P. María-Mojica

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.

Heavy metals in tissues from loggerhead turtles (Caretta caretta) from the southwestern Mediterranean (Spain)

Cadmium, lead, copper, and zinc were measured in tissues of 21 loggerhead turtles (Caretta caretta) from the southwestern Mediterranean coastline. Mean concentrations (dry weight) of essential elements (Zn and Cu) were 107 and 21.6 microg/g in liver, 27.9 and 3.8 microg/g in kidney, 65.4 and 5.0 microg/g in pectoral muscle, 11.1 and 3.45 microg/g in brain, and finally 19.2 microg/g and undetected in bone, respectively. Mean concentrations of heavy metals (Cd and Pb) were 23.4 and 2.8 microg/g in liver, 31.5 and 0.5 microg/g in kidney, 0.2 and 0.3 microg/g in pectoral muscle, 0.2 and 0.7 microg/g in brain, and undetected and 1.2 microg/g in bone, respectively. Metal concentrations were similar to other studies conducted on Mediterranean turtles. However, cadmium concentrations varied widely among individuals, which has been associated with potential sources of cadmium in Mediterranean Sea. This is the first study into metal accumulation in tissues of loggerhead turtle from Spanish Mediterranean coastline.

Raptor Ecotoxicology in Spain: A Review on Persistent Environmental Contaminants

Abstract Initial studies on the pressure from environmental contaminants on raptor populations in Spain date back to the 1980s, and they have been carried out from a range of viewpoints using a range of sentinel raptor species. However, there is no national monitoring scheme, and therefore the research carried out has been sporadic both spatially and temporally. The exposure to metals has not varied over time, except in the case of lead, whose concentration in eggs and tissues has diminished. In general, the concentrations of metals detected in raptor samples from Spain are generally low and not sufficient to produce toxic effects. Excepting DDT and DDE, most organochlorine-based pesticides in raptors from Spain have diminished over the last 2 decades. The concentrations of DDE found in the eggs of various species could in part explain problems in the reproductive success of raptors in Spain.

Organochlorine residues in booted eagle (Hieraaetus pennatus) and goshawk (Accipiter gentilis) eggs from southeastern Spain

Most organochlorine (OC) use has been banned in Spain, but these compounds are persistent and may still adversely affect predatory birds. Data generally are lacking, however. Residues of hexachlorobenzene, alpha-hexachlorocyclohexane, lindane, hexachloro-octahydro-epoxy-dimethanonaphthalene, DDT, dichlorodiphenyldichloroethane, dichlorodiphenyldichloroethylene (DDE), and polychlorinated biphenyls (PCBs) were measured in 22 failed eggs of booted eagles (Hieraaetus pennatus) and goshawks (Accipiter gentilis) from southeastern Spain; both species are declining in this region. Hexachloro-octahydro-epoxy-dimethanonaphthalene, DDE, and sum PCB congener concentrations were significantly higher in booted eagle than in goshawk eggs, and an inverse relationship was found between shell thickness and DDE concentrations in booted eagles. Organochlorides may have been associated with the failure of some booted eagle eggs, but concentrations in booted eagle and goshawk eggs decreased over the period during which populations have dwindled. Thus, although OCs may be a contributory factor, they are unlikely to be the primary cause of the recent population declines in southeastern Spain.

Changes in blood pesticide levels in booted eagle (Hieraaetus pennatus) associated with agricultural land practices.

We estimated the degree of exposure to lindane and endosulfan in the blood of booted eagle nestlings (Hieraaetus pennatus) (1999-2003), in order to assess the usefulness of these samples as a unit for monitoring changes in exposure as a result of shifts in agricultural practices and the implementation of legal measures. The highest blood lindane concentrations were obtained 1 year prior to its prohibition by the European Union. Subsequent to that year, the drop in blood concentrations was dramatic. Furthermore, endosulfan blood concentrations follow a progression coinciding with an increase in olive, grape and plum-tree crops. We conclude that concentrations of organochlorine pesticides in the blood of booted eagle nestlings may be used to monitor the use of those pesticides over a particular agricultural region and alert the authorities of possible environmental or health risks.

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.

Detection of strychnine by gas chromatography-mass spectrometry in the carcase of a Bonelli's eagle (Hieraaetus fasciatus).

