Age and sex differences in pharmaceutical contamination in a keystone scavenger.

Pharmaceutical contaminants have a recognized negative impact on wildlife health. However, there are still many knowledge gaps on the factors influencing exposure and metabolic processing of compound mixtures as a function of season and individual characteristics such as age and sex. We evaluated age and sex differences in a set of seventeen compounds, including eleven antibiotics, five NSAIDs and caffeine, evaluated by HPLC-MS-TOF analysis in griffon vultures (Gyps fulvus) from central Spain. Pharmaceutical cocktails (up to 10 compounds simultaneously) were found in all individuals. Lincomycin was detected in all individuals, and fluoroquinolones were found at high frequencies, while NSAIDs were at low frequencies and concentrations, including flumixin meglumine, which can be lethal to vultures. A higher total number of compounds and sum of concentrations, as well as prevalence and concentration of several of the pharmaceuticals tested was found in females than in males for both nestlings and adults. This is the first study to present evidence of sex differences in the pharmacokinetics of dietary drug contaminants in a vulture species. Chronic exposure to "medications" in entire populations can potentially have sub-lethal health effects that affect fitness differently according to age and sex, with demographic implications for population viability. Specifically, if females have higher mortality after fledging due to high pharmaceutical contamination, this should be considered when modelling the population dynamic of this species for conservation purposes.

Mercury Exposure in Birds of Prey from Norway: Relation to Stable Carbon and Nitrogen Isotope Signatures in Body Feathers.

Mercury (Hg) and stable carbon and nitrogen isotope ratios were analysed in body feathers from nestlings of white-tailed eagles (Haliaeetus albicilla) (WTE; n = 13) and Northern goshawks (Accipiter gentilis) (NG; n = 8) and in red blood cells (RBC) from NG (n = 11) from Norway. According to linear mixed model, species factor was significant in explaining the Hg concentration in feathers (LMM; p < 0.001, estimate (WTE) = 2.51, 95% CI = 1.26, 3.76), with concentrations higher in WTE (3.01 ± 1.34 µg g-1 dry weight) than in NG (0.51 ± 0.34 µg g-1 dry weight). This difference and the isotopic patterns for each species, likely reflect their diet, as WTE predominantly feed on a marine and higher trophic-chain diet compared to the terrestrial NG. In addition, Hg concentrations in RBCs of NG nestlings were positively correlated with feather Hg concentrations (Rho = 0.77, p = 0.03), supporting the potential usefulness of nestling body feathers to biomonitor and estimate Hg exposure. Hg levels in both species were generally below the commonly applied toxicity threshold of 5 µg g-1 in feathers, although exceeded in two WTE (6.08 and 5.19 µg g-1 dry weight).

Developing a European network of analytical laboratories and government institutions to prevent poisoning of raptors.

Many cases of wildlife poisoning in Europe have been reported causing population declines, especially in raptors. Toxicovigilance and risk assessment studies are essential to reinforce the knowledge of the number of illegal poisoning cases and the substances involved in these crimes. Many researchers and projects in different institutions have suggested the creation of a network to improve communication and share information between European countries. This article presents the results of the Short-Term Scientific Mission titled "Developing a Network of Analytical Labs and Government Institutions" supported by the COST Action European Raptor Biomonitoring Facility (CA16224), which aims to initiate a network of veterinary forensic toxicology laboratories, in order to improve communication among laboratories to prevent wildlife poisoning, especially in raptors. For this purpose, a questionnaire was designed and sent by email to 119 laboratories in Europe. It contained 39 questions on different topics (e.g. laboratory activities, analytical information). A total of 29 responses were received. Most participant laboratories work on veterinary forensic toxicology research and external cases at the same time, which provides a robust overview of the actual situation in the field. Analytical techniques and data collection methods should be harmonised, and communication between laboratories is encouraged to create a more effective network. The present study established contact between laboratories as an initial step to create a European network and compiled basic data to identify strengths and weaknesses that will help harmonise methodologies across Europe and increase pan-European capacities.

Wildlife poisoning: a novel scoring system and review of analytical methods for anticoagulant rodenticide determination.

Anticoagulant rodenticides (ARs) are commonly used to control rodent populations and frequently involved in wildlife and domestic animal poisoning. These poisoning cases (especially for ARs) are a challenge for forensic toxicologists, and adequate post-mortem examination and toxicological analyses become essential for a proper diagnosis. Publications describing different analytical methods for AR analysis in biological samples are growing, and a clear compilation of the overall picture is needed to standardize methodologies in future research. This review aims to compile and compare the analytical procedures applied for AR determination in the literature. Using this information, a scoring system was developed for those techniques using liver and blood as matrices, and the techniques were ranked considering different criteria (i.e. sample amount required, recoveries, limits of quantification (LOQs), number of ARs analysed, points of the calibration curve and multi-class methods). This review shows an overview of the main methods used for AR analysis in forensic toxicology and will help to elucidate future directions to improve multi-residue techniques to detect the ARs involved in wildlife lethal poisoning.

