Behavioral Responses of Imidacloprid-Dosed Farmland Birds to a Simulated Predation Risk.

Sublethal exposure to imidacloprid and other neonicotinoid insecticides may affect the neurological functions of birds. As such, behavior may be compromised. Here, we tested experimentally the effects of 1 and 6 mg/kg bw of imidacloprid on the antipredator behavioral responses of the red-legged partridge (Alectoris rufa) to simulated predator threats. Sixty-six partridges were challenged in groups or individually to intra- and interspecific alarm calls, to a raptor silhouette (aerial predation risk), and to a fox model (terrestrial predation risk). Antipredator behaviors were recorded as active (escape, active vigilance) and passive (passive vigilance, crouching, and freezing) responses. Latency in response to the stimuli, percentage of individuals who responded, response duration, speed of active responses, and vocalizations were measured. In experiments with partridges in the group, crouching against simulated predation risk lasted less time in birds treated with 6 mg a.i./kg bw than in control birds. In the experiments with individual partridges, passive vigilance against the intraspecific alarm lasted longer in birds treated with 6 mg a.i./kg bw than in control birds. The observed hyperreactivity to the predatory threat after a sublethal imidacloprid exposure can have consequences on survival under field conditions, where predation is a main driver of population dynamics.

Mercury-Modulated Immune Responses in Arctic Barnacle Goslings (Branta leucopsis) upon a Viral-Like Immune Challenge.

Historical mining activities in Svalbard (79°N/12°E) have caused local mercury (Hg) contamination. To address the potential immunomodulatory effects of environmental Hg on Arctic organisms, we collected newborn barnacle goslings (Branta leucopsis) and herded them in either a control or mining site, differing in Hg levels. An additional group at the mining site was exposed to extra inorganic Hg(II) via supplementary feed. Hepatic total Hg concentrations differed significantly between the control (0.011 ± 0.002 mg/kg dw), mine (0.043 ± 0.011 mg/kg dw), and supplementary feed (0.713 ± 0.137 mg/kg dw) gosling groups (average ± standard deviation). Upon immune challenge with double-stranded RNA (dsRNA) injection, endpoints for immune responses and oxidative stress were measured after 24 h. Our results indicated that Hg exposure modulated the immune responses in Arctic barnacle goslings upon a viral-like immune challenge. Increased exposure to both environmental as well as supplemental Hg reduced the level of natural antibodies, suggesting impaired humoral immunity. Hg exposure upregulated the expression of proinflammatory genes in the spleen, including inducible nitric oxide synthase (iNOS) and interleukin 18 (IL18), suggesting Hg-induced inflammatory effects. Exposure to Hg also oxidized glutathione (GSH) to glutathione disulfide (GSSG); however, goslings were capable of maintaining the redox balance by de novo synthesis of GSH. These adverse effects on the immune responses indicated that even exposure to low, environmentally relevant levels of Hg might affect immune competence at the individual level and might even increase the susceptibility of the population to infections.

Diet and Spatial Ecology Influence Red-Legged Partridge Exposure to Pesticides Used as Seed Treatment.

Seed treatment with pesticides is an extended agricultural practice with a high risk to granivorous birds that consume those seeds. To characterize that risk, it is necessary to understand the ecological factors that determine the exposure chances of birds to treated seeds. We investigated how pesticide uptake by red-legged partridges was related to cultivated plant ingestion and to the use of recently sown fields. We analyzed pesticide residues in 144 fecal samples from 32 flocks and determined the plant diet composition using DNA metabarcoding. Habitat use was studied through the monitoring of 15 GPS-tagged partridges. We confirmed, through the analysis of seeds, that >80% of cereal fields from the area had seeds treated with triazole fungicides. Tebuconazole was detected in 16.6% of partridges' feces. During the sowing season, cultivated plants accounted for half of the plant diet, but no association was found between cultivated plant consumption and pesticide intake. GPS tracking revealed that tebuconazole was detected in feces when partridges had recently used sown fields, whereas nonexposed partridges showed no overlap with recently sown areas. Our results highlight the need to incorporate field ecology into the characterization of pesticide exposure to improve the efficacy of environmental risk assessment.

Direct evidence of poison-driven widespread population decline in a wild vertebrate.

Toxicants such as organochlorine insecticides, lead ammunition, and veterinary drugs have caused severe wildlife poisoning, pushing the populations of several apex species to the edge of extinction. These prime cases epitomize the serious threat that wildlife poisoning poses to biodiversity. Much of the evidence on population effects of wildlife poisoning rests on assessments conducted at an individual level, from which population-level effects are inferred. Contrastingly, we demonstrate a straightforward relationship between poison-induced individual mortality and population changes in the threatened red kite (Milvus milvus). By linking field data of 1,075 poisoned red kites to changes in occupancy and abundance across 274 sites (10 × 10-km squares) over a 20-y time frame, we show a clear relationship between red kite poisoning and the decline of its breeding population in Spain, including local extinctions. Our results further support the species listing as endangered, after a breeding population decline of 31% to 43% in two decades of this once-abundant raptor. Given that poisoning threatens the global populations of more than 2,600 animal species worldwide, a greater understanding of its population-level effects may aid biodiversity conservation through increased regulatory control of chemical substances. Our results illustrate the great potential of long-term and large-scale on-ground monitoring to assist in this task.

HEALTH ASSESSMENT OF CAPTIVE BEARDED VULTURES (GYPAETUS BARBATUS) IN CATALONIA (SPAIN).

