Unexpected high PCB and total DDT levels in the breeding population of red
kite (Milvus milvus) from Doñana National Park, south-western Spain
Belén Gómara a , María José González a , Raquel Baos b , Fernando Hiraldo b ,
Esteban Abad c , Josep Rivera c , Begoña Jiménez a,⁎
a
Department of Instrumental Analysis and Environmental Chemistry, Institute of Organic Chemistry, CSIC. Juan de la Cierva 3, 28006 Madrid, Spain
b
Department of Applied Biology, Estación Biológica de Doñana, CSIC. Avda. Ma Luisa s/n, Pabellón del Peru, 41013 Sevilla, Spain
c
Department of Ecotechnologies, IIQAB. CSIC. Jordi Girona 18-26, 08034 Barcelona, Spain
Received 4 January 2007; accepted 16 July 2007
Available online 28 August 2007
Abstract
This study provides information on the current status of contamination by organochlorines (DDTs, PCBs, PCDDs and PCDFs) in the declining red
kite (Milvus milvus L.) population breeding in the Doñana National Park (DNP), south-western Spain. Analyses were performed in addled eggs
collected between 1999 and 2001. DDE concentrations ranged from 0.1 to 33.5 μg/g ww, representing more than 86% of the total DDTs. Of the
samples studied, 50% showed DDE levels above those associated with reproductive impairment in other raptor species. Concentrations of ortho
PCBs (average 36.8 μg/g ww ± 37.7) in 50% of the eggs were much higher than levels reported to cause reduced hatching success, embryo mortality,
and deformities in birds (N 20 μg/g ww). It is remarkable that average ortho PCB and DDE concentrations showed an increase of one order of
magnitude compared to previous data for the species during the 80s. Total PCDD/Fs showed levels in the low pg/g range (7.2–42 pg/g ww), having
PCDDs and PCDFs similar contributions in most samples. Total mean TEQs were 238 pg/g (ww), being the range 7.02–667 pg/g (ww). Spatial
variation within DNP was observed for PCBs, DDTs, as well as for TEQs. Since some eggs exceeded the NOEL (67%) and LOEL (33%) reported for
other raptor species, we would expect the red kite to experience detrimental effects to dioxin-like toxicity. Our results suggest that organochlorine
contaminants should be regarded as an element of concern in the population under study, in addition to other conservation problems already reported.
Further investigations should be undertaken to identify potential sources of these chemicals in DNP, and to find out if organochlorine contamination is
present in other predator species in the area, as well as their potential health effects on individuals and/or populations.
Keywords: PCDDs; PCDFs; PCBs; TEQs; DDTs; Eggs; Raptors; Red kite; Doñana National Park; Spain
1. Introduction
Several case studies reported in humans and wildlife
demonstrate that contamination by organochlorine compounds
has become a generalised problem (Colborn, 1995). After the
70s, a number of investigations revealed some organochlorines
as the main cause of certain wildlife population declines
(Peakall et al., 1975; Fyfe et al., 1988). Nowadays contamination by this class of compounds continues to be an element of
concern in top predators such as raptors, with a particular
concern posed by species at risk (Merino et al., 2005; Jiménez
⁎ Corresponding author. Tel.: +34 91 5622900x431; fax: +34 91 5644853.
E-mail address: bjimenez@iqog.csic.es (B. Jiménez).
:
et al., 2007). The red kite (Milvus milvus), is a medium-sized
raptor species endemic to the western Palearctic, the Iberian
Peninsula being the southern edge of its distribution. The three
largest populations (in Germany, France and Spain, which
together hold more than 75% of the global population) all
declined during the period 1990–2000 (Seoane et al., 2003),
and overall the species declined by almost 20% during the
decade of the 90s, being catalogued as Near Threatened (NT) at
global-European scale (BirdLife International, 2005). In Spain,
the species showed an overall decline in the breeding population
of up to 43% for the period 1994 to 2001–02, and surveys of
wintering birds in 2003–04 suggest a similarly large decline in
core wintering areas (BirdLife International, 2005). This decline
has been mainly attributed to human persecution, especially
through illegal poisoning (Viñuela, 2005), however additional
factors related to environmental pollution (e.g., chlorinated
pollutants) have not been widely studied and their potential role
in the population decline of the species remains unknown. A
recent study concerning red kites from the Island of Menorca
(Spain) reported high levels of PCBs in failed eggs, suggesting
that a more detailed study to clear up possible deleterious effects
of PCBs on this species should be done (Jiménez et al., 2007).
