A First Record of Organochlorine Pesticides in Barn Owls (Tyto alba) from Portugal: Assessing Trends from Variation in Feather and Liver Concentrations.

We evaluated feathers as a non-destructive biomonitoring tool documenting organochlorine pesticides (OCP) in liver and checked possible trends in pesticide use in two areas based on OCP concentrations in barn owls (Tyto alba). We measured the concentrations of 16 OCP in 15 primary feathers and 15 livers from barn owl carcasses collected on roadsides in Tagus Valley and Évora regions, south Portugal. Total OCP mean concentration was 8 120 ng g-1 in feathers and 178 ng g-1 in livers. All compounds were detected in feathers while in livers δ-HCH, endosulfan sulphate, p,p'-DDT and p,p'-DDD were not detected. The high ß-HCH and heptachlor concentrations in feathers most likely derived from external endogenous contamination. P,p'-DDE was the OCP with the highest hepatic concentration. Both matrices indicated an exposure to recently released heptachlor. The differing OCP concentrations between Tagus Valley and Évora seem to reflect differences in land-use and pesticide use histories of the two locations, and/or faster degradation of OCP in the Tagus area.

Development of a QuEChERS method for simultaneous analysis of antibiotics in carcasses for supplementary feeding of endangered vultures.

Antibiotics have been beneficial for human and animal health. However, an excessive use in livestock and a deficient management of the carcasses can lead to adverse effects in the scavengers that ingest them, especially in "supplementary feeding sites" (SFS). The aim of this study was to assess the potential risk of exposure to antibiotics for an endangered population of Cinereous vultures (Aegypius monachus) from southeastern Portugal. Hence, a multi-residue method based on QuEChERs was adapted and validated to analyse, in small volumes of tissues, the most frequent antibiotics used in livestock. The method was applied to 87 samples of liver, muscle and kidney from 7 goats and 25 sheep disposed in SFS. According to questionnaires to farmers, the animals had not been treated with antibiotics, but analyses showed residues in 29% of the samples. Antibiotics were more frequent in goats (42.9%) than in sheep (24.2%), and oxytetracycline and trimethoprim were the most common (both 13.8%). Oxytetracycline, the most common antibiotic for livestock in Portugal, showed the highest concentration (1452.68 ng g-1). To our knowledge, this is the first study of presence of antibiotics in carrion from SFS. The concentrations of antibiotics in carrion do not seem to pose a risk of acute intoxication for adult Cinereous vultures. However, subtle and likely chronic exposure with unknown health consequences may occur, which requires more research. Moreover, the results of this first study can be used in future studies to assess the risk for avian scavengers.

Osmotic gradient ektacytometry: A valuable screening test for hereditary spherocytosis and other red blood cell membrane disorders.

INTRODUCTION: New generation osmotic gradient ektacytometry has become a powerful procedure for measuring red blood cell deformability and therefore for the diagnosis of red blood cell membrane disorders. In this study, we aim to provide further support to the usefulness of osmotic gradient ektacytometry for the differential diagnosis of hereditary spherocytosis by measuring the optimal cutoff values of the parameters provided by this technique. METHODS: A total of 65 cases of hereditary spherocytosis, 7 hereditary elliptocytosis, 3 hereditary xerocytosis, and 171 normal controls were analyzed with osmotic gradient ektacytometry in addition to the routine red blood cell laboratory techniques. The most robust osmoscan parameters for hereditary spherocytosis diagnosis were determined using receiver operating characteristic curve analysis. RESULTS: The best diagnostic criteria for hereditary spherocytosis were the combination of decreased minimal elongation index up to 3% and increased minimal osmolality point up to 5.2% when compared to the mean of controls. Using this established criterion, osmotic gradient ektacytometry reported a sensitivity of 93.85% and a specificity of 98.38% for the diagnosis of hereditary spherocytosis. CONCLUSION: Osmotic gradient ektacytometry is an effective diagnostic test for hereditary spherocytosis and enables its differential diagnosis with other red blood cell membrane diseases based on specific pathology profiles.

Influence of a Former Mining Area in the Heavy Metals Concentrations in Blood of Free-Living Mediterranean Pond Turtles (Mauremys leprosa).

