Silvia Espín

Effects of heavy metals on biomarkers for oxidative stress in Griffon vulture (Gyps fulvus).

Metals are involved in the formation of reactive oxygen species (ROS) which may result in metal-related oxidative stress that can lead to oxidative damage to lipids, DNA and proteins. It is necessary to understand the mechanisms of metal toxicity in wild birds, and the concentrations that cause effects on oxidative stress biomarkers. The aim of this study is to assess the concentrations of lead (Pb), cadmium (Cd), mercury (Hg), copper (Cu) and zinc (Zn) with regards to oxidative stress in blood samples of 66 Griffon vultures (Gyps fulvus) from two areas of the Autonomous Community of Valencia (East of Spain). The two study areas (Alcoy n=36 and Cinctorres n=30) were selected as random locations of interest that had not yet been studied, and are feeding stations where supplementary food, mainly of pork origin, is provided for vultures. Given that the two study areas are not considered polluted sites, we expected to find low metal concentrations. However, there are no known threshold concentrations at which metals can affect antioxidant systems, and low metal levels may have an effect on antioxidant biomolecules. In this study, since sampling was done at the beginning of the hunting season, the low Pb levels found in most Griffon vultures from Alcoy and Cinctorres (median=12.37 and 16.26μg/dl, respectively) are suggestive of background levels usually found in vultures that feed on pork carcasses all year round. The ingestion of game meat with bullet fragments in carcasses or with Pb shots embedded in the flesh could be the cause of the high blood Pb concentrations found in three vultures from Cinctorres (83, 290 and 362μg/dl). Griffon vultures feeding in Cinctorres had enhanced CAT and GST activities and tGSH concentrations, which may be interpreted as protective response against the higher TBARS levels. This study provides threshold concentrations at which metals affect antioxidant system derived from 66 samples of Griffon vulture. Blood Cd concentrations greater than 0.05μg/dl produced an induction of 33% in GPx and of 44% in CAT activity in erythrocytes of vultures from Alcoy. Hg concentrations in blood higher than 3μg/dl produced an induction of 10% in SOD activity. Concentrations of Pb above 15µg/dl in blood produced an inhibition of 12.5% in GPx and 11.3% in CAT activity, and a TBARS induction of 10.7% in erythrocytes of Griffon vultures.

Feathers as a Biomonitoring Tool of Polyhalogenated Compounds: A Review.

Feathers have many advantages that make them an excellent nondestructive tool for monitoring polyhalogenated compounds (PHCs). This paper proposes a review on the PHCs in feathers and factors influencing the pollutant load. Special attention has given to external contamination and the main analytical methods used to detect these compounds in feathers. Some authors have found strong and significant correlations between the concentrations of PHCs in feathers and internal tissues, providing positive expectations for their future use in the field of ecotoxicology. However, changes in diet, time elapsed between the previous molt period and sampling, sample size, and/or external contamination have been suggested as possible causes to explain the lack of correlations reported in some studies. Further studies with newly grown feathers and blood samples would be required in order to clarify this issue. Although atmospheric deposition has been reported as cause of external contamination, preening oil seems to be the most relevant factor contributing to this process. Unfortunately, washing techniques tested to date are not able to effectively remove the surface contamination from barbs and shafts, and therefore, it is necessary to develop methods able to discriminate between internal and external contamination. Finally, in this review, deposition rate is proposed as a measurement unit, as this allows comparisons between different parts of the same feather, as well as between different feathers.

Oxidative stress biomarkers in Eurasian eagle owls (Bubo bubo) in three different scenarios of heavy metal exposure.