At this point, ecologically sensitive areas present a particular
interest since in general, they represent important refuges for
threatened wildlife species. An example of this is Doñana National
Park (DNP), a 50,720 Ha protected area in south-western Spain
that holds a unique biological diversity in Europe, especially the
Marshes (García-Novo and Marín, 2006). Doñana Marshes (also
known as Guadalquivir Marshes) is world-renowned for its dense
predator populations (Valverde, 1967), including some of the most
endangered species of the world, such as the Spanish imperial
eagle (Aquila adalberti) and the Iberian lynx (Lynx pardinus), and
is one of the major wintering and stopover sites for waterbirds
breeding throughout the Western Palearctic (García-Novo and
Marín, 2006). In the Guadalquivir Marshes, a relatively small
breeding population of red kites, whose number of pairs declined
from 41 in 1987 to 19–20 in 2000 (Máñez, 2001), coexists with
migratory birds from Central Europe during winter (Heredia et al.,
1991). The red kite is listed as a species at critical risk of extinction
at regional scale (Máñez, 2001), being the resident population of
Doñana almost the unique breeding nucleus remaining in
Andalusia (Viñuela, 2005). Recently, Sergio et al. (2005) have
reported illegal poisoning and high rates of competition and nest
predation as some of the main conservation problems of this
population, but contamination was not evaluated in their study.
The present study provides information on the actual status of
contamination by organochlorines (DDTs, PCBs, PCDDs and
PCDFs) in the breeding red kite population of Doñana National
Park, which could be relevant when adopting strategies to preserve
the conservation status of the species in this area.
2. Materials and Methods
(Deventer, The Netherlands). Acetone and toluene were purchased from SDS
(Peypin, France). PCDD/F congeners were obtained from Wellington
Laboratories (Ontario, Canada) and PCBs and DDTs were obtained from
Dr. Ehrenstorfer (Augsburg, Germany).
2.4. Analytical procedure
Sample treatment involved three steps as previously described in detail by
Merino et al. (2005). Basically, eggs extraction was carried out using a solid
phase matrix dispersion procedure. Then, clean up was performed using
multilayer columns filled with neutral silica, silica modified with sulphuric
acid, and silica modified with potassium hydroxide. The final fractionation step
was achieved using Supelclean™ Supelco ENVI™-Carb tubes (Bellefonte, PA,
USA). Three fractions were collected: The first fraction contained the bulk of
PCBs and DDTs; the second and third fractions contained non-ortho substituted
PCBs and PCDD/Fs, respectively. Congener separation and quantification of
ortho PCBs and DDTs were carried out by high resolution gas chromatography
(HRGC) using a Hewlett Packard 6890 gas chromatograph equipped with a
63
Ni electron capture micro-detector (Palo Alto, CA, USA) as described by
Gómara et al. (2002). Non-ortho PCB congeners were determined by GC
coupled to an ion trap detector (ITD) in the tandem mass spectrometry (MS/
MS) operation mode as reported by Gómara et al. (2006) using a Varian CP3800 gas chromatograph coupled to a Saturno 2000 ion trap detector (Palo
Alto, CA, USA). Resolution and quantification of PCDDs and PCDFs were
performed by high resolution gas chromatography coupled with high resolution
mass spectrometry (HRGC-HRMS) on a GC 8000 series gas chromatograph
(Carlo Erba Instruments, Milan, Italy) equipped with a CTC A 200S auto
sampler (Water Instruments, Manchester, UK) and coupled to an Autospec
Ultima mass spectrometer (Micromass, Manchester, UK), using a positive
electron ionization source and operating in the selected ion monitoring mode at
10,000 resolving power (10% valley definition), as previously described by
Merino et al. (2005). Quantification of non-ortho PCBs and PCDD/Fs was
carried out by the isotopic dilution technique following procedures from EPA
(U.S. EPA, 1994).
