Endocrine parameters and heavy metals in storks mixed models (GLMMs) using the MIXED Procedure of SAS (SAS Institute 2001), which simultaneously accounts for random effects, ﬁxed effects, and covariates. When necessary, dependent variables were log10-transformed to obtain a normal distribution. Concentrations of heavy metals and As were log10-trans- formed, when necessary, and were included in the models as covariates. We controlled for the potential effects of colony location, brood size, age, sex, and body condition (Blas et al. 2005, 2006) by including them in the models as either fixed factors (colony location, brood size, sex) or covariates (age, body condition). All of the two-way interactions between met- als, As, and the rest of the variables were also tested. We considered baseline corticosterone concentration and time (in minutes following capture) to reach the maximum recorded lev- els as covariates in the analysis performed on the induced response to stress. T4 and T3 lev- els were also taken into account as appropriate in the models to test thyroid status. Brood size was categorized as either single- (one-chick nests) or multiple-chick broods (two- or three- chicks nests), because we found no differences in either baseline or stress-induced cortico- sterone levels between nestlings from two- and three-chick nests (Blas et al. 2005). To avoid pseudoreplication derived from the lack of independence in values from nestlings within the same brood and sharing the same colony, we included the nest as a random variable (nested into colony; Littell et al. 1996). The models were constructed by a stepwise forward procedure, testing the signiﬁcance of each variable and adding only the variable that resulted in a better ﬁt to the model (Baos et al. 2006). Final (i.e., minimum adequate) models retained only those terms showing a signiﬁcant effect on hormone concentrations at a > 5% rejection probability. All tests were two-tailed. Results Heavy metals and As levels. Concentrations of Cu, Zn, and As did not differ between loca- tions (Table 1). Pb levels were significantly higher in nestling storks from the reference colony (Table 1), although only one individual from CC (4.3 %; n = 23) and none from DdA showed Pb concentrations > 200 µg/L, the value reported to cause sublethal effects in birds (Franson et al. 1996). All blood Cd con- centrations were < LOD (0.10 µg/L) in both locations; therefore, Cd was not considered in subsequent analyses. Relationships between hormone concentra- tions, heavy metals. and As levels. Baseline corticosterone. We found no signiﬁcant rela- tionships between heavy metals or As and baseline corticosterone levels, either tested alone (F < 2.78; p > 0.110) or through inter- actions with colony location, brood size, age, sex, and body condition (F < 1.60; p > 0.222). Only colony location (F1,32 = 35.91; p < 0.0001) and brood size (F1,23 = 6.03; p = 0.022) were related to baseline levels of corticosterone, with birds from CC showing higher hormone concentrations (mean ± SD, 30.6 ± 11.6 ng/mL; n = 23) than those from DdA (14.1 ± 7.3 ng/mL; n = 35), and single nestlings showing higher levels (32.3 ± 17.3 ng/mL; n = 10) than birds from multiple- chick broods (18.2 ± 9.4 ng/mL; n = 48). Stress-induced corticosterone. Maximum corticosterone concentration was positively related to blood Pb levels (F1,20 = 10.93; p = 0.004; Table 2) in both colony locations (interaction term, Pb × colony, F1,19 = 0.58; p = 0.456), and singletons had higher levels of Table 1. Mean ± SD (range) of heavy metals and arsenic (µg/L or mg/L wet weight) in blood of nestling white storks according to colony location. Colony locationa CC (reference) DdA (exposed) Cu (µg/L) 359.5 ± 55.5 (233–451) n = 23 357.2 ± 