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 contamina- tion 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: (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
2.1. Sampling
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
(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
63Ni 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 CP-
3800 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).
3. Results and discussion
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
levels reported in previous studies conducted in the 80s in the same species. Earlier studies by González et al. (1983) in addled eggs from
13,562 517–109,933
sources of DDT besides residues of technical DDT used in agriculture
PCBs
DDTs (ng/g, f.w.)
DDE 9316 10,888 4711 3654 152–33,549
TDE 0.20 0.37 0 0.11 0.03–1
DDT 207 202 129 103 3–608
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 / Arithmetic mean / SD / Median / Geometric mean / Range2378 TCDF / 2.12 / 2.20 / 1.35 / 1.09 / 0.07–7.15
12378 PeCDF / 0.84 / 0.65 / 0.56 / 0.62 / 0.17–2.19
23478 PeCDF / 6.59 / 3.60 / 7.12 / 5.53 / 1.75–12.5
123478 HxCDF / 0.66 / 0.41 / 0.55 / 0.56 / 0.20–1.77
123678 HxCDF / 0.46 / 0.21 / 0.40 / 0.42 / 0.18–0.93
234678 HxCDF / 0.31 / 0.17 / 0.30 / 0.25 / b 0.04–0.66
123789 HxCDF / 0.10 / 0.06 / 0.09 / 0.09 / b 0.03–0.21
1234678 HpCDF / 0.24 / 0.31 / 0.18 / 0.14 / b 0.02–1.17
1234789 HpCDF / 0.12 / 0.09 / 0.09 / 0.10 / b 0.04–0.32
OCDF / 0.24 / 0.23 / 0.13 / 0.17 / b 0.06–0.75
2378 TCDD / 2.45 / 1.46 / 2.27 / 1.95 / 0.39–4.62
12378 PeCDD / 2.85 / 1.34 / 3.01 / 2.54 / 1.12–5.18
123478 HxCDD / 0.55 / 0.20 / 0.55 / 0.51 / 0.21–0.85
123678 HxCDD / 1.81 / 0.87 / 1.77 / 1.62 / 0.68–3.77
123789 HxCDD / 0.26 / 0.30 / 0.18 / 0.19 / 0.08–1.20
1234678 HpCDD / 0.68 / 0.37 / 0.54 / 0.61 / 0.31–1.45
OCDD / 2.71 / 2.38 / 1.70 / 1.61 / b 0.06–6.80
Total PCDDs / 11.3 / 5.53 / 9.84 / 9.90 / 3.21–18.7
Total PCDFs / 11.7 / 6.76 / 10.63 / 9.80 / 4.02–23.3
Total PCDD/Fs / 23.0 / 12.0 / 19.34 / 19.9 / 7.22–42.0
TEQs (pg/g, f.w.)
TEQs PCDDs / 5.38 / 2.72 / 5.33 / 4.66 / 1.81–8.70
TEQs PCDFs / 8.95 / 5.61 / 8.39 / 7.29 / 2.80–20
TEQs mono-ortho PCBs / 101 / 109 / 72 / 41 / 1.48–320
TEQs non-ortho PCBs / 122 / 110 / 95 / 64 / 0.81–327
Total TEQs / 238 / 220 / 171 / 137 / 7.02–667
above those reported to cause detrimental effects in reproduction