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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 (southwestern Spain) and in a reference site. We used a standardized capture, handling, and restraint protocol to determine both baseline and maximum plasma corticosterone. Circulating levels of thyroxine (T4) and triiodothyronine (T3) were also measured.ResultsNo 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 single 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 survival, 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. [ABSTRACT FROM AUTHOR]

Top-predators serve as sentinels of ecosystem health due to their sensitivity to environmental contamination. However, our understanding on how contaminants affect individual fitness is still scarce, especially for long-lived species. Here, we measured blood concentrations of 4 metals (Cd, Cu, Pb, and Zn) and one metalloid (As) in black kite (Milvus migrans) adults from Doñana National Park (SW Spain) following a major mine spill. Besides temporal variation (1998-2001) in metal and As concentrations, we tested how metal and As profiles changed across individuals in relation to their sex, age or breeding status, and examined whether metal and As concentrations affected individuals' fitness (breeding success and mortality). We found that, overall, blood concentrations of Pb, Cd and As increased throughout the study period in black kites, mirroring the progressive increase previously reported for their main prey. Both sex (Cu and Zn) and breeding status (Zn and Pb) affected element concentrations. Non-breeding (floater) females had higher levels of Zn than their breeding counterparts. The same pattern of higher contamination in floaters was observed for Pb, which might be related to differences in diet and foraging activity between breeders and floaters. The percentage of the individuals with Pb concentrations that exceeded toxicity thresholds (> 200 μg/L) was relatively high (15.4 %). Moreover, Pb concentrations were negatively correlated to males' breeding success. We found no clear evidence to support metal and As effects on survival or life expectancy after accounting for environmental and individual sources of variation. Our results highlight the importance of long-term studies of marked individuals in wild populations; detailed knowledge of ecological processes relevant to these populations, combined with measures of contaminant exposure at individual level, provides opportunities to enhance our understanding of its fitness effects and potential demographic consequences., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Accidents at mines involving stored tailings have produced catastrophic environmental damage. In April 1998 the dam of the Aznalcóllar mine tailings pond in the surroundings of the Doñana National Park (southwestern Spain) broke, discharging into the Guadiamar River more than 6 million m 3 of toxic mud and acidic water with high concentrations of heavy metals and arsenic. We used the Eurasian otter (Lutra lutra) as sentinel species to assess the potential impact of the toxic spill on the river ecosystems and their recovery with time by studying the spatial and temporal variation (1999-2003, 2006) of selected trace element (Cu, Zn, Cd, Pb and As) concentrations in feces. Throughout the sampling period, the highest heavy metal and As levels were found in the most spill-affected reaches of the Guadiamar River (i.e., the Middle and, to a lesser extent, the Lower reaches), pointing out the mining accident as the main origin of the contamination. Overall, levels of trace elements decreased with the time elapsed since the toxic spill, except for Cd (F 1,352  = 0.29, P = 0.59). However, rebounds for some elements (Pb, As, and Cu) were also observed, especially in the Middle and Lower reaches of the river, which might be attributed to the residual contamination in abiotic compartments and/or new inputs from industrial and agricultural activities in the nearby areas. Concentrations were relatively high when compared to those reported for both our reference area (Guadalete River) and other metal-polluted zones. We found that the estimated amounts of Pb and As ingested during the first years after the spill in the Guadiamar Middle reach would be high enough to cause reproductive issues. This could affect the local population recovery, although evidence on distribution range and numbers suggests otherwise, with thriving populations at regional scale. Our results support the role of otters as sentinel species for biomonitoring contaminants and thus to evaluate fluvial ecosystem health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background/objective: Exposure to environmental contaminants may result in reduced reproductive success and long-lasting population declines in vertebrates. Emerging data from laboratory studies on model species suggest that certain life-stages, such as development, should be of special concern. However, detailed investigations of long-term consequences of developmental exposure to environmental chemicals on breeding performance are currently lacking in wild populations of long-lived vertebrates. Here, we studied how the developmental exposure to a mine spill (Aznalcóllar, SW Spain, April 1998) may affect fitness under natural conditions in a long-lived bird, the White Stork (Ciconia ciconia)., Methodology: The reproductive performance of individually-banded storks that were or not developmentally exposed to the spill (i.e. hatched before or after the spill) was compared when these individuals were simultaneously breeding during the seven years after the spill occurred (1999-2005)., Principal Findings: Female storks developmentally exposed to the spill experienced a premature breeding senescence compared with their non-developmentally exposed counterparts, doing so after departing from an unusually higher productivity in their early reproductive life (non-developmentally exposed females: 0.5 ± 0.33SE fledglings/year at 3-yr old vs. 1.38 ± 0.31SE at 6-7 yr old; developmentally exposed females: 1.5 ± 0.30SE fledglings/year at 3-yr old vs. 0.86 ± 0.25SE at 6-7 yr old)., Conclusions/significance: Following life-history theory, we propose that costly sub-lethal effects reported in stork nestlings after low-level exposure to the spill-derived contaminants might play an important role in shaping this pattern of reproduction, with a clear potential impact on population dynamics. Overall, our study provides evidence that environmental disasters can have long-term, multigenerational consequences on wildlife, particularly when affecting developing individuals, and warns about the risk of widespread low-level contamination in realistic scenarios.