Mercury Exposure in Birds Linked to Marine Ecosystems in the Western Mediterranean.

Mercury (Hg), particularly as methylmercury (MeHg), is a nonessential, persistent, and bioaccumulative toxic element with high biomagnification capacity and is considered a threat to marine environments. We evaluated total Hg concentrations in liver, kidney, and brain in 62 individuals of 9 bird species linked to marine ecosystems from western Mediterranean admitted in a Wildlife Rehabilitation Center (WRC) (Alicante, Spain, 2005-2020). Age- and sex-related differences in Hg levels, as well as the cause of admission to the WRC, were also evaluated in certain species. The species studied were: northern gannet (Morus bassanus), European shag (Phalacrocorax aristotelis), great cormorant (Phalacrocorax carbo), osprey (Pandion haliaetus), Balearic shearwater (Puffinus mauretanicus), yellow-legged gull (Larus michahellis), razorbill (Alca torda), common tern (Sterna hirundo), and black-headed gull (Chroicocephalus ridibundus). Concentrations in feathers of 27 individuals, and concentrations in internal tissues in 7 other individuals of 7 different species were also reported but not statistically evaluated due to the limited number of samples. Results suggest that individuals were chronically exposed to Hg through diet. The differences in Hg concentrations among species may be explained by their diet habits. Mercury concentrations strongly correlated between tissues (r = 0.78-0.94, p < 0.001, n = 61-62). Some individuals of certain species (i.e., European shag, northern gannet, and great cormorant) showed Hg concentrations close to or above those described in the literature as causing reproductive alterations in other avian species. Consequently, certain individuals inhabiting western Mediterranean could be at risk of suffering long-term, Hg-related effects. Some of the species evaluated are listed within different categories of threat according to the International Union for Conservation of Nature (IUCN) and are endangered at a national level, so this study will provide valuable information for assessors and authorities in charge of the management of the environment and pollution.

Mercury and Organochlorine Pesticides in Tissues of Loggerhead Sea Turtles (Caretta caretta) Stranded Along the Southwestern Mediterranean Coastline (Andalusia, Spain).

Nineteen loggerhead sea turtles (Caretta caretta) stranded along the southwestern Mediterranean coastline (Andalusia) were used in this study. A total of 68 samples of fat (n = 18), liver (n = 15), kidney (n = 13), pectoral muscle (n = 19), and brain (n = 3) were analysed for total mercury (Hg) and organochlorine pesticides [OC: ∑Dichlorodiphenyltrichloroethanes (∑DDT), ∑Hexachlorocyclohexane (∑HCH), ∑Heptachlor, ∑Drins and ∑Endosulfan]. These loggerhead sea turtles showed tissue Hg and OC concentrations similar to or lower than those reported in other studies. Few growth-related variations in Hg or OC levels in relation to straight carapace length were found, probably because the specimens were mostly juveniles. This study will help to fill the gap on spatio-temporal exposure data and ascertain the real world-wide picture of the contamination levels in loggerhead sea turtles.

Effects of calcium supplementation on oxidative status and oxidative damage in great tit nestlings inhabiting a metal-polluted area.

Calcium has been proposed to diminish metal toxicity by the modulation of the oxidative stress. This study explores the effects of Ca availability and metal exposure on oxidative stress biomarkers in great tit (Parus major) nestlings. Nests were supplemented with Ca (Ca-supplemented group) or not supplemented (Control group) in a metal-polluted and a background zone in SW Finland. Metal concentrations were analyzed from feces. We analyzed antioxidants (tGSH, GSH:GSSG ratio, CAT, GST, GPx, SOD), protein carbonylation and lipid peroxidation in red cells of nestlings. Ca-supplemented and fast-growing nestlings showed higher CAT activity to cope with reactive oxygen species (ROS) generated during intensive growth and metabolism. SOD and GPx (the latter not statistically significant) were more active in the polluted area, possibly reflecting higher ROS production in nestlings from this zone due to the enhanced metal exposure and smaller size. Antioxidant levels changed over the range of metal concentrations depending on the Ca levels in plasma, suggesting that higher Ca levels stimulate antioxidants and mitigate the impacts of metals. Ca supplementation may improve nestling traits and reproductive output when antioxidants are enhanced in a situation of oxidative challenge. Therefore, Ca should be considered in future studies assessing metal exposure and effects on wild birds.

