continuous variables
considered was significant at the level chosen
Ž5%.. The various models predict a different behaviour for each species according to the metal
being considered. For example, the glossy ibis was
the species least contaminated by Cu or Zn; similarly, the white stork was the species with the
highest concentration of Cu in blood, whereas Zn
was the ninth in the order of Zn concentration
coming after the gadwall, yellow-legged gull,
pochard, black kite, mallard, spoonbill, grey heron,
and great crested grebe.
In the models that did not include species
ŽModel 2., the factors and continuous variables
that are significant at the 5% level varied from
one metal to another ŽTables 4 and 6.. In all
cases the models explained a good part of the
deviance, except in the case of As. Nevertheless,
for all the metals the deviance explained was
always less in Model 2 than Model 1. For Zn, the
Model 2 included only trophic position, predicting
the highest values in the carrion consumers and
the lowest values in the invertebrate predators. In
the case of Pb, Model 2 included trophic position
and sex as significant; this model predicts a lower
concentration of Pb in females from each trophic
level. For Cd, Model 2 includes trophic position
and two continuous variables, days and weight, as
significant, with metal concentration increasing as
weight and possible days of exposure to the spill
decrease. For Cu, Model 2 includes one factor,
trophic position, and one continuous variable,
weight; within each trophic level, the Cu concentration in blood tends to increase with bird weight.
For Co, only trophic position is significant; Model
2 predicts the greatest concentration in invertebrate predators and the smallest concentration in
fish predators. For As, Model 2 includes two
significant variables, trophic position as a factor
and days as a continuous variable, with the metal
concentration increasing as the possible days of
exposure to the spill decrease. The Generalised
Linear Models show that when the species to
which an individual belongs is considered separately from its trophic position, in all the metals
studied species is the variable that most contributes to explaining the variability of metal concentration in blood. This may be linked with the
physiological characteristics of species related to
their ability to absorb and excrete metals. In
some species of mammals the level of metal contamination has been related more to inability to
excrete metals than to their trophic position. Nevertheless, we cannot discard the possibility that
the importance of species is linked with ecological
factors such as longevity, ability to move elsewhere, differences in microhabitat, and feeding
habits, which were not considered in this study
and yet are known to affect the level of contamination of an individual.
5. Conclusions
Among the various metals studied, some Žsuch
as Zn and Cu. tended to be present in all the
individuals, whereas others Žsuch as Sb and Tl.
were not detected in any of the specimens examined. In the case of Zn and Cu Žthe only
metals for which blood values proceeding from
studies of contaminated areas were found. only
two species showed individuals with higher levels
than the reference values. The reference limit for
Zn was exceeded by 20% of the gadwall specimens, while 3% of the white stork specimens had
Model 2
Constant
Carrion consumers
Omnivorous
Birds and mammals
predator
Fish and crayfish
predator
Fish predator
Invertebrate predator
Days
Sex Žfemale.
Weight
Residual deviance
Degrees of freedom
Zn
Pb
Cd
Cu
Co
As
Estimated
parameter
S.E.
Estimated
parameter
S.E.
Estimated
parameter
S.E.
Estimated
parameter
S.E.
Estimated
parameter
S.E.
Estimated
parameter
S.E.
1.322
0.144
0.000
—0.137
0.0697
0.1266
0.0697
0.0975
—2.088
—2.349
0.000
—1.010
0.4659
1.0240
0.4659
0.5393
—4.892
—1.507
0.000
1.092
0.2074
0.2469
0.2074
1.1886
—1.615
0.465
0.000
—0.199
0.0982
0.1669
0.0982
0.1268
—4.784
1.447
0.000
—0.930
0.1907
0.3516
0.1907
0.2670
—4.758
—0.621
0.000
—0.402
0.2666
0.3235
0.2666
0.2462
—0.480
0.0798
—1.618
0.4963
—0.991
0.1927
—0.070
0.1210
0.609
0.2174
—0.155
0.2119
—0.562
—1.016
—
—
—
308.73
162
0.1442
0.0926
0.000
—0.453
—
—0.635
—
386.64
101
0.4659
0.5130
0.2810
0.2008
0.0019
0.408
—0.596
—
—
0.0003
317.83
165
0.4083
0.1379
—2.123
1.636
—
—
0.00004
—
126.29
170
0.4013
0.2559
—0.237
—0.200
—0.0077
—
—
151.81
169
0.3685
0.2312
0.0025
S.E.: Standard Error.
—0.419
0.555
—0.0204
0.2581
—
—0.0001
1418.18
161
0.0001
V. Benito et al. / The Science of the Total Eni ironment 242 (1999) 309—323
Table 6
Generalised Linear Models with normal distribution of errors and identity link for each of the metals analysed in blood of birds captured in Doñana National Park.
The variables studied have been trophic level, sex, days of exposure and weight. The variable species has been omitted in all cases
321
322
V. Benito et al. / The Science of the Total Eni ironment 242 (1999) 309—323
higher levels of Cu than the reference values. In
the cases of Pb and Cd, the levels of a considerable number of individuals were higher than the
reference values for birds in uncontaminated areas. However, with the data obtained it is not
possible to give an opinion on whether the concentration of metals found in blood had been
affected by the spill or not, since this may be
influenced by the physiological response of the
species to distinct metals, and by the greater or
lesser bioavailability of those metals. The reference values should also be interpreted with care,
since they do not refer to the same types of
species as those analysed. Furthermore, there is
no data for levels of metals and arsenic in blood
before the spill to facilitate a comparison with the
values obtained. The possible presence of metal
before the spill must also be considered. In addition, the concentration of some elements in blood,
such as arsenic, may be a poor indicator of the
level of contamination of an individual.
Nevertheless, given the toxicity of the metals
and arsenic still present in the area around
Doñana, it is advisable to continue long-term
monitoring of metals and the metalloid As in
blood, since many additional data are required to
establish sublethal effects in the species studied.
Owing to the individual variation found, a large
number of species should be included in the
protocol for monitoring the effects of the spill on
bird fauna in Doñana. We recommend the inclusion of threatened species feed in the marshes
ŽBlanco and González, 1992.. When the selection
has been made, it is necessary to bear in mind the
trophic level.
Acknowledgements
Funds for this study were provided by the Consejo Superior de Investigaciones Cientı́ficas
ŽCSIC. for which the authors are deeply indebted.
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