The occurrence of the emerging chlorinated flame retardant Dechlorane Plus (DP) and three of its possible degradation products was investigated in white stork eggs from two colonies in Spain. The average DP concentrations were 401 pg/g wet weight (w.w.) for the urban/industrial colony and 105 pg/g w.w. for the rural colony. One possible degradation product, anti-[DP-1Cl], was found in approximately 10% of the samples. No significant stereoisomer enrichments were detected in any colony based on the average anti-DP fractional abundances found which agrees with previous studies in herring gulls. The relationship between DP and PBDE contents in both colonies was explored leading to different scenarios, i.e. no correlation was found in the urban colony but they were statistically correlated in the rural colony. To the best of our knowledge, this is the first study to report DP in a species from a terrestrial food web, and also to report a DP degradation product in biota., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Polybrominated diphenyl ethers (PBDEs) are ubiquitous pollutants for which there is still a lack of knowledge about the environmental behavior and fate of the higher brominated congeners (octa- to deca-BDEs). In this study, the PBDE content and congener profiles in failed eggs from two colonies of white stork (Ciconia ciconia) in Spain were studied. The average total PBDE concentration was 1.64ng/g (wet weight, w.w.) for the rural colony and 9.08ng/g (w.w.) for the urban colony. Higher brominated BDEs dominated the congener profiles of both colonies. Of particular interest was the determination of BDE-209 as the dominant congener accounting for 44.1% and 38.6% of the total PBDE content in the rural and urban colonies, respectively. BDE-202, considered an indicator of BDE 209 debromination, was detected in 83% and all of the samples from rural and urban colonies, respectively. The observed congener profile in which BDE-207>BDE-208>BDE-206 does not correspond to any known technical PBDE mixture and is evidence for possible BDE-209 degradation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Site-quality is a major determinant of fitness but its effect can be confounded by individual quality, a relationship that has been little studied in large, long-lived vertebrates. The fitness effects of quality estimates depend on the assumption of co-variation between individual and territory quality and can be framed as five working hypotheses: no effect on fitness, exclusive effect of individual quality, exclusive effect of site quality, and independent or interactive effects of the two. We explored such a framework using a medium-sized raptor, the black kite Milvus migrans, as a model species. Individual and territory quality co-varied, but the strength of the relationship varied across different estimates of individual quality (age, body size, or mass residuals). Short-term production of fledglings was related to the independent effects of both individual and territory quality. However, longer-term production of recruits was related solely to territory quality. The disappearance of individual quality effects over the long-term may be caused by antagonistic selective pressures acting during different stages of the life cycle. Our results contribute to a growing appreciation of the long-term fitness-benefits of advantages experienced in early life and highlight the importance of a long-term perspective in studies assessing the effects of individual and territory quality. In our case study, prioritizing sites for conservation on the basis of territory quality may be a feasible pathway to maintain the viability of the population. However, scenarios where such a method could be inefficient have been previously reported, suggesting caution in its application. More studies are needed to understand the generality of the efficiency of priority-setting approaches based on site quality.

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 microg/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 microg/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 (>20 microg/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.