The objective of this study was to assess metal exposure in the Mediterranean Pond Turtle (Mauremys leprosa) inhabiting a watercourse in an ancient mining district polluted by different metals ("Rambla de Las Moreras", southeastern Spain) and included in the Ramsar Convention. For this purpose, mercury (Hg), lead (Pb), copper (Cu), zinc (Zn) and cadmium (Cd) were analysed in blood samples from 42 M. leprosa healthy adults (27 males and 13 females). The highest median concentrations were found for Zn, followed by Cu, Pb, Hg and Cd (366, 33, 9, 0.83 and 0.14 µg/dL, respectively). Although the literature regarding toxic metals in freshwater turtles is relatively scarce, Pb may cause deleterious effects in our population. In general, males presented higher levels than females, which could be due to maternal transfer during egg formation. The significant correlations between Cu-Cd and Cu-Hg suggest the implication of an efficient mechanism of detoxification involving metallothioneins.

Per- and polyfluoroalkyl substances in plasma and feathers of nestling birds of prey from northern Norway.

Plasma samples from nestlings of two top predators, White-tailed eagle (Haliaeetus albicilla) and Northern goshawk (Accipiter gentilis) from northern Norway were analysed for a wide range of per- and polyfluoroalkyl substances (PFASs). Body feathers from the White-tailed eagles were also analysed and significant associations between specific PFASs in blood plasma and body feathers were found (0.36

Validation of a QuECheRS method for analysis of neonicotinoids in small volumes of blood and assessment of exposure in Eurasian eagle owl (Bubo bubo) nestlings.

Neonicotinoid pesticides have gained great interest in the last years both for agricultural and domestic use. Since the information on their environmental distribution or the effects derived from exposure to ecosystems and biota is scarce, new analytical techniques are being developed for monitoring studies. In this sense, two extraction techniques based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methodology to detect the neonicotinoids authorised in Spain (acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, nitenpyram and thiamethoxam) were adapted and compared: a) using acetate buffer (AB); and b) using citrate buffer (CB). For detection and quantification, high performance liquid chromatography coupled with time of flight mass spectrometry (HPLC/TOF-MS) was used. The CB method provided a wide range of recoveries (68-134%) and accuracy (4-9%). The AB method provided good recoveries (59-76%, 59% corresponded to clothianidin) precision (4-11%) linearity (0.987-0.998%) and limit of quantification (2-10ng/mL) for all the compounds. To test the effectiveness of the technique, we analysed 30 blood samples of free-ranging nestlings of Eurasian eagle owl (Bubo bubo). The only compound detected, in one nestling from a dry land farming area, was imidacloprid, with a concentration of 3.28ng/mL. To our knowledge, this is the first study of neonicotinoid pesticides in free-ranging birds of prey using non-destructive samples, providing the first insight for biomonitoring studies. Further studies, including toxicokinetics and toxicodynamics, are recommended to assess the risk for these species.

Tracking pan-continental trends in environmental contamination using sentinel raptors-what types of samples should we use?

Biomonitoring using birds of prey as sentinel species has been mooted as a way to evaluate the success of European Union directives that are designed to protect people and the environment across Europe from industrial contaminants and pesticides. No such pan-European evaluation currently exists. Coordination of such large scale monitoring would require harmonisation across multiple countries of the types of samples collected and analysed-matrices vary in the ease with which they can be collected and the information they provide. We report the first ever pan-European assessment of which raptor samples are collected across Europe and review their suitability for biomonitoring. Currently, some 182 monitoring programmes across 33 European countries collect a variety of raptor samples, and we discuss the relative merits of each for monitoring current priority and emerging compounds. Of the matrices collected, blood and liver are used most extensively for quantifying trends in recent and longer-term contaminant exposure, respectively. These matrices are potentially the most effective for pan-European biomonitoring but are not so widely and frequently collected as others. We found that failed eggs and feathers are the most widely collected samples. Because of this ubiquity, they may provide the best opportunities for widescale biomonitoring, although neither is suitable for all compounds. We advocate piloting pan-European monitoring of selected priority compounds using these matrices and developing read-across approaches to accommodate any effects that trophic pathway and species differences in accumulation may have on our ability to track environmental trends in contaminants.

Contaminants in the southern tip of South America: Analysis of organochlorine compounds in feathers of avian scavengers from Argentinean Patagonia.