The main aim of the present study is the assessment of oxidative stress related to metals in the Eurasian eagle owl (Bubo bubo) from three areas (agricultural and rural area, industrial area, and mining area) of Murcia, Southern Spain. Mean blood metal concentrations were Cd=0.07±0.21, Pb=3.27±5.21, Cu=10.62±4.77, Zn=311.47±67.14, Hg=2.32±3.83 μg/dl wet weight. Although individuals from the mining area had significant higher Pb and Hg concentrations, and significant lower glutathione-S-transferase (GST) and catalase (CAT) activities in red blood cells (RBC); the lack of differences in oxidative damage to membrane lipids (TBARS) among areas suggests that the antioxidant capacity of the different populations is able to deal with oxidant species and maintain TBARS levels in the same amount. Despite the low levels of metals, several oxidative stress biomarkers were correlated with metal concentrations. This study provides threshold concentrations at which metals cause effects on the antioxidant system in Eagle owls. Blood Cd concentrations greater than 0.3 μg/dl produced an inhibition in GPx (32%) and CAT (26%) activity in RBC. However, Cd concentrations higher than 0.02 μg/dl were enough to produce an inhibition of these enzymes. Regarding Pb levels, blood concentrations above 2 μg/dl produced an inhibition of 8% and 10.5% in GPx and CAT activities, respectively, in RBC. A depletion of 16% and 4% in tGSH levels was associated with Pb concentrations higher than 15 and 3 μg/dl, respectively, in individuals from the ancient mine site. In addition, Pb concentrations above 2 and 10 μg/dl produced a TBARS induction of 10% and 28%, respectively, in individuals from both the industrial and the mining area. Finally, Hg concentrations greater than 3 and 10 μg/dl resulted in a TBARS induction of 102% and 190%, respectively, in Eurasian eagle owls from the industrial area. Our findings show that Pb may produce effects on oxidative stress biomarkers in Strigiformes at lower concentrations than those typically accepted for considering physiological effects in Falconiformes (20 µg/dl in blood). In addition, we provide new data on Hg and Cd concentrations related to effects in the antioxidant system.

A review on exposure and effects of arsenic in passerine birds

UNLABELLED Arsenic (As) is a metalloid of high concern because of its toxic effects for plants and animals. However, it is hard to find information on this metalloid in passerines. This review presents a comprehensive overview of As exposure and effects in birds, and more particularly in passerines, as a result of an extensive search of the literature available. Internal tissues are the most frequently analyzed matrices for As determination in passerines (37.5% of the reviewed studies used internal tissues), followed by feathers and eggs (32.5% each), feces (27.5%), and finally blood (15%). A clear tendency is found in recent years to the use of non-destructive samples. Most studies on As concentrations in passerines have been done in great tit (Parus major; 50%), followed by pied flycatcher (Ficedula hypoleuca; 22.5%). Some factors such as diet and migratory status are crucial on the interspecific differences in As exposure. More studies are needed to elucidate if intraspecific factors like age or gender affect As concentrations in different tissues. The literature review shows that studies on As concentrations in passerines have been done mainly in the United States (30%), followed by Belgium (22.5%), and Finland (20%), making evident the scarce or even lack of information in some countries, so we recommend further research in order to overcome the data gap, particularly in the southern hemisphere. Studies on humans, laboratory animals and birds have found a wide range of effects on different organ systems when they are exposed to different forms of As. This review shows that few field studies on As exposure and effects in passerines have been done, and all of them are correlative so far. Arsenic manipulation experiments on passerines are recommended to explore the adverse effects of As in free-living populations at similar levels to those occurring in the environment. CAPSULE This review summarizes the most interesting published studies on As exposure and effects in passerines.

Factors that influence mercury concentrations in nestling Eagle Owls (Bubo bubo)

Mercury (Hg) is a global pollutant that bioaccumulates and biomagnifies in food chains, and is associated with adverse effects in both humans and wildlife. The Hg levels detected in blood obtained from Eagle Owl (Bubo bubo) chicks in Southeast Spain (Murcia) can be considered low (mean Hg concentration in blood from 2006 to 2012 was 36.83 ± 145.58 μg/l wet weigh, n=600), and it is therefore unlikely that Hg pollution can negatively affect their breeding. Positive correlation (r=0.339, p<0.001, n=229) was found to exist between the Hg concentrations in the blood and back feathers of the chicks. We provide a regression equation that could be helpful to estimate blood Hg levels when analyzing Hg concentrations in back feathers. Blood Hg concentrations in Eagle Owls have shown positive correlations with Hg levels in rabbit muscles, more evident in nests from the Northern area (r=0.600, p=0.014, n=16), where rabbits are the main prey of Eagle Owls. The best Linear Mixed Model to explain variations in blood Hg concentrations in nestling Eagle Owls includes year and location within the mining area as variables. The variable year is assigned the largest value of relative importance, followed by the location in the ancient mining sites and then the zone. Rainfalls may have an effect on the temporal differences in the blood Hg concentrations of nestling Eagle Owls. Although the studied region is not considered Hg polluted, the Hg levels were higher in Eagle Owls and European Rabbits from areas within the ancient mining sites as compared to those in the entire region. This result shows that spatial differences in Hg concentrations in Eagle Owls may be affected by local contamination, and that the role of diet composition may be less significant.