Quality assurance criteria were based on the application of the quality
control and quality assurance measures, which included the analysis of blank
samples covering the complete analytical procedure. Additional evaluation to
ensure good quality data was obtained by the participation in several
intercalibration studies covering a wide variety of biotic matrices. The results
were consistent with the consensus means given by the inter-laboratory
organizations (NIST/NOAA, 2003; Becher et al., 2004; Becher et al., 2005).
Concentrations are expressed on a wet weight (ww) basis. 2,3,7,8-TCDD
equivalents (TEQs) were estimated for PCDD/F congeners and dioxin-like
PCBs with an assigned TEF value, based on the bird toxic equivalency factors
(TEFs) reported in 1998 by the World Health Organization (Van den Berg et al.,
1998).
2.1. Sampling
3. Results and discussion
Failed eggs from the population of red kite breeding at Doñana National
Park (south-western Spain) were used to investigate the organochlorine
contaminants load. Twelve addled eggs, found intact at the nest, were sampled
between 1999 and 2001. Eggs were obtained from ten different nests. Samples
were kept frozen at − 20 °C until analysis. The egg content was freeze dried prior
to analysis. On average, lipid percent was 8 and percent moisture was 79.
2.2. Residue analyses
The contaminants examined were DDTs (DDT and its two main metabolites,
DDE and TDE), PCBs (ortho PCBs # 28, 52, 95, 101, 123 + 149, 118, 114, 153,
132 + 105, 138, 167, 156, 157, 180, 170, 189, 194; non-ortho PCBs # 81, 77,
126, 169) and all the 2,3,7,8-substituted PCDDs and PCDFs.
2.3. Reagents and standards
All reagents used for the analysis were of trace analysis grade. Hexane,
sulphuric acid (95%–97%) and silica gel were supplied by Merck Co.
(Darmstadt, Germany) and granular anhydrous sodium sulphate by J.T. Baker
Table 1a shows data concerning DDT and PCB concentrations of
each sample analyzed. Total mean DDTs were 9.5 ± 11.1 μg/g, being the
range 0.2–34 μg/g. Average DDE concentration was 9.3 ± 10.8 μg/g,
ranging from 0.1 to 33.5 μg/g and representing more than 86% of the
total DDTs. 50% of the eggs had levels of this metabolite higher than
those associated with reproductive impairment in bald eagles
(Haliaeetus leucocephalus) (6 μg/g of DDE, Elliot and Harris, 2001/
2002).
Concentrations of ortho PCB congeners ranged from 0.5 to 110 μg/g
(average 36.8 ± 37.7 μg/g). PCB 153 and 180 were the main contributors
to total concentrations, accounting both with a 60%, followed by PCB
138 with a percentage contribution of 15% (Fig. 1). Concentrations of
ortho PCBs in 50% of the eggs were much higher than levels
(N 20 μg/g ww) reported to cause reduced hatching success, embryo
mortality, and deformities in raptor species (Elliot and Harris, 2001/
2002). Furthermore, average values found in the present study for ortho
PCBs, as well as DDTs, are almost one order of magnitude higher than
Table 1a
PCB and DDT concentrations in red kite eggs, expressed in ng/g and pg/g on a
f resh weight basis (f.w.)
Congener
Arithmetic SD
mean
Ortho PCBs (ng/g, f.w.)
28
55
52
35
95
102
101
960
123 + 149
133
118
992
114
87
153
11,023
132
7
105
126
138
5783
183
1729
167
146
156
608
157
56
180
10,513
170
2407
189
86
194
1973
Σ ortho PCBs 36,822
Median Geometric
mean
72
30
103
1175
148
1111
80
11,455
21
121
6103
1777
140
739
51
10,527
2497
129
1969
37,696
21
22
63
440
70
729
61
8298
0
79
4340
1188
103
322
41
8359
1821
32
1617
29,114
26
22
51
245
59
365
40
4063
19
63
2147
586
65
201
28
3741
861
28
759
13,562
Non-ortho PCBs (pg/g, f.w.)
57 a
46 a
81
77
113
111
126
1122
1064
169
160
130
Σ non-ortho
1438
1236
PCBs
44
61
840
150
1179
22 a
75
454
53
812
DDTs (ng/g, f.w.)
DDE
9316
TDE
0.20
DDT
207
Σ DDTs
9524
a
n = 9.