basic compounds. The sample was homogenised, acidified with a solution of 35 per cent hydrochloric acid and centrifuged. The supernatant was collected for extraction using diethyl ether as the solvent, and both ethereal and aqueous phases were obtained. The aqueous phase was then alkalinised using sodium hydroxide and extracted with chloroform in order to obtain the final basic extract for analysis. The ethereal phase was used to obtain the neutral and acid extracts: first it was alkalinised using sodium bicarbonate and immediately extracted with diethyl ether to obtain neutral extract. The remaining aqueous phase was acidified with hydrochloric acid and extracted with diethyl ether in order to obtain the final acid extract. Organic solvents of the final basic (chloroform), neutral and acid (diethyl ether) extracts were then evaporated at 40°C to a 0·5 ml final volume using a gentle stream of nitrogen. The extracts were analysed by a gas chromatography-mass spectrometry (GC-MS) system, which consisted of an Agilent 5973 N mass spectrometer and Agilent 6890 N gas chromatograph. The capillary column used was a HP-5MS. The carrier gas was helium and its flow rate was 1 ml/minute. The oven temperature was increased from 60°C (held for one minute) to 310°C (held for two minutes) at 10°C/minute. Ionisation was performed with the electron impact at 70 eV. Three chromatograms were run, for the acid, neutral and basic extracts. Mass spectra corresponding to all chromatographic peaks were compared with those included in the NBS75K.L spectrum library. Analytical results of the extracts from animals used as controls were all negative. Two strychnine isomers were detected in the neutral extract from the Bonelli’s eagle carcase; they had retention times of 14·63 and 14·75 minutes, respectively (Fig 1). Comparison of the mass spectrum with the NBS75K.L library revealed 99 per cent similarity with strychnine (Fig 2). A third peak, at 15·69 minutes, also showed 50 per cent similarity with strychnine. Marques and others (2000) also detected two isomers of strychnine using GC-MS; however, the extraction and purification procedures used by these authors were specific for strychnine and, therefore, more laborious and expensive. In the period 1994 to 1996, strychnine was the most frequent toxin used in poisoned baits in south-east Spain (Maria-Mojica and others 1998); in the last review produced by the authors’ laboratory, strychnine and aldicarb occupied the first positions in the ranking of poisoned baits (MotasFIG 1: Chromatographic peaks of strychnine in extracts of tissue samples taken from the decomposed carcase of a Bonelli’s eagle (Hieraaetus fasciatus) A bu nd an ce ( m V ) 250,000

In vitro evaluation of cell death induced by cadmium, lead and their binary mixtures on erythrocytes of Common buzzard (Buteo buteo).

Cadmium and lead are persistent and ubiquitous metals that can cause several deleterious effects in living beings. Apoptosis and necrosis are two types of cell death that can be found after in vivo and in vitro exposure to these metals. In this study, isolated red blood cells from living captive Common buzzard (Buteo buteo) were exposed in vitro to different concentrations of lead, cadmium, and the mixture lead-cadmium in a proportion of 1:10 (similar to that found in previous field studies). Data obtained from dose-response curves were used to evaluate the interactive effects of metal mixtures on cell viability. In general, except for the exposure to NOEC, additivity was the most frequently observed response. As described in human, after in vitro exposure, lead was highly accumulated in buzzard erythrocytes, while cadmium accumulation was scarce. Finally, the type of cell death (apoptosis or necrosis) induced by the exposure to different concentrations of these heavy metals and their mixtures was evaluated in the red blood cells. Apoptosis was found to be the main type of cell death observed after cadmium and/or lead exposure. However, this exposure caused an increase in lysis or necrosis, especially if red blood cells were exposed to high doses.

Interspecific differences in the antioxidant capacity of two Laridae species exposed to metals

The main aim of the present study was to assess the concentration of metals (Pb, Cd, Hg, Cu and Zn) in blood, and elucidate their potential effects on oxidative stress biomarkers in red blood cells of Audouins gull (Ichthyaetus audouinii) and Slender-billed gull (Chroicocephalus genei) chicks in Southeastern Spain. For this purpose, total glutathione (GSH) content, antioxidant enzymes activities (glutathione peroxidase, GPx; superoxide dismutase, SOD; catalase, CAT and glutathione-S-transferase, GST), and lipid peroxidation (TBARS) were analyzed. In general, metal exposure in both species can be considered low, except for Hg in Audouins gull. Our findings show higher antioxidant levels in Audouins gull than in Slender-billed gull; probably due to a combination of different basal antioxidant capacity between species, and to an up-regulation of the antioxidant system in Audouins gull as a response to the higher Hg, Cu and Zn concentrations. This could reduce the production of TBARS keeping them at lower levels than those found in Slender-billed gull. In spite of this, the significantly higher Hg levels found in Audouins gull (13.6µg/dl wet weight) in comparison to Slender-billed gull (2.7µg/dl), likely related to their different diet and the former consuming discarded fish, were able to produce lipid peroxidation in this species. The positive effect of Hg on SOD activity in Slender-billed gull, and of Pb on GSH levels in Audouins gull, could reflect the necessity of the organism to upregulate these antioxidants to balance the increased oxidative stress caused by metals. The degree of metal exposure seems to be essential in the response of the antioxidant system, which may suffer up or down-regulations depending on metal concentrations. This study supports the interactive effects of metals on oxidative stress biomarkers, the complexity of the antioxidant system and the close cooperation between antioxidants, which requires the study of several metals and biomarkers to evaluate oxidative stress and damage in wild birds.