Baos et al. Materials and Methods Bird species and study area. The white stork is a large (2,200–4,400 g), long-lived wading bird that breeds from North Africa to north- ern Europe. At Iberian latitudes, it has a pro- longed breeding season (from February to July), with chicks hatching after 33–34 days of incubation and depending on both adults for food and shelter during approximately 75 days before ﬂedging (Cramp and Simmons 1980). Fieldwork was conducted in two colonies in southwestern and midwestern Spain during spring 2000. The exposed colony was situated in the Dehesa de Abajo (DdA; Puebla del Río, Sevilla, southwestern Spain), in the vicinity of Doñana National Park and only 1 km from the area affected by the Aznalcóllar mine spill in 1998 (Baos et al. 2006). In this colony, white storks breed in open nests at the top of wild olive trees. During the study, about 300 pairs of storks bred at DdA, one of the largest colonies in the western Palearctic (Pastor et al. 2004). The ecologic features of the exposed colony that may influence the results of our study, such as its size (number of breeding pairs), density of breeders, nestlings’ diet [mainly crayﬁsh; see Blas et al. (2005) for details], and its unique location in relation to anthropogenic disturbance (McNabb 2000; Mullner et al. 2004; Silverin 1998), seriously limited the number of colonies we could use as reference. In addition, a reference colony would have to be located far enough away to ensure that there would not be overlap in the use of habitat during the breeding season. Considering all of these requirements, the most suitable colony for a reference was in Cáceres province (CC), about 230 km north of DdA. The reference colony, approximately 50 breeding pairs, was in a scattered oak forest surrounded by crop ﬁelds and close to a per- manent water source, with crayfish present. Although there were no existing data on metal contamination for this colony, it was located in a natural area far from urban environments and other apparent sources of pollution. Nevertheless, actual metal levels were meas- ured in birds from both colonies for a proper characterization of metal contamination. Field procedure and blood sampling. During the ﬁrst week of June 2000, randomly selected nests of single-chick broods (n = 5 in each colony), and two- or three-chick broods (DdA, n = 15; CC, n = 10) were sampled at both colonies. Blood sampling for hormone and metal residue determinations were per- formed for either the single nestling or, in the case of two- and three-chick nests, for both the oldest and the youngest birds within a brood (DdA, n = 35; CC, n = 23 birds). Storks underwent a standardized capture, handling, and restraint protocol known to elicit an increase in circulating corticosterone in birds (Wingﬁeld and Kitaysky 2002). Blood samples 1498 (~ 1 mL) were taken from the brachial vein within the ﬁrst minute after capture, and again at 2, 10, 30, and 45 min [see Blas et al. (2005) for detailed description of the sampling proto- col]. A greater amount of blood (2–3 mL) was taken 45 min after capture, transferred to a separate vial (1–2 mL), and frozen immediately at –80°C for determination of heavy metals and As. All birds were individually marked with metal and plastic bands, and their wing- chord length and body mass were measured before they were returned to the nest. Blood samples for hormone determination were kept on ice until centrifuged (3,000 rpm for 10 min) the same day of capture, and plasma was frozen and stored at –80°C for radioimmunoassay (RIA). Thyroid hormones were quantified in plasma samples taken 2 min postcapture. The age of nestlings, estimated following the method described by Chozas (1983), ranged between 24 and 59 days. We calculated a body condition index as the individual’s residual value from a reduced major axis regres- sion of log10-body mass on log10-wing-chord length, following the method of Green (2001). Sex determination was performed through DNA analyses on blood (Ellegren 1996). This project was approved by the Spanish Animal Care Committee for research involving animals, and the permission to capture nestling white storks at DdA and CC was given by the environmental agencies of the governments of Andalusia and Extremadura, respectively. Birds were treated humanely and with regard for alleviation of suffering during handling. Determination of corticosterone and thyroid hormones. We used RIAs to quantify plasma concentrations of corticosterone and thyroid hormones (T4, T3). In all assays, sam- ples were measured in duplicate, and internal control samples containing known amounts of the hormones were included to ensure that the accuracy of each assay was high and to determine between-assay and within-assay variability. Serial dilutions of samples pro- duced displacement curves that were parallel with each hormone standard curve, indicating that the RIAs were suitable for measuring cir- culating hormone levels in storks. For cortico- sterone, samples from the present study were analyzed in the same series of RIAs reported previously (Blas et al. 2005); the assay proce- dures and parameters were detailed in the same study. Circulating thyroid hormones are mostly bound to plasma proteins, with only a small amount circulating as free (unbound) hor- mones. We determined total amounts (i.e., bound plus unbound) of T4 and T3 in unex- tracted serum following procedures and reagents described by Smits et al. (2002). These RIAs use 150 µg 8-anilino-1-naphthalene sul- fonic acid in each assay tube, and barbital buffer to eliminate interference of serum bind- ing proteins. Samples were diluted 4- to 10-fold prior to use in the assays and meas- ured near the midrange of the standard curves. This was well above the minimum limits of detection (LOD) of 62 pg/mL of plasma for T3 and 372 pg/mL of plasma for T4. All sam- ples were measured in a single T3 or T4 RIA; the percent coefficient of variation (%CV) within the T3 RIA was 7.4%, whereas the %CV within the T4 RIA was 6.3%. Antiserum and purified hormones used for standards were purchased from Sigma Chemical Company (St. Louis, MO, USA); labeled steroids were purchased from New England Nuclear (Mandel Scientiﬁc, Guelph, Ontario, Canada). Determination of heavy metals and As. We followed the methodology of Benito et al. (1999) for determination of Pb, Zn, Cu, Cd, and As. Blood samples (1 mL) were prepared using a 30-min ultrasonic extraction with Triton X-100 (Merck Farma y Química, S.A., Valencia, Spain). Zn was analyzed by flow injection-ﬂame-atomic absorption spectrome- try using an atomic absorption spectrometer (model 3300) equipped with a ﬂow injection analysis system for atomic spectroscopy (FIAS- 400) and an autosampler (AS-90; PerkinElmer Hispania, S.A., Madrid, Spain). Pb, As, Cu, and Cd were measured using a longitudinal AC Zeeman (AAnalyst 600) atomic absorption spectrometer equipped with a transversely heated graphite atomizer and pyrolitic graphite-coated tubes with inserted L´vov platform (PerkinElmer Hispania, S.A.). Calibration was performed following the method of additions standard curve (Benito et al. 1999). The LODs were 0.23 mg/L for Zn, 2 µg/L for Pb, 24 µg/L for As, 13 µg/L for Cu, and 0.10 µg/L for Cd. Analytical results under the LOD were set to half the respective LOD. The precision as measured by triplicate analysis, expressed as the relative SD, was always < 10%. The range of recovery evaluated by spiking blood samples with each of the ele- ments was 85–115%. We established accuracy for Pb and Cd by analyzing certiﬁed reference sample of bovine blood (CRM-195; Institute for Reference Materials and Measurements, Geel, Belgium). For As, and Cu, we used a cer- tified reference sample, TORT-2 (lobster hepatopancreas; National Research Council of Canada, Ottawa, Ontario, Canada). The International Atomic Energy Agency (Vienna, Austria) provided an A-13 reference sample of animal blood for Zn accuracy. The values obtained for all of these elements were consis- tent with the certiﬁed reference values. Statistical analyses. To test whether heavy metals and As were related to hormone levels, we analyzed baseline and maximum recorded corticosterone and plasma T4 and T3 concen- trations separately through general linear VOLUME 114 | NUMBER 10 | October 2006 • Environmental Health Perspectives