The aim of this study was to assess the exposure to organochlorine compounds (OC) in 91 primary wing feathers of avian scavengers, Turkey vulture (Cathartes aura), American black vulture (Coragyps atratus) and Southern crested caracaras (Polyborus plancus) from the southern tip of South America, in the Argentinean Patagonia. We analyzed for a series of OC including hexachlorocyclohexane (HCH) isomers, endosulfan, aldrin, dieldrin, endrin, dichlorodiphenyltrichloroethane (p,p'-DDT), dichlorodiphenyldichloroethane (p,p'-DDD), dichlorodiphenyldichloroethylene (p,p'-DDE), heptachlor and heptachlor-epoxide. This is the first study on OC in feathers of three terrestrial top carnivores from South America. OC concentrations found in the studied species were much higher than those found in feathers of raptors from Europe and Asia, which likely indicate their high use in the region, specifically in agriculture, and other possible uses of OC in this area. ∑HCH had the highest median concentration, followed by ∑Drins, ∑DDT, ∑Heptachlor, and ∑Endosulfan, similar to those reported in several food samples in Argentina. On the other hand, differences in OC profiles between species and areas may be related to feeding and migratory habits, as well as the molt period. Three individuals showed ∑DDT (DDT, DDD and DDE) concentrations in feathers related to sublethal effects. However, this comparison should be used with caution due to problems with extrapolating such data across tissues and species.

An overview of existing raptor contaminant monitoring activities in Europe.

Biomonitoring using raptors as sentinels can provide early warning of the potential impacts of contaminants on humans and the environment and also a means of tracking the success of associated mitigation measures. Examples include detection of heavy metal-induced immune system impairment, PCB-induced altered reproductive impacts, and toxicity associated with lead in shot game. Authorisation of such releases and implementation of mitigation is now increasingly delivered through EU-wide directives but there is little established pan-European monitoring to quantify outcomes. We investigated the potential for EU-wide coordinated contaminant monitoring using raptors as sentinels. We did this using a questionnaire to ascertain the current scale of national activity across 44 European countries. According to this survey, there have been 52 different contaminant monitoring schemes with raptors over the last 50years. There were active schemes in 15 (predominantly western European) countries and 23 schemes have been running for >20years; most monitoring was conducted for >5years. Legacy persistent organic compounds (specifically organochlorine insecticides and PCBs), and metals/metalloids were monitored in most of the 15 countries. Fungicides, flame retardants and anticoagulant rodenticides were also relatively frequently monitored (each in at least 6 countries). Common buzzard (Buteo buteo), common kestrel (Falco tinnunculus), golden eagle (Aquila chrysaetos), white-tailed sea eagle (Haliaeetus albicilla), peregrine falcon (Falco peregrinus), tawny owl (Strix aluco) and barn owl (Tyto alba) were most commonly monitored (each in 6-10 countries). Feathers and eggs were most widely analysed although many schemes also analysed body tissues. Our study reveals an existing capability across multiple European countries for contaminant monitoring using raptors. However, coordination between existing schemes and expansion of monitoring into Eastern Europe is needed. This would enable assessment of the appropriateness of the EU-regulation of substances that are hazardous to humans and the environment, the effectiveness of EU level mitigation policies, and identify pan-European spatial and temporal trends in current and emerging contaminants of concern.

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.

A modification of QuEChERS method to analyse anticoagulant rodenticides using small blood samples

El uso de rodenticidas anticoagulantes es el método más frecuentemente utilizado para el control de plagas de roedores. Debido a sus características físico-químicas y particular mecanismo de acción, la utilización de estos compuestos en zonas rurales puede suponer un riesgo de intoxicación secundaria de sus depredadores. Para evaluar el riesgo a estos compuestos para la fauna silvestre, especialmente en aves rapaces que se alimentan de roedores, se llevan a cabo los programas de biomonitorización. Se ha desarrollado un método rápido, fácil y económico que permita el análisis de pequeños volúmenes de muestra de sangre. En el presente trabajo se han comparado tres diferentes modificaciones de la metodología "QuEChERS", y posteriormente uno de ellos fue elegido para la detección y cuantificación de estos compuestos. El proceso previo al análisis de los extractos incluye dos pasos sencillos: La muestra es extraída usando un solvente orgánico y una solución salina y, posteriormente, el sobrenadante es purificado usando una técnica de extracción en fase solida dispersiva. La detección y cuantificación de los rodenticidas anticoagulantes se realizó por cromatografía líquida acoplada a un detector de masas Agilent 1100 VL con trampa de iones y fuente de electrospray para ionización. La técnica finalmente elegida permite una recuperación entre 72-134% para los siete rodenticidas objeto de estudio (warfarina, cumatetralilo, difenacoum, clorofacinona, brodifacoum, bromadiolona, difacinona), la cual es superior a la obtenida con otras técnicas que analizan compuestos similares. Además, la sensibilidad de esta técnica es mayor a la que ofrecen otras técnicas. Con el fin de comprobar la utilidad de la técnica validada, se analizaron un total de 50 muestras de sangre de búho real (Bubo bubo) capturados en su hábitat natural (AU)