Osteoclasts are large, multinucleated cells, which originate from the fusion of macrophages. They play a central role in bone development and remodeling via the resorption of bone and are thus important mediators of bone loss, which leads to osteoporosis. IL-1R-associated kinase (IRAK)-M is a pseudokinase, which acts as a negative modulator of innate immune responses mediated by TLRs and IL-1R. Recently, it has been reported that IRAK-M also participates in the control of macrophage differentiation into osteoclasts. In addition, it was shown that IRAK-M knockout mice develop a strong osteoporosis phenotype, suggesting that down-regulation of this molecule activates osteoclast-mediated bone resorption. We studied the effect of the osteoporosis-inducing glucocorticoid, 6-methylprednisolone (6-MP), on IRAK-M expression in osteoclasts. Our results showed that osteoclasts, derived from THP-1 and RAW cells as well as human blood monocytes, differentiated into osteoclasts, express high levels of IRAK-M at mRNA and protein levels. In addition, 6-MP down-regulates IRAK-M expression, which correlates with an increased activation of bone resorption. These findings suggest a mechanism of corticosteroid-induced osteoporosis and open new avenues for treating this endemic disease of Western societies.

In 1998, the Aznalcóllar mine tailings dyke in southwestern Spain broke, flooding the Agrio-Guadiamar river system with acid tailings up to the borders of one of the largest breeding colonies of white storks in the western Palearctic, Dehesa de Abajo. Over the following years, a high proportion of nestlings developed leg defects not seen before the spill, prompting this study. Nestlings with deformed legs had significantly lower plasma phosphorous (P) and higher Ca:P ratios than non-deformed cohorts in the first two years, but in the third year, when more, younger birds were studied, plasma P ranged from much higher to much lower in the affected colony compared with reference birds. Coefficients of variation for phosphorous were 19% and 60%, in reference and contaminated colonies, respectively. Storks from the contaminated colony were unable to control P levels and Ca:P ratios within the narrow limits necessary for normal bone development.

Studies of birds from Doñana (southwestern Spain) after the Aznalcóllar mining accident (April 1998) have reported high levels of genetic damage when compared to conspecifics from reference areas. However, potential relationships between DNA damage and metal pollution have not yet been reported. The aim of the present study was to investigate the current levels of Zn, Pb, As, Cu, and Cd and to determine if they were associated with the genetic damage observed in free-living, nestling white storks (Ciconia ciconia) and black kites (Milvus migrans) born in the Doñana area after the mining spill. Blood concentrations of heavy metals and of As were quantified and DNA damage (comet assay) was determined in 258 storks and 132 kites monitored during a four-year period (1999-2002). Correlations between these elements and genetic damage varied between species and throughout years within species. Some elements did not show any relationship with DNA damage (e.g., Pb), whereas others had a significant correlation (e.g., As in storks, and Cu and Cd in kites) or only marginal statistical effects (e.g., Zn and Cd in storks, and As in kites) in some years but not in others. These results suggest that nestling white storks and black kites were affected, in part, by the elements studied, but they alone do not satisfactorily explain the observed DNA damage. Moreover, our results show that species-specific differences should be carefully considered when planning schemes for pollution monitoring, and highlight the need for including the temporal scale into the study of the pollutants effects in the wild.

The post-natal development of the adrenocortical response to stress was investigated in European white storks. Sixty wild nestlings aged 24-59 days old were subjected to a standardized capture and restraint protocol, and the time-course pattern of the response to stress was assessed through determination of circulating corticosterone in blood samples collected at five fixed times during the 45-min period following capture. The time course of the response was best fit to a third-order function of handling time, and showed a strong effect of age. Although age did not affect baseline titers and all birds showed a positive post-capture increase in circulating corticosterone, age had a positive effect on the relative increase from baseline titer, the recorded time to reach maximum level, and the acute concentration after 10 min following capture and restraint. While young nestlings displayed very little response to capture, the response near fledging resembled the typical adrenocortical pattern widely reported in fully developed birds. Our results concur with those found in altricial and semi-altricial species, and suggest that non-precocial birds follow a similar mode of development of the hypothalamic-pituitary-adrenal (HPA) axis. The fact that HPA sensitivity to stress is functional suggests that young storks gradually develop emergency responses of adaptive value and are able to overcome acute perturbations in spite of their parental dependence, at least during the last two-thirds of post-natal development. According to the Developmental Hypothesis, such gradual changes would allow nestlings to respond to perturbations as a function of the specific behavioral and physiological abilities of their age. The potential sources of stress that nestlings have to face during development (i.e., weather conditions, dietary restrictions, and social competition) are discussed according to developmental changes in behavioral and physiological abilities.