Razorbills (Alca torda) as bioindicators of mercury pollution in the southwestern Mediterranean.

Levels of mercury (Hg) were analyzed in the tissues of 50 Razorbills (Alca torda), from the Mediterranean area, which had drowned in fishing nets. The mercury distribution pattern in tissues was similar to those of other studies (liver > feather vane > kidney > muscle > brain > feather shaft), with mercury concentrations of 2.85 ± 0.90, 2.66 ± 1.60, 2.23 ± 0.87, 1.54 ± 0.54, 1.48 ± 0.54 and 1.30 ± 0.76 mg/kg (dry weight), respectively. It could be considered that Razorbills in the southwestern Mediterranean were chronically exposed to relatively low levels of MeHg, probably below 0.5 ppm, via dietary intake. We have proposed prediction equations for brain and kidney Hg concentrations using feather shafts as non-invasive samples. This work provides a solid understanding of Razorbill Hg exposure both in their wintering and breeding grounds, and shows that this species can be useful for assessing marine environmental health in the Mediterranean area.

Delta-aminolevulinic acid dehydratase (δALAD) activity in four free-living bird species exposed to different levels of lead under natural conditions.

The purposes of this study were: (1) to determine the δALAD activity and δALAD ratio in blood of four free-living bird species (Griffon vulture, Eagle owl, Slender-billed gull and Audouins gull); (2) and to investigate the correlations between δALAD activity/ratio and Pb concentrations in blood samples. A decrease was observed in δALAD activity in Griffon vultures and Eagle owls exposed to Pb. In addition, negative relationships were found between δALAD ratio or δALAD activity and Log blood Pb levels in Griffon vultures and Eagle owls, and these relationships were stronger in areas with the highest Pb exposure. We provide equations that may be helpful to estimate δALAD activity and δALAD ratio using blood Pb concentrations. Regarding gull species, δALAD activity found in the present study may be considered the normal activity in Slender-billed gull and Audouins gull species, since very low blood Pb concentrations and no correlations were found in these species. Although both δALAD activity and δALAD ratio are sensitive biomarkers of Pb exposure and effect in birds, the use of δALAD ratio may improve the results. Besides, this study provides blood threshold concentrations at which Pb bears effects on δALAD enzyme (5µg/dl in Eagle owl; 8µg/dl in Griffon vulture; and probably >2µg/dl in Slender-billed gull and Audouins gull). Our findings show that Eagle owl seems to be more sensitive to δALAD enzymatic inhibition by Pb than Griffon vultures. Eagle owls and Griffon vultures exhibited up to 79% and 94% decrease in δALAD activity when blood Pb concentrations exceeded 19 and 30µg/dl, respectively. Regarding the effects related with δALAD inhibition, significant negative correlations were found between δALAD activity and hematocrit in Eagle owls and Griffon vultures, which may be related to compensatory response associated with a decrease in δALAD activity. In addition, an effect on creatine kinase activity and total proteins in plasma was found in Griffon vultures. The significant negative correlations found between δALAD activity and tGSH in Griffon vulture, and between δALAD activity and Log CAT activity in Eagle owls, may be related to a protective response of antioxidant system against reactive oxygen species (ROS). The negative relationship found between δALAD activity and TBARS levels in Griffon vulture is probably related to an induction of lipid peroxidation by ROS that may be generated by δALA accumulation when δALAD activity is depressed. δALAD activity and δALAD ratio in blood are suggested as important nondestructive biomarkers for Pb exposure and effect for future biomonitoring studies in Griffon vulture and Eagle owl. Further studies are recommended to provide new data on Pb concentrations at which δALAD activity is affected in different wild bird species and to elucidate why different species tolerate Pb in different ways.

Effects of calcium supplementation on growth and biochemistry in two passerine species breeding in a Ca-poor and metal-polluted area.