The use of anticoagulant rodenticides is the most common method to control rodent plagues. Due to their physicochemical characteristics and particular mechanism of action, the application of these compounds in rural areas can pose a risk of secondary poisoning for their predators. In order to evaluate the risk of these compounds for wildlife, especially raptors that feed on rodents, biomonitoring programmes are undertaken. A fast, easy and low cost technique was needed to analyse small volumes of blood samples. Therefore, three different modifications of QuEChERS methodology have been compared, and one of them selected to detect and quantify these compounds. The process prior to analysis of the extracts involves two simple steps: the sample is extracted and partitioned using an organic solvent and salt solution. The supernatant is then cleaned using a dispersive solid phase extraction (dSPE) technique. Detection and quantification of the anticoagulant rodenticides were performed by LC-MSMS on an Agilent 1100 VL Series ESI/LC/MSD, with an electrospray ionisation (ESI) source and ion trap analyser. The method finally chosen provides a 72-134% recoveries for the seven rodenticides (warfarin, coumatetralyl, brodifacoum, bromadiolone, difenacoum, chlorophacinone, diphacinone), higher than in other methods to analyse similar compounds. Sensitivity of our method is also higher than in other methods. In order to prove the utility of the technique, a total of 50 blood samples of free-living Eagle owls (Bubo bubo) were analysed (AU)

Organohalogen exposure in a Eurasian Eagle owl (Bubo bubo) population from Southeastern Spain: temporal-spatial trends and risk assessment.

Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and organochlorine insecticides (OCs) were analysed in 58 Eurasian Eagle owl (Bubo bubo) unhatched eggs collected between 2004 and 2009 in Southeastern Spain. Levels of p,p'-DDE were found to be higher than in eggs laid by other European owls in the same decade, probably due to the greater agricultural activity in our study area. Compared to other European raptors, exposure to PCBs can be considered intermediate, but low to PBDEs. Land use differences and prey availability were the rationale to divide the study area in two subareas in further assessments. Temporal trends of HCB, p,p'-DDE, ß-HCH, PCBs and PBDEs were significantly different in each subarea, generally increasing over time in the Southern but decreasing or remaining stable in the Northern. On the contrary, levels of cyclodienes tended to decrease in both subareas. Dietary shifts with a greater amount of birds are suggested as a cause for increasing organochlorine loads in raptors. This may explain the increasing trend in the Southern territories. However, due to the proximity of most of these nests to Cartagena, an important industrial city, increasing environmental pollution cannot be ruled out. Although average levels of the compounds analysed are below threshold levels, 17% of the samples exceeded 400 pg g(-1)ww (wet weight), the LOAEC for Total TEQs. Moreover, a negative correlation between TEQ concentrations and the metabolizable fraction of PCBs (F(prob)=0.0018) was found when TEQs values were above 10 pg g(-1)ww. This could be indicative of hepatic enzymes induction in the birds exposed at higher concentrations, which are mainly breeding in the Southern subarea. These females could be suffering from Ah-receptor-related toxic effects, some of which have been related to altered bird reproduction. Finally, a significant negative correlation between p,p'-DDE levels and eggshell thickness (r=-0.469, p<0.001) was observed, with about 17% of eggshell thinning for eggs with p,p'-DDE levels above 100 µg g(-1)lw. The persistence of this degree of thinning over a period of time has been related to population declines in other raptor species.

Blood lead levels and δ-ALAD inhibition in nestlings of Eurasian Eagle Owl (Bubo bubo) to assess lead exposure associated to an abandoned mining area.