In the Aznalcóllar mining accident (April 1998), nearly six million cubic meters of toxic wastes were spilled in the surroundings of the Doñana National Park (southwestern Spain). The present study focused on the likely effects of metal pollution on the immune system of nestling white storks (Ciconia ciconia) and black kites (Milvus migrans) sampled in the nearby area. Using the phytohaemagglutinin skin test, we examined cell-mediated immune response (CMI) in relation to Pb, Zn, Cu, Cd, and As concentrations in blood of 281 nestling white storks and of 89 black kites. The former species was monitored along a four-year period (1999, 2001-2003), while black kites were sampled in 1999. Overall, average levels of heavy metals and As were relatively low when compared to those reported for birds in metal-polluted areas. Copper showed a negative effect on CMI in both species, although the relationship was significant only for white storks in 2002. We found no evidence that environmental exposure to Pb, Zn, As, and Cd had any effect on nestlings' CMI. Interannual consistency is revealed as an important factor, supporting the need of long-term studies when assessing the immunotoxic effects of metal exposure in the wild.

Although blood monocytes possess significant cytotoxic activity against tumor cells, tumor-infiltrating monocytes are commonly deactivated in cancer patients. Monocytes pre-exposed to tumor cells show significantly decreased expression levels of TNF-alpha, IL-12p40, and IL-1R-associated kinase (IRAK)-1. Activation of the Ser/Thr kinase IRAK-1 is an important event in several inflammatory processes. By contrast, another IRAK family member, IRAK-M, negatively regulates this pathway, and is up-regulated in cultures of endotoxin-tolerant monocytes and in monocytes from septic patients within the timeframe of tolerance. In this study, we show that IRAK-M expression is enhanced at the mRNA and protein level in human monocytes cultured in the presence of tumor cells. IRAK-M was induced in monocytes upon coculturing with different tumor cells, as well as by fixed tumor cells and medium supplemented with the supernatant from tumor cell cultures. Moreover, blood monocytes from patients with chronic myeloid leukemia and patients with metastasis also overexpressed IRAK-M. Low concentrations of hyaluronan, a cell surface glycosaminoglycan released by tumor cells, also up-regulated IRAK-M. The induction of IRAK-M by hyaluronan and tumor cells was abolished by incubation with anti-CD44 or anti-TLR4 blocking Abs. Furthermore, down-regulation of IRAK-M expression by small interfering RNAs specific for IRAK-M reinstates both TNF-alpha mRNA expression and protein production in human monocytes re-exposed to a tumor cell line. Altogether, our findings indicate that deactivation of human monocytes in the presence of tumor cells involves IRAK-M up-regulation, and this effect appears to be mediated by hyaluronan through the engagement of CD44 and TLR4.

In 1998, a mine tailings dyke in southwestern Spain broke, flooding the Agrio-Guadiamar river system with acid tailings up to the borders of one of the largest breeding colonies of white storks in the western Palearctic, Dehesa de Abajo. Over the following years, a high proportion of nestlings developed leg defects, prompting this study. Ten fledglings with leg deformities from the spill area were compared with 11 normal storks of the same year class from another region far from the spill. However, metals were analyzed as a continuum rather than by site, as reference birds also contained high levels of metals. Gross pathology of the legs was supported by histopathology, which showed that bone remodeling activity was greater in the deformed storks, which also had more irregular subperiosteal bone, and tended to have higher residual islets of cartilage in their metaphyses, which, in turn were related to metal contaminant residues. Both Ca and P in bone were affected independently by metals. Deformed birds had lower serum bone alkaline phosphatase. Bone malformations, measured by leg asymmetry, was only partially explained by bone metals, indicating that a combination of factors was involved with the abnormal development in these young storks.