Several studies provide evidence of calcium (Ca)-limited reproduction in birds. A Ca-supplementation experiment was carried out in 2014 in a Ca-poor area associated with metal pollution in SW Finland. We aimed to evaluate the relationship between Ca availability and heavy metal exposure in free-living passerines, and to compare Ca levels in plasma and feces and the effects of Ca supplementation and metals on breeding, nestling growth, and plasma biochemistry in great tits and pied flycatchers. Although the Ca supplement was used by parents, in general the treatment had limited effects on growth and biochemistry, suggesting that parents are capable of finding sufficient Ca-rich foods to allow nestlings to grow properly. Snail shells were an abundant Ca source in the moderately polluted zone for pied flycatcher, and great tits likely combines the intake of snail shells and other anthropogenic Ca-rich items. Great tits had higher Ca concentrations in feces and plasma than pied flycatcher nestlings, suggesting that they need and sustain higher Ca levels and seem to be more opportunistic in search for Ca than pied flycatcher, since they consumed more of the supplemented Ca. Negative effects of pollution in nestling size and fledgling number were found in great tit. This species may suffer especially from the lower food quality and quantity in the polluted area. The pied flycatcher seems to be adapted to low Ca availability and they can successfully breed when metal concentrations are not too high. Our results show that great tits and pied flycatchers may employ different strategies in response to low Ca availability.

Effects of experimental calcium availability and anthropogenic metal pollution on eggshell characteristics and yolk carotenoid and vitamin levels in two passerine birds.

The maternal investment into egg quality depends on the condition of the female, the quality of the mate, and the quality of the environment. In that sense, availability of nutrients and exposure to pollutants are essential parameters to consider. The main aim of this study is to assess the effects of calcium (Ca) availability and anthropogenic metal pollution on early-stage reproduction in two passerine species, great tits (Parus major) and pied flycatchers (Ficedula hypoleuca), inhabiting a Ca-poor and metal-polluted area in SW Finland. Both species were able to obtain sufficient Ca for eggshell formation, and metal pollution was below the level of having negative effects in the egg size and eggshell characteristics. However, metal polluted environment negatively affected yolk lutein and vitamin D3 levels in both species, probably because of a lower access to carotenoid-rich diet and higher metal interference with vitamin D3 metabolism. The higher levels of vitamin D3 in yolks in the unpolluted zone could also be due to upregulated D3 levels as a response to the lower natural Ca availability. Yolk carotenoids and vitamin D3 were positively associated with nestling growth and size, supporting their importance for the appropriate chick development. The interspecific differences in yolk nutrient concentrations possibly reflect the different growth rate of these species. Pied flycatchers are likely adapted to low Ca availability through an efficient vitamin D3 metabolism, but their Ca intake could be close to a deficient level.

Effects of dietary lead exposure on vitamin levels in great tit nestlings – An experimental manipulation ☆

Exposure to metal pollution negatively affects animal physiology, including nutrient metabolism, but in the wild an effect can seldom be attributed to a single metal. Moreover, little is known about how the metabolism of vitamins, essential micronutrients for developing juveniles, is affected by toxic metals. Therefore we experimentally investigated the effects of lead (Pb), a widespread toxic metal, on four fat-soluble vitamins A (total and retinol), D3, E (total and α-tocopherol) and K and carotenoids (lutein, zeaxanthin and unidentified) in great tit (Parus major) nestlings. In addition to a control group where no Pb was provided, two Pb-dosed groups were compared to a metal exposed group in the vicinity of a Ni-Cu smelter. We examined whether Pb treatment affects vitamin homeostasis and how the response of Pb-treated birds relates to that of a population under industrial exposure of Pb and other metals. For this purpose, vitamin and carotenoid levels were quantified with UPLC-MS from plasma of 7 days-old nestlings. All metal exposed groups showed increased vitamin A and retinol levels. However, vitamin levels were not directly associated with fecal Pb levels, with the exception of retinol, which was positively correlated with fecal Pb. Alpha-tocopherol, lutein and zeaxanthin levels were positively associated with body mass and wing growth rate. To conclude, Pb exposure increased plasma vitamin A and retinol levels while the levels of other vitamins and carotenoids rather reflected secondary pollution effects via differences in habitat and diet quality at the smelter site. Our findings suggest Pb exposed nestlings may allocate the vitamins needed for growth and development to fight the physiological stress thus compromising their fitness.