Baos et al. Both models suggest that for a given level of T3 (which was similar between colonies), birds from DdA had higher levels of the pre- cursor T4 (Figure 2). Discussion Heavy metals and As levels. Blood levels of essential metals Cu and Zn were similar between colony locations, and generally lower than those reported in bird populations from other metal-polluted areas (Beyer et al. 2004; Van Eeden and Schoonbee 1996). Contrary to our expectations, nestlings from the reference colony (CC) had higher blood Pb levels than those sampled in DdA. Nevertheless, Pb levels > 200 µg/L, the value reported to cause sub- lethal effects in birds (Franson 1996), were found in only one nestling from CC, with the remaining individuals from both CC and DdA having “background” concentrations. When working with wildlife, one major difficulty rests with the establishment of an absolute con- trol condition, when in fact the best option may be a reference population for comparison. Although no previous studies on any pollutant had been conducted in the reference location, the stork colony was in a natural area far from apparent sources of contamination. This result supports the argument of Norris (2000) that the widespread distribution of many potential endocrine-modulating substances makes it difﬁcult, if not impossible, to ﬁnd a true con- trol population. Blood levels of As were found at concentrations below those reported in birds from contaminated areas (Burger and Gochfeld 1997) and were similar between the two sampled locations. Relationships between hormone concentra- tions, heavy metals, and As levels. Our results showed a positive association between Pb and the stress-induced response elicited after 45 min of handling and restraint regardless of colony location. Of particular importance is that this relationship occurred at Pb levels below those normally associated with detri- mental health effects in birds (Franson 1996). Recent experimental and field studies on passerines exposed to Pb have reported no 2.0 )L 1.5 m / g n (T3 1.0 0.5 CC DdA 2 4 6 12 14 16 8 10 T4 (ng/mL) Figure 2. Relationship between plasma T4 and T3 concentrations in nestling white storks from CC (reference) and DdA (exposed) colonies. 1500 effect on the stress response (Eeva et al. 2003, 2005; Snoeijs et al. 2005); however, these stud- ies measured corticosterone levels just after capture (i.e., baseline concentrations) for which we also did not ﬁnd a signiﬁcant relationship. Lead ranks second on the list of top 20 hazardous substances from the 2005 CERCLA (Comprehensive Environmental Response, Compensation, and Liability Act) Priority List [Agency for Toxic Substances and Disease Registry (ATSDR) 2005]. The wides- pread use of lead arsenate as a pesticide in the early 20th century left a broad legacy of soil and groundwater contamination. Recent or past lead-shot hunting, mining, and smelting activities and industrial effluents are other sources of Pb that have made this element one of the most common metals in contaminated ecosystems (ATSDR 1999). Exposure to Pb is associated with neurobehavioral, hematologic, nephrotoxic, and reproductive effects in birds and other animals (Scheuhammer 1987). Experimental studies in mammals have shown that both adult and developmental Pb exposure elevate baseline corticosterone levels (Cory-Slechta et al. 2004; Der et al. 1977; Vyskocil et al. 1990); however, similar effects have not been reported on stress-induced response in either mammals or avian species. Therefore, we cannot discount other factors potentially correlated with Pb (e.g., pesticide exposure) that may contribute to some degree to the positive relationship we found in storks between Pb and the stress response. Experimental studies are needed to establish a cause–effect relationship. In the present study we also found that the association between Pb and the stress-induced response was greater for birds from single-chick broods than for those from multiple-chick broods. In a previous article (Blas et al. 2005), we reported that single nestlings were reared in nests that experienced brood reduction, which suggested lower parental quality. Reduced attendance by young or inexperienced parents may lead singletons to suffer from environ- mental stressors other than Pb (e.g., a greater exposure to harsh weather conditions). This in turn may explain both their higher levels of maximum corticosterone (Blas et al. 2005) and the stronger relationship between their induced response to stress and Pb. In a recent experi- mental study on rats, Cory-Slechta et al. (2004) provide evidence that Pb exposure and stress interact. Furthermore, the fact that both stress and Pb target brain mesocorticolimbic dopaminergic systems (e.g., Barrot et al. 2000; Cory-Slechta et al. 1997) provides a biological basis for such an interaction. Di Giulio and Scanlon (1985) reported that the highest plasma and adrenal concentrations of cortico- sterone were observed in ducks receiving the highest level of dietary cadmium when they were food-restricted. Similarly, Wayland et al. (2002) found signiﬁcant correlations between renal Cd concentration and the stress response of female common eiders (Somateria mollissima borealis) during the incubating period, when they are reported to fast, whereas no effects were detected in prenesting females. In this sense, although it has to be interpreted with caution because of the small sample size, our result would support the argument that contaminants acting in concert with other stres- sors may have a greater impact on individuals or populations than would be elicited by either the contaminants or other stressors acting alone. Although temporary increases in cortico- sterone are believed to be beneﬁcial, serving to redirect individuals’ behavior and physiology to foraging or self-maintenance and to enhance assimilation of energy during the stressful period, chronic or frequent activation of the stress response can be deleterious, resulting in growth suppression, reproductive dysfunction, and immunosuppression (Wingfield and Kitaysky 2002). The brain is also a target of the adverse effects of prolonged elevation of glucocorticosteroids (Kerr et al. 1991), with deleterious effects that may include impair- ment of cognitive function (Kitaysky et al. 2003). Thus, a compromised HPA function by Pb, alone or in conjunction with other envi- ronmental stressors, could have far-reaching consequences in terms of altered behavioral and metabolic processes necessary for survival. Few studies have reported the effects of metal pollution on the homeostasis of thyroid hormones in birds, especially in natural settings (Leatherland 2000). In the present study, nei- ther heavy metals nor As were related to circu- lating levels of thyroid hormones. However, lower levels of T4 were found in the location with higher concentrations of Pb (CC, the ref- erence site). Although this result may suggest that circulating levels of T4 are susceptible to Pb exposure in nestling storks, colony location and not Pb levels accounted for differences in plasma T4. The studies reporting Pb effects on circulating thyroid hormones have focused on heavily exposed subjects, often with other manifestations of lead poisoning (Robins et al. 1983; but see also Refowitz 1984). Thus, a possible explanation for the lack of signiﬁcant relationships between Pb and plasma thyroid hormones is that metal levels measured in nestling storks were too low to exhibit a signif- icant correlation. Alternatively, location- dependent intrinsic (e.g., genetic) or extrinsic (e.g., environmental) factors other than varia- tion in Pb, including pollutants other than those analyzed (e.g., polychlorinated biphenyls, dioxins, pesticides) could potentially account for our results. Conclusions The present study provides, to our knowl- edge, the ﬁrst evidence that Pb may adversely VOLUME 114 | NUMBER 10 | October 2006 • Environmental Health Perspectives