Spatiotemporal variations of organochlorine pesticides in an apex predator: Influence of government regulations and farming practices.

BACKGROUND: Intensification of agricultural practices has caused several negative effects to the environment. The use of fertilizers and pesticides may alter geochemical cycles or cause direct wildlife intoxication. Detrimental effects of organochlorine pesticides (OCPs) have forced the authorities to ban or restrict its use. This study evaluates the variation in levels of OCPs in a sentinel species in relation to changes in government regulations and the spatial configuration of agricultural practices around the nests. METHODS: Between 2003 and 2007, we analysed OCP levels in 256 blood samples of Eurasian Eagle-owl (Bubo bubo) chicks nesting in area of intensive commercial agriculture with historical frequent use of pesticides, in South-eastern Spain. We studied year-to-year variations in OCP concentrations and their relation with land use configuration around raptor nests by Generalized Linear Mixed Models (GLMM). RESULTS: OCPs were detected in 100% samples surveyed in 2003 and 2004, while dropped to 27% in 2005, 6.8% in 2006 and 6.3% in 2007, coinciding with the ban of OCPs. The presence of the main OCPs was related to agricultural practices. In particular, endosulfan and lindane were related to irrigated crops and urban areas, while DDT-related compounds and dieldrin were associated with dry land farming. CONCLUSIONS: OCP concentrations in blood samples of Eurasian Eagle-owls may respond quickly to the implementations of new regulations about the use of agricultural products. This raptor was confirmed as a good sentinel species allowing rapid detection of changes in pesticides use.

Lead exposure in common shelduck (Tadorna tadorna): Tracking the success of the Pb shot ban for hunting in Spanish wetlands.

Levels of lead (Pb) were analyzed in tissues of 25 Common shelduck (Tadorna tadorna) from La Albufera Natural Park (NP) in Valencia (Eastern Spain), which were found dead due to a botulism outbreak. The objective was to assess Pb exposure in waterfowl, fourteen years after the implementation of the Real Decreto 581/2001, which banned the use of Pb ammunition for hunting activities in internationally relevant wetlands. For this purpose, this study measured the actual ingestion of lead pellets in Common shelduck from La Albufera de Valencia and provide data on Pb residue concentrations in different tissues of this species breeding in this NP. The Pb distribution pattern in tissues was bone > kidney > liver > brain > muscle, with Pb concentrations of 778.96 ±â€¯998.64, 122.40 ±â€¯89.18, 92.08 ±â€¯57.65, 18.90 ±â€¯16.30 and 15.13 ±â€¯11.85 ng/g (wet weight), respectively. This work presents, as far as we are aware, the first published data on Pb concentrations in Common shelduck. The Pb levels detected were lower than those reported in other studies on Anatid species from Spanish wetlands, which may be explained by this ban. Moreover, lead concentrations found were below those levels for which any observable effect has been described for acute or chronic lead exposure.

Estrogenic activity of zearalenone, α-zearalenol and ß-zearalenol assessed using the E-screen assay in MCF-7 cells.

Mycotoxins, including zearalenone (ZEA), can occur worldwide in cereals. They can enter the food chain and cause several health disorders. ZEA and its derivatives (α-zearalenol, α-ZOL and ß-zearalenol, ß-ZOL) have structural analogy to estrogen, thus they can bind to estrogen receptors (ERs). In order to characterize the estrogenic activity of ZEA, α-ZOL and ß-ZOL, the proliferation of ER-positive human breast cancer cells (MCF-7) exposed to these mycotoxins was measured. After exposure at levels ranging from 6.25 to 25 µM, cell proliferation was evaluated by using the E-Screen bioassay. In accordance with previous studies, our results show the estrogenic activity of ZEA, α-ZOL and ß-ZOL in MCF-7 cells. This effect is related to ZEA and its metabolites being flexible enough to bind to mammalian ERs. The relative proliferative effect (RPE) ranged from 10% to 91%. The α-ZOL induced the highest proliferative effect due to its higher affinity for the ERs compared to the other mycotoxins.

Trace element concentrations in feathers and blood of Northern goshawk (Accipiter gentilis) nestlings from Norway and Spain.