Bearded vulture (Gypaetus barbatus) populations are declining worldwide primarily due to anthropogenic factors. A captive breeding program has been established in Spain, a country with one of the largest free-living populations in Europe, to further enhance the conservation efforts of this emblematic species. However, captive vulture populations can be exposed to different risks through food, such as drug residues and antimicrobial-resistant (AMR) bacteria. Health surveillance of species involved in captive breeding programs is important to face introduction of healthy animals in situ and to obtain baseline clinical data. The objective of this study was to assess the general health status of bearded vultures held in captivity in Catalonia (northeastern Spain) by carrying out hematologic, biochemical, toxicologic, and bacteriologic analyses. A total of 16 bearded vultures were sampled; the data obtained from one vulture, with a chronic tibiotarsal fracture, were excluded from the statistical analysis. Hematologic and biochemical parameters of the bearded vultures were mostly within the range of standard values as stated in previous studies. Basal feather and serum corticosterone levels were analyzed and described for the first time in this species. A total of 15 Escherichia coli isolates were obtained that were resistant to fluoroquinolones (80%), tetracycline (60%), trimethoprim and ampicillin (40%), sulfamethoxazole (33%), and colistin (20%), with 40% of them being multidrug resistant. Three of 15 isolates were carriers of the mcr-1 gene. Only the injured bird previously treated with enrofloxacin was positive for fluoroquinolone residues. Periodic monitoring for the presence of AMR bacteria would be recommended in captive breeding programs as a preventive action to establish future therapies.

Nonspecific protection of heat-inactivated Mycobacterium bovis against Salmonella Choleraesuis infection in pigs.

Trained immunity is the capacity of innate immune cells to produce an improved response against a secondary infection after a previous unrelated infection. Salmonellosis represents a public health issue and affects the pig farming industry. In general, vaccination against salmonellosis is still facing problems regarding the control of distinct serovars. Therefore, we hypothesized that an immunostimulant based on heat inactivated Mycobacterium bovis (HIMB) could have an immune training effect in pigs challenged with Salmonella enterica serovar Choleraesuis (S. Choleraesuis) and decided to explore the amplitude of this non-specific immune response. For this purpose, twenty-four 10 days-old female piglets were randomly separated in three groups: immunized group (n = 10) received orally two doses of HIMB prior to the intratracheal S. Choleraesuis-challenge, positive control group (n = 9) that was only challenged with S. Choleraesuis, and negative control group (n = 5) that was neither immunized nor infected. All individuals were necropsied 21 days post-challenge. HIMB improved weight gain and reduced respiratory symptoms and pulmonary lesions caused by S. Choleraesuis in pigs. Pigs immunized with HIMB showed higher cytokine production, especially of serum TNFα and lung CCL28, an important mediator of mucosal trained immunity. Moreover, immunized pigs showed lower levels of the biomarker of lipid oxidation malondialdehyde and higher activity of the antioxidant enzyme superoxide dismutase than untreated challenged pigs. However, the excretion and tissue colonization of S. Choleraesuis remained unaffected. This proof-of-concept study suggests beneficial clinical, pathological, and heterologous immunological effects against bacterial pathogens within the concept of trained immunity, opening avenues for further research.

Factors influencing lead, mercury and other trace element exposure in birds from metal mining areas.

Non-ferrous metal mining is considered one of the largest sources of toxic metal released to the environment and may threaten ecosystems, notably biota. We explored how birds that inhabit non-ferrous metal mining sites are exposed to mercury, lead, and other trace elements by analyzing their feathers and verifying which factors may influence element concentrations in feathers. We sampled a total of 168 birds, representing 26 species, with different feeding habits and migration patterns in a non-polluted reference site and two historical metal mining areas: Almadén, which is considered one of the most heavily mercury-contaminated sites worldwide, and the Sierra Madrona mountains where lead has been mined since ancient times. The quantification of aluminum (Al), arsenic (As), barium (Ba), beryllium (Be), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), mercury (Hg), magnesium (Mg), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), selenium (Se), thorium (Th), thallium (Tl), uranium (U), vanadium (V) and zinc (Zn) was performed by inductively coupled plasma mass spectrometry (ICP-MS). Feather analysis revealed contamination by Hg and Pb, in Almadén and Sierra Madrona, respectively. We found that granivorous birds had the lowest feather Hg levels compared to those found in omnivorous, insectivorous, and piscivorous species, whereas feather Pb was about twice as high in granivores and omnivores, than in insectivorous and piscivorous birds. We also found differences among study sites in 13 elements and confirmed the influence of feather age, migratory patterns of the birds, and external deposition of elements, on metal concentrations in the feathers. Our results highlight that despite the cessation of metal mining in the study areas, local avifauna are being exposed to Hg and Pb from abandoned mines and old tailings sites, indicating that appropriate measures are needed to protect biota from overexposure to these toxic metals.

Pilot monitoring scheme of water pollutants in Important Bird and Biodiversity Areas.

In this study we have established a monitoring scheme to determine the presence and distribution of widely used pharmaceuticals, pesticides, organophosphate esters (OPEs) and perfluoroalkyl substances (PFAS) in water bodies from Important Bird and Biodiversity Areas (IBAs) from Spain. The monitoring scheme included the georeferenced sampling of rocky mountain, Atlantic forest, riparian forest, Mediterranean forest, agricultural, inland aquatic and coastal aquatic IBAs, with the aim to evaluate the impact of widely used chemicals in those aquatic resources. Water samples were extracted using a generic solid-phase extraction protocol and analyzed by 3 