Avian biologists and toxicologists use tests of immune function to evaluate health or quality in birds. Nestlings are widely studied members of the population because of the logistical ease of working with them, and because of their vulnerability to environmental contaminants. Current immunological techniques are designed for domestic poultry and are far from ideal, since poultry are precocial (developmentally mature at hatching), while many wild species are altricial (developmentally immature, i.e. blind, naked and totally dependent at hatching). The purpose of this study was to identify a sensitive means of evaluating in vivo antibody responsiveness in nestling American kestrels. Two antigens, sheep red blood cells (SRBC), and dinitrophenol-keyhole limpet hemocyanin (DNP-KLH), were used to stimulate a B cell mediated response. Antibody production was measured using a hemagglutination assay (SRBC), or an enzyme-linked immunosorbent assay (DNP-KLH). Two formulations of the antigen DNP-KLH were compared. DNP-KLH stimulated a stronger and more consistent antibody response in nestling kestrels than did SRBCs.

A total of 330 white storks (Ciconia ciconia) and 138 black kites (Milvus migrans) were blood sampled during four consecutive years in an area heavily contaminated as a consequence of a massive spillage of toxic acid mining waste rich in heavy metals that impacted on the Doñana National Park (south western Spain), in April 1998. The alkaline single cell gel electrophoresis (Comet) assay was performed as a genotoxicity test, in order to assess whether the high level of DNA damage first detected by us 1 year after the disaster was still present in birds in each of the successive 3 years. Our results clearly show that, when compared with control individuals from non-polluted areas, white storks and black kites born in the contaminated area for a period of up to 4 years after the toxic accident have suffered an increase of at least 2- to 10-fold in the level of their genetic damage through the study period. Taken as a whole, these observations seem to indicate that the toxic spill still appears to be affecting the wildlife 4 years after the mining disaster and that attempts at cleaning up the waste have proved ineffective based on DNA damage detection.

White storks (Ciconia ciconia) fed in contaminated waters resulting from the Aznacollar acid mining-sludge spillage into the R. Guadiamar, which feeds the eastern flank of the Guadalquivir marshes (Doñana), S.W. Spain. The sludge was rich in a range of toxic elements, and in organic pollutants such as the aromatic amines. Storks did not exhibit elevated metals in their blood immediately following the accident, but chick blood collected the year following the accident showed genotoxic damage compared to the controls. In this study lead isotope analysis was used to assess if the storks had ingested sludge-derived contaminants. The sludge lead isotope ratio was distinct from that of the Doñana sediments. The stork blood lead isotope ratios exactly matched that of the sludge. It was concluded that the storks had ingested sludge-derived contaminants. A detailed study of the lead contamination along the R. Guadiamar and the R. Guadalquivir (of which the Guadiamar is a tributary) was also conducted to place the white stork colony lead exposure in the context of the spatial contamination of the storks' habitat.

Single cell gel electrophoresis, the so-called "Comet" assay, was performed as a genotoxicity test in white storks sampled in an area heavily contaminated after the ecological disaster in south western Spain. This disaster occurred as a consequence of a massive toxic spillage of acid waste rich in heavy metals that impacted on the Doñana National Park. The importance of this protected area as a breeding and wintering site for many endangered bird species makes this analysis of DNA damage of special interest. Our results clearly show that white storks born in the contaminated area 1 year after the toxic spill bear a high burden of genetic damage as compared with control individuals. The possible implications for future survival as well as reproductive rate are discussed.

In April 1998, an ecological disaster resulting from a massive toxic spill of mining acid waste rich in heavy metals posed a serious threat to the Doñana National Park in southwestern Spain. This especially important protected area is the nesting and breeding site for many endangered bird species; white storks (Ciconia ciconia) and black kites (Milvus migrans) are considered the more representative. The suitability of the Comet assay as a biomarker for genotoxic analysis in environmental biomonitoring has been recently validated in studies using different sentinel organisms such as fish, amphibians, rodents, or mollusks. Birds preying on a variety of invertebrate and vertebrate species in the marshlands are appropriate for evaluating the potential deleterious effects of the toxic spill on wildlife of the Dofiana area. Our study on wetland birds high on the aquatic trophic chain sampled within a few months after the toxic spill in the area around Doñana National Park has shown the accumulation of heavy metals. Fourteen months after the mine waste spill, blood samples from white storks and kites collected in the neighborhood of the park and from control birds at reference areas for comparison were examined by fluorescence image analysis after lymphocyte isolation, and by subsequent alkaline single-cell gel (SCG) electrophoresis, known as the Comet assay. Our results indicate that the exposed birds had a significantly increased level of genotoxic damage compared with control animals from noncontaminated locations.