Information on trace element pollution in the terrestrial environment and its biota is limited compared to the marine environment. In the present study, we collected body feathers and blood of 37 Northern goshawk (Accipiter gentilis) nestlings from Tromsø (northern Norway), Trondheim (central Norway), and Murcia (southeastern Spain) to study regional exposure, hypothesizing the potential health risks of metals and other trace elements. Blood and body feathers were analyzed by a high resolution inductively coupled plasma mass spectrometer (HR-ICP-MS) for aluminum (Al), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), cadmium (Cd), mercury (Hg) and lead (Pb). The influence of regional differences, urbanization and agricultural land usage in proximity to the nesting Northern goshawks was investigated using particular spatial analysis techniques. Most trace elements were detected below literature blood toxicity thresholds, except for elevated concentrations (mean ± SD µgml-1 ww) found for Zn (5.4 ± 1.5), Cd (0.00023 ± 0.0002), and Hg (0.021 ± 0.01). Corresponding mean concentrations in feathers (mean ± SD µgg-1 dw) were 82.0 ± 12.4, 0.0018 ± 0.002, and 0.26 ± 0.2 for Zn, Cd and Hg respectively. Multiple linear regressions indicated region was a significant factor influencing Al, Zn, Se and Hg feather concentrations. Blood Cd and Hg concentrations were significantly influenced by agricultural land cover. Urbanization did not have a significant impact on trace element concentrations in either blood or feathers. Overall metal and trace element levels do not indicate a high risk for toxic effects in the nestlings. Levels of Cd in Tromsø and Hg in Trondheim were however above sub-lethal toxic threshold levels. For holistic risk assessment purposes it is important that the concentrations found in the nestlings of this study indicate that terrestrial raptors are exposed to various trace elements.

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 Audouin's 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 Audouin's gull. Our findings show higher antioxidant levels in Audouin's 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 Audouin's 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 Audouin's 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 Audouin's 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.

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 Audouin's 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 Audouin's 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 Audouin's 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.

Comparative Long-term Study of a Large Series of Patients with Invasive Ductal Carcinoma and Invasive Lobular Carcinoma. Loco-Regional Recurrence, Metastasis, and Survival.

Our aim was to compare histologic and immunohistochemical features, surgical treatment and clinical course, including disease recurrence, distant metastases, and mortality between patients with invasive ductal carcinoma (IDC) or invasive lobular carcinoma (ILC). We included 1,745 patients operated for 1,789 breast tumors, with 1,639 IDC (1,600 patients) and 145 patients with ILC and 150 breast tumors. The median follow-up was 76 months. ILC was significantly more likely to be associated with a favorable phenotype. Prevalence of contralateral breast cancer was slightly higher for ILC patients than for IDC patients (4.0% versus 3.2%; p = n.s). ILC was more likely multifocal, estrogen receptor positive, Human Epidermal Growth Factor Receptor-2 (HER2) negative, and with lower proliferative index compared to IDC. Considering conservative surgery, ILC patients required more frequently re-excision and/or mastectomy. Prevalence of stage IIB and III stages were significantly more frequent in ILC patients than in IDC patients (37.4% versus 25.3%, p = 0.006). Positive nodes were significantly more frequent in the ILC patients (44.6% versus 37.0%, p = 0.04). After adjustment for tumor size and nodal status, frequencies of recurrence/metastasis, disease-free and specific survival were similar among patients with IDC and patients with ILC. In conclusion, women with ILC do not have worse clinical outcomes than their counterparts with IDC. Management decisions should be based on individual patient and tumor biologic characteristics rather than on lobular versus ductal histology.

Selective sentinel lymph node biopsy after neoadjuvant chemotherapy in breast cancer: results of the GEICAM 2005-07 study.

INTRODUCTION: A controversial aspect of breast cancer management is the use of sentinel lymph node biopsy (SLNB) in patients requiring neoadjuvant chemotherapy (NCT). This paper discusses the detection rate (DT) and false negatives (FN) of SLNB after NCT to investigate the influence of initial nodal disease and the protocols applied. METHODS: Prospective observational multicenter study in women with breast cancer, treated with NCT and SLNB post-NCT with subsequent lymphadenectomy. DT and FN rates were calculated, both overall and depending on the initial nodal status or the use of diagnostic protocols pre-SLNB. RESULTS: No differences in DT between initial node-negative cases and positive cases were found (89.8 vs. 84.4%, P=.437). Significant differences were found (94.1 vs. 56.5%, P=0,002) in the negative predictive value, which was lower when there was initial lymph node positivity, and a higher rate of FN, not significant (18.2 vs. 43.5%, P=.252) in the same cases. The axillary study before SLNB and after the NCT, significantly decreased the rate of FN in patients with initial involvement (55.6 vs 12.5, P=0,009). CONCLUSIONS: NCT means less DT and a higher rate of FN in subsequent SLNB, especially if there is initial nodal involvement. The use of protocols in axillary evaluation after administering the NCT and before BSGC, decreases the FN rate in these patients.

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.