In order to biomonitor lead contamination in Southeastern Spain, 218 blood samples from 28 to 30-day old Eurasian Eagle Owl chicks (Bubo bubo) born between 2003 and 2007 were analysed. In general, mean lead levels showed that chicks were exposed to background concentrations. However, mean levels in chicks born in an ancient and abandoned mining site ("Sierra Minera Cartagena-La Union") or in their surroundings (Geometric mean (GM) = 5.83 µg/dl, range 0.49-25.61 µg/dl), an area highly polluted by lead and other metals, were significantly higher (p < 0.001) than the rest of the population (GM = 1.66 µg/dl, range = Non detected-18.37 µg/dl). Because δ-ALAD activity is considered the best biomarker for lead exposure and effect in birds, the activity of this enzyme was also evaluated and correlated with lead levels in blood. In this study, low levels of blood lead inhibited δ-ALAD, even when lead concentrations were lower than the limits described by other authors in raptors. Adverse effects caused by this inhibition may occur when blood lead levels were above 15 µg/dl, although only eight chicks presented these concentrations in their blood. Sampling site also influenced enzymatic activity, since it decreased about 60% in the polluted area in relation to the rest. For all these reasons, further research regarding risk assessment for lead exposure in Eagle Owls nesting in the polluted area is advisable. Our results suggest that the Eurasian Eagle Owl can be considered a suitable sentinel animal for monitoring lead contamination and δ-ALAD activity can be used as a sensitive biomarker for lead exposure and effect in this species.

Blood delta-ALAD, lead and cadmium concentrations in spur-thighed tortoises (Testudo graeca) from Southeastern Spain and Northern Africa.

Mediterranean spur-thighed tortoise (Testudo graeca) is actually included in the IUCN as vulnerable species. Its main European population is located in southeastern Spain. Although a great deal of information has been acquired on the internal medicine and survey and even parasitological fauna on these animals, there are no references about contaminants levels in this species. The objectives of this study were to compare the levels of two metals (cadmium and lead) in the blood of spur-thighed tortoises from two different populations, one from Southeastern of Spain (n = 22) and the other from North of Africa (n = 39), kept in captivity at the Santa Faz Recuperation Centre (Alicante, Spain) and to investigate the relationship between their blood levels of lead and their blood delta-aminolevulinic acid dehydratase (delta-ALAD) activity. Blood lead and cadmium concentrations were higher in tortoises from African than in those from Spain. Moreover, a negative and significant correlation (P < 0.05) was found between delta-ALAD activity and blood lead levels, indicating the suitability of this enzyme as biomarker for lead in this species.

Aspectos clínicos y forenses del envenenamiento de aves silvestres: diferencias entre aldicarb y estricnina / Clinical and forensic aspects of the poisoning of wild birds: differences between aldicarb and strychnine

Aldicarb y estricnina son frecuentemente usados para envenenar fauna silvestre y doméstica. La estricnina se utilizó ampliamente hace algunos años hasta que fue prohibido su uso en 1994, siendo sustituida por otros plaguicidas carbamatos como el aldicarb o el carbofurano. En los últimos años se ha producido un aumento de envenenamientos por estricnina. Los síntomas de la intoxicación por estricnina y carbamatos están ampliamente descritos en mamíferos, pero no en aves. En la mayoría de los casos forenses la única información disponible es el análisis químico-toxicológico del cadáver o del cebo. Esta carencia de información es debida, en gran medida, a un inadecuado manejo y procesado de la escena del crimen, la inadecuada formación de los agentes para tomar y embalar las muestras, y/o la falta de formación toxicológica y/o forense de los veterinarios. La escena del crimen es solo uno de los componentes de la investigación pero necesita ser coordinada con los otros aspectos de la investigación. La escena del crimen debe ser estudiada hasta el más mínimo detalle para establecer un correcto diagnóstico. En este trabajo, utilizando como referencia el envenenamiento por aldicarb y estricnina, enfatizamos algunos aspectos clínicos y forenses que deben ser tenidos en cuenta en la investigación del envenenamiento de aves silvestres

Aldicarb and strychnine are frequently used to poison wild and domestic animals. Strychnine was widely used until it was banned in 1994, being substituted for carbamates such as aldicarb or carbofuran. Recently, an increase of deliberated poisonings using strychnine has been observed. Symptoms of aldicarb and strychnine poisonings have been widely described in mammals, however they have been poorly reported in birds. In most forensic cases the only available data is the chemical analysis realized on baits or tissues from dead animals. This scarcity of information is due usually to an inadequate management and processing of the crime scene, an unsuitable training of the first officer to collect and package the physical evidences, and/or unsuitable toxicological and forensic training of the veterinarians. Is should be recognized the crime scene is only one component of an investigation and needs to be coordinated with other aspects of the investigation. The crime scene should be investigated down to the last detail in order to establish an accurate diagnosis. In this work, using aldicarb and strychnine poisonings as references, we emphasize some clinical and forensic aspects which should be taken into account in the investigation of poisoning of wild birds