41.1 (247–426) n = 34 Pb (µg/L) 90.7 ± 51.0 (6–214) n = 23 44.4 ± 33.6 (ND–126) n = 35 Zn (mg/L) 2.8 ± 0.5 (1.8–3.8) n = 22 2.9 ± 0.4 (2.1–3.7) n = 35 As (µg/L) 48.6 ± 46.8 (ND–205) n = 20 34.5 ± 51.4 (ND–248) n = 32 ND, below the LOD (see “Materials and Methods” for details). aResults of GLMMs testing for differences between colony locations using normal error and identity link function (nest = random factor): Cu, F1,33 = 0.05, p = 0.829; Pb, F1,33 = 7.59, p = 0.010; Zn, F1,33 = 0.31, p = 0.579; As, F1,31 = 3.11, p = 0.088. Table 2. Summary of results from the GLMM explaining stress-induced response (i.e., maximum cortico- sterone concentration after 45 min of handling and restraint) in nestling white storks (n = 58 birds). Effect Intercept Baseline corticosterone Age Brood size Pb (log) Pb (log) × brood size All other effects Estimate 16.660 0.246 0.349 –38.160b 3.596 25.941b SE 7.666 0.113 0.134 16.484 2.892 9.601 F-Value (df) 2.17a (32) 4.69 (1,20) 6.74 (1,20) 5.36 (1,20) 10.93 (1,20) 7.30 (1,20) < 2.80 p-Value 0.037 0.043 0.017 0.031 0.004 0.014 > 0.113 df, degrees of freedom. The estimated effect, SE, F-values, and associated probabilities are shown for those variables that signiﬁcantly improved the ﬁt of the model. at-Test. bEstimate corresponds to brood size = 1. corticosterone (53.3 ± 14.6 ng/mL; n = 10) than nestlings from multiple-chick broods (42.9 ± 9.3 ng/mL; n = 48; F1,20 = 5.36; p = 0.031; Table 2). The interaction term Pb × brood size was also signiﬁcant (F1,20 = 7.30; p = 0.014), suggesting that Pb had a greater effect on the stress-induced corticosterone of single nestlings than on those of multiple- chick broods (Table 2, Figure 1). Neither Cu, Zn, nor As levels alone or as part of inter- action terms showed any signiﬁcant relation- ship with maximum corticosterone values (F < 2.80; p > 0.113). Age was positively related to the stress-induced corticosterone secreted after applying our handling and restraint protocol (F1,20 = 6.74; p = 0.017; Table 2). The final model also included a positive association between baseline and max- imum corticosterone concentrations (F1,20 = 4.69; p = 0.043; Table 2), but neither sex, body condition, nor time elapsed to reach the maximum hormone concentration showed a signiﬁcant effect. Thyroid hormones. The analysis of plasma T3 levels revealed that neither heavy metals, As concentrations, brood size, sex, age, body con- dition, nor any of the interaction terms accounted for differences in hormone levels (F < 3.34; p > 0.085). Plasma T4 concentrations were positively related to T3 (F1,21 = 13.20; p = 0.002) as expected, given that T4 is the pre- cursor of active T3. Although absolute T3 levels were similar between colonies (CC, 1.05 ± 0.3 ng/mL, n = 23; DdA, 1.09 ± 0.4 ng/mL, n = 35), when controlling for T4 the model showed a signiﬁcant effect of location (F1,31 = 4.67; p = 0.039). Regarding the variables affecting T4 con- centrations, only colony location had a signiﬁ- cant effect, being higher in DdA (9.5 ± 3.2 ng/mL; n = 33) than in CC nestlings (5.3 ± 1.5 ng/mL; n = 22; F1,31 = 40.41; p < 0.0001). No remaining variables or interactions were significant in the final model accounting for variation in plasma T4 (F < 3.19; p > 0.089). Single-chick brood Multiple-chick brood )L 1.9 m / (ng 1.8 e n o tre 1.7 s o c i tro 1.6 c m um1.5 i x a M1.4 0 0.5 2.0 2.5 1.0 Pb (μg/L) 1.5 Figure 1. Relationships between Pb levels (log-µg/L) in blood and the stress-induced response (i.e., maxi- mum corticosterone concentration, log-ng/mL) in nestling white storks from single chick-broods (n = 10) and multiple-chick broods (n = 48) after 45 min of handling and restraint (from both colonies). Environmental Health Perspectives • VOLUME 114 | NUMBER 10 | October 2006 1499