10,888
0.37
202
11080
4711
0
129
4872
3654
0.11
103
3795
Range
2–235
2–91
4–303
6–3298
3–501
21–3197
0.9–218
161–33,785
b 0.02–74
4–320
89–17,535
17–5095
4–358
5–2135
2–137
128–30,211
33–7467
3–391
32–5985
517–109,933
b 0.03–128
15–393
0.8–3074
0.2–405
16–3730
152–33,549
0.03–1
3–608
177–34,158
levels reported in previous studies conducted in the 80s in the same
species. Earlier studies by González et al. (1983) in addled eggs from
red kites collected in 1981, reported mean PCB concentrations of 2 ppm
on a fresh weight basis. That study also reported DDE at low ppm levels
(0.4 ppm) and DDT with a mean value of 0.3 ppm. Current PCB levels
are also much higher than average values of 1.15 μg/g found in black
kite (Milvus migrans) eggs collected in 1998 in the same area (Jiménez
et al., 2000). In contrast to black kites, an ecologically similar sympatric
raptor breeding at Doñana, and although the diet of the two species is
similar (Veiga and Hiraldo, 1990), breeding red kites are year-round
residents in the Guadalquivir Marshes, being thus more exposed to the
local sources of pollutants. Research studies conducted in red kite eggs
from the Mediterranean Island of Menorca, where the breeding
population have also experienced an overall decline during the last
decade (De Pablo, 2004) also revealed PCB concentrations above the
threshold value reported for negative effects (Jiménez et al., 2007),
while this was not the case for DDE, as found in Doñana. Since legal use
of the pesticide DDT was banned in Spain in 1977 (Zumbado et al.,
2005), it was expected that both DDT and its main metabolite DDE,
should show a decreasing tendency since then, especially DDT.
However, the present study reveals the opposite trend in the case of
DDE, i.e., a high increase in its average levels compared to samples
collected in the 80s, and similar DDT levels to those reported two
decades ago. This may indicate the existence of currently unknown
sources of DDT besides residues of technical DDT used in agriculture
before 1977 that deserve further investigation.
Besides the high concentrations found, it is also important to note
that both DDE and ortho PCB levels were highly variable among
samples (standard deviations were in both cases higher than average
values; see Table 1a). Since we knew the exact location where each
addled egg was collected, we tried to identify common patterns of
variation among sampling locations. Results showed that DDE and
ortho PCB concentrations were significantly higher in eggs collected
from the southern part of Doñana National Park (nests located nearest
to the Atlantic coast) compared to samples from both the Biological
Reserve (BR, at the core of the park) and the northern part of the park
grouping together (U-Mann Whitney test, Z values ≥ 2.55, p ≤ 0.011,
Table 2). No differences between sampling locations were found in
relation to the frequency of eggs with ortho PCB and DDE levels
Fig. 1. Relative contribution of individual congeners of ortho-PCBs to total ortho-PCB levels in red kite eggs from Doñana, Spain.
Table 1b
Concentrations of 2,3,7,8-substituted PCDDs and PCDFs and calculated TEQs for PCBs and PCDD/Fs in red kite eggs, expressed in pg/g on a fresh weight basis (f.w.)
Congener
2378 TCDF
12378 PeCDF
23478 PeCDF
123478 HxCDF
123678 HxCDF
234678 HxCDF
123789 HxCDF
1234678 HpCDF
1234789 HpCDF
OCDF
2378 TCDD
12378 PeCDD
123478 HxCDD
123678 HxCDD
123789 HxCDD
1234678 HpCDD
OCDD
Total PCDDs
Total PCDFs
Total PCDD/Fs
TEQs (pg/g, f.w.)
TEQs PCDDs
TEQs PCDFs
TEQs mono-ortho PCBs
TEQs non-ortho PCBs
Total TEQs
Arithmetic mean
SD
Median
Geometric mean
Range
2.12
0.84
6.59
0.66
0.46
0.31
0.10
0.24
0.12
0.24
2.45
2.85
0.55
1.81
0.26
0.68
2.71
11.3
11.7
23.0
2.20
0.65
3.60
0.41
0.21
0.17
0.06
0.31
0.09
0.23
1.46
1.34
0.20
0.87
0.30
0.37
2.38
5.53
6.76
12.0
1.35
0.56
7.12
0.55
0.40
0.30
0.09
0.18
0.09
0.13
2.27
3.01
0.55
1.77
0.18
0.54
1.70
9.84
10.63
19.34
1.09
0.62
5.53
0.56
0.42
0.25
0.09
0.14
0.10
0.17
1.95
2.54
0.51
1.62
0.19
0.61
1.61
9.90
9.80
19.9
0.07–7.15
0.17–2.19
1.75–12.5
0.20–1.77
0.18–0.93
b 0.04–0.66
b 0.03–0.21
b 0.02–1.17
b 0.04–0.32
b 0.06–0.75
0.39–4.62
1.12–5.18
0.21–0.85
0.68–3.77
0.08–1.20
0.31–1.45
b 0.06–6.80
3.21–18.7
4.02–23.3
7.22–42.0
5.38
8.95
101
122
238
2.72
5.61
109
110
220
5.33
8.39
72
95
171
4.66
7.29
41
64
137
1.81–8.70
2.80–20
1.48–320
0.81–327
7.02–667
above those reported to cause detrimental effects in reproduction
(Fisher exact test: ortho PCBs, p = 0.21; DDE, p = 0.06).
Concerning non-ortho PCBs, concentrations were in the range
0.01–3.7 ng/g (average 1.4 ± 1.2 ng/g), being PCB 126 the most
abundant congener as reported in this and other raptor species (Jiménez
et al., 2007). Total PCDD/Fs showed levels in the low pg/g range
(7.22–42 pg/g), having PCDDs and PCDFs similar contributions in
most samples (Table 1b). Most of the 2,3,7,8-substituted congeners
Fig. 2. Relative contribution of each individual 2,3,7,8-substituted dibenzo-p-dioxin and 2,3,7,8-substituted dibenzofuran to the sum of the concentrations of
polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in red kite eggs from Doñana, Spain.
Table 2
Median values and range (in brackets) of ortho PCBs, non-ortho PCBs, DDE, DDT, PCDDs, PCDFs and TEQs (wet weight basis) calculated for eggs (n = 12) of Red
kites (Milvus milvus) from southern Doñana National Park (southern DNP) and both the Biological Reserve (BR) and northern Doñana National Park (northern DNP)
Southern DNP (n = 4)
BR and northern DNP (n = 8)
Σ ortho
PCBs
μg/g
Σ non-ortho
PCBs
ng/g
DDE
μg/g
DDT
μg/g
78.33 ⁎
(43.87–109.93)
5.47
(0.52–58.08)
3.08 ⁎ ⁎
(1.79–3.73)
0.57
(0.02–1.29)
20.91 ⁎
(7.32–33.55)
1.56
(0.15–14.74)
0.46 ⁎ ⁎
(0.26–0.61)
0.07
(0.003–0.24)
Total
PCDDs
pg/g
Total
PCDFs
pg/g
Total
TEQs
pg/g
16.1
(9.1–18.7)
8.53
(3.2–18.6)
15.3
(8.1–23.3)
6.84
(4–19.9)
527.5 ⁎ ⁎
(292–667)
100.45
(7–201)
U-Mann Whitney test: ⁎ p b 0.05; ⁎ ⁎ p b 0.01.
were detected in all samples, with some exceptions. 1,2,3,7,8,9HxCDF, 1,2,3,4,6,7,8-HpCDF and OCDF were under the limit of detection (LOD) in most cases.
The 2,3,4,7,8-PeCDF was the most abundant congener, contributing with almost a 30% (Fig. 2). Among PCDDs, the most abundant
congeners were 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and OCDD accounting with a 10% each one (Fig. 2).
We found no differences between sampling locations either in PCDD
or PCDF levels (U-Mann Whitney test, Z-values ≤ 1.71, p ≥ 0.089,
Table 2), although average concentration of non-ortho PCBs was again
higher in the southern DNP (U-Mann Whitney test, Z-value = 2.72,
p = 0.007, Table 2). Although no detailed information is available
regarding differences between sampling locations in prey items collected
during the breeding season, studies on winter ecology of radiotagged
birds showed that adult resident kites remain in the proximity of their nest
all year round, with small home ranges when foraging, especially in the
case of females (Heredia et al., 1991). Therefore, it seems likely that
differences observed between the southern DNP and both the BR and
northern DNP are due to different type and/or proportion of prey items,
with individuals breeding nearest the coastal line preying on more
contaminated food than birds nesting inland. In addition, it should be
considered that foraging habits of the red kite include visits to rubbish
dumps during winter (Heredia et al., 1991), which could also contribute to
the contaminants load, especially PCBs, found in the species. Clearly,
further investigation is needed in order to identify the particular sources of
exposure to these chemicals and the origin of the spatial variation detected
within the DNP.
In Fig. 3, it can be observed that, with the exception of one sample
(Mm10), non-ortho PCBs were the major contributors to calculated
TEQs, with a percentage of 50%, ortho PCBs contributing with a 43%
and the remaining 7% contributed by PCDD/Fs. Total mean TEQs in
red kites were 238 pg/g, being the range 7.02–667 pg/g, with samples
collected in the southern part of the Park showing, again, the highest
values (Z-value = 2.72, p = 0.007, Table 2).
The no observed effect level (NOEL) and lowest observed effect level
(LOEL) based on CYP1A induction in bald eagles (H. leucocephalus)
has been reported by some authors to be 100 and 210 pg/g TEQs,
respectively (Elliot et al., 2001). Although species-sensitivity may play a
role, applying these guidelines to red kites in our study, we would expect
the species to experience detrimental effects to dioxin-like toxicity, since
some eggs exceeded the NOEL (67%) and LOEL (33%). It should be
remarked that all samples collected in the southern DNP showed levels
above LOEL (Fisher exact test: p = 0.002). No differences were found
between sampling locations regarding NOEL (Fisher exact test: p = 0.21).
4. Conclusions
The high PCB and DDE concentrations found in red kite eggs
from Doñana could represent a serious element of risk for the
population breeding in this area of particular interest. It is
remarkable that 50% of the samples showed ortho PCB
concentrations higher than a threshold value of 20 μg/g suggested
for reproductive effects of PCBs. Half of the samples showed
DDE concentrations above the threshold level associated with
reproductive impairment in other raptor species, such as the bald
eagle. Even more remarkable than the high PCB and DDE
concentrations reported, is the increase of one order of magnitude
Fig. 3. Relative contribution of the sum of ortho polychlorinated biphenyls (ortho-PCBs), non-ortho polychlorinated biphenyls (non-ortho-PCBs), polychlorinated
dibenzofurans (PCDFs) and polychlorinated dibenzo-p-dioxins (PCDDs) to total toxic equivalent quantities (TEQs) in red kite eggs from Doñana, Spain.
observed in the concentrations of these organochlorines when
compared to samples of the species collected two decades ago.
With the information available to date, it is unknown whether
the detrimental effects reported in other bird species at the levels
found in our study are contributing to the decline of the breeding
red kite population in DNP. However, our results suggest that
organochlorine contaminants should be regarded as an element of
concern in the population under study, in addition to other
conservation problems already reported. Moreover, since Doñana
is an important refuge for wintering red kites from Central
Europe, this concern should be extended to other European
populations, which may also be exposed to these persistent
organic pollutants while wintering in the marshes.
Therefore, special attention should be given to the sources and
fate of these chemicals in DNP. In this regard, further investigations
should be undertaken to find out if organochlorine contamination
is present in other predator species in the area, as well as their
potential health effects on individuals and/or populations.
Measures in order to minimise the exposure to these chemicals
should be applied by the local and regional authorities in order to
preserve the conservation status of the red kite, listed as a species
at critical risk of extinction at regional scale, and Near Threatened
at global-European scale. Despite the high level of protection of
this area, Doñana seems to be far from an isolated pristine
environment. Pollution monitoring studies are strongly encouraged in this and other protected areas.
Acknowledgements
This study was funded by Consejería de Medio Ambiente de
la Junta de Andalucía. We thank F.G. Vilches, J.M. Terrero, G.
García, A. Sánchez, M.C. Medina, H. Lefranc, F.J. Hernández,
and S. Cabezas for their assistance during fieldwork. B. Gómara
wishes to thank Ministerio de Educación y Ciencia and CSIC for
her grant. R. Baos received financial support from the Spanish
Ministerio de Educación y Ciencia through a FPU fellowship.
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