Research Adrenocortical Response to Stress and Thyroid Hormone Status in Free-Living Nestling White Storks (Ciconia ciconia) Exposed to Heavy Metal and Arsenic Contamination Raquel Baos,1 Julio Blas,2 Gary R. Bortolotti,2 Tracy A. Marchant,2 and Fernando Hiraldo1 1Department of Applied Biology, Estación Biológica de Doñana, Sevilla, Spain; 2Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada BACKGROUND/OBJECTIVE: Endocrine parameters have proven useful in the detection of early or low-level responses to pollutants. Although most of the studies on endocrine modulation have been focused on processes involving gonadal steroids, contaminants may target other parts of the endocrine system as well. In this study we examined the adrenocortical stress response and thyroid hormone status in free-living nestling white storks (Ciconia ciconia) in relation to heavy metals (zinc, lead, copper, cadmium) and arsenic levels in blood. METHODS: Fieldwork was conducted in an area polluted by the Aznalcóllar mine accident (south- western Spain) and in a reference site. We used a standardized capture, handling, and restraint pro- tocol to determine both baseline and maximum plasma corticosterone. Circulating levels of thyroxine (T4) and triiodothyronine (T3) were also measured. RESULTS: No effects of metals or As were found on baseline corticosterone, but maximum levels of corticosterone were positively related to Pb in both locations. This relationship was stronger in sin- gle nestlings than in birds from multiple-chick broods, which suggests a greater impact of Pb on more stressed individuals. Metal pollution did not affect plasma T4 or T3 levels, although thyroid status differed with location. CONCLUSIONS: Because a compromised hypothalamus–pituitary–adrenal (HPA) function can have far-reaching consequences in terms of altered behavioral and metabolic processes necessary for sur- vival, our results suggest that birds exposed to sublethal Pb levels may be at risk through an altered adrenocortical stress response, and further support the idea that HPA axis-related end points might be useful indicators of metal exposure and potential toxicity in wild animals. KEY WORDS: Aznalcóllar, corticosterone, free-living birds, handling-restraint protocol, lead, metal pollution, stress response, thyroxine, triiodothyronine, white stork. Environ Health Perspect 114:1497–1501 (2006). doi:10.1289/ehp.9099 available via http://dx.doi.org/ [Online 11 July 2006] In nature, animals can be used as bioindicators to provide an early warning of potential adverse, contaminant-related effects on organ- isms or populations themselves, on organisms or populations that prey upon them, and as sentinels for exposure and effects on humans (Fox 2001). Endocrine assessment is a useful diagnostic tool in the detection of early or low-level responses to pollutants, responses that may precede more significant health problems (Kendall et al. 1998). Although most of the studies on endocrine disruption or modulation have been focused on reproductive problems and behavioral abnormalities related to repro- duction, contaminants may target other parts of the endocrine system more commonly than they disrupt processes involving gonadal steroids (Damstra et al. 2002; Hinson and Raven 2006). The hypothalamus–pituitary– adrenal (HPA) axis is an important system that regulates and integrates many physio- logic functions in response to environmental stressors (Wingfield and Kitaysky 2002). Activation of the HPA axis during a stress response results in glucocorticoid secretion from the adrenal glands (mainly corticos- terone in birds). This, in turn, initiates several important physiologic changes including effects on intermediary metabolism, growth, immune function, and inﬂammatory responses (reviewed by Kitaysky et al. 2003; Sapolsky et al. 2000). Thyroid hormones— thyroxine (T4) and triiodothyronine (T3)—also play an important role in metabolism and exert pro- found effects on avian development (both dif- ferentiation and growth) (McNabb 2000). The impairment of adrenal or thyroid function by contaminant exposure may occur in the absence of gross toxicologic effects and may be critical to developing individuals, with severe consequences such as reduced growth, reduced cognitive capabilities, and impaired immune function (Schantz and Widholm 2001; Smits et al. 2002; Vos et al. 2000). This may result in permanent alterations in the functional capacity of these individuals, with negative long-term consequences to survival and overall fitness (McArthur et al. 1983). Although abnormal glucocorticoid concentra- tions and impaired functioning of the HPA axis, as well as thyroid modulation, have been linked to exposure to different classes of pollu- tants (Leatherland 2000; Lorenzen et al. 1999; Mayne et al. 2004, 2005; Smits et al. 2002; Vos et al. 2000; Vyskocil et al. 1990), little is known about the effects of sublethal metal exposure on the adrenal and thyroid systems in free-living birds (Wayland et al. 2002, 2003). Particularly lacking are studies of endocrine modulation in altricial species (Scannes and McNabb 2003). Environmental Health Perspectives • VOLUME 114 | NUMBER 10 | October 2006 We conducted a ﬁeld study to examine the relationships between exposure to heavy metals (zinc, copper, lead, cadmium) and arsenic and the adrenocortical response to stress in wild nestling white storks (Ciconia ciconia) hatched in the area affected by the Aznalcóllar mine accident (southwestern Spain) (Grimalt et al. 1999), and in a reference location for compari- son. We also evaluated the potential effects of metal pollution on thyroid hormone status in these two groups. Previous studies in the area affected by the mine spill have reported higher genotoxic damage in nestling storks than in controls, with damage having increased 2- to 10-fold between 1999 and 2002 (Pastor et al. 2004). Additionally, nestling storks hatched in the years after the accident had skeletal deformities that were partially related to metal pollution (Smits et al. 2005). We used a standardized capture, han- dling, and restraint protocol to assess the adrenocortical response to stress (Wingfield and Kitaysky 2002), and both baseline and maximum plasma corticosterone, as well as plasma T4 and T3 levels were studied in rela- tion to heavy metals and As concentrations in blood. The effects of other potentially con- founding factors affecting endocrine status, such as location, brood size, age, body condi- tion, and sex, were also considered. Address correspondence to R. Baos, Department of Applied Biology, Estación Biológica de Doñana (CSIC), Avda Mª Luisa s/n Pabellón del Perú, Apdo 1056, E-41013 Sevilla, Spain. Telephone: (34) 95 423 23 40. Fax: (34) 95 462 11 25. E-mail: raquel@ ebd.csic.es We thank A. Román, A. Tamayo, J. Ramírez, M.C. Quintero, J.L. Arroyo, B. Jiménez, R. Tobar, C. Alonso, C. Sánchez, R. Álvarez, R. Rodríguez, G. García, and N. Pastor for assistance during ﬁeld- work; J.C. Núñez, J.L. Tella, and S. Cabezas for pro- viding logistic support and access to the breeding colonies; V. Benito for analyzing metals; and R. Jovani for providing valuable comments on earlier drafts of the manuscript. This work was supported by Empresesa de Gestion Medioambiental, S.A., the European Union (i3P), and the Isabel María López Martínez Memorial Scholarship in a grant to J.B.; the Spanish Ministerio de Educación y Cultura in a postdoctoral fellowship to J.B. and a predoctoral F.P.U fellowship to R.B., and a Natural Sciences and Engineering Research Council of Canada grant to G.R.B. The Junta de Andalucía also provided funds to support this research. The authors declare they have no competing ﬁnancial interests. Received 14 February 2006; accepted 11 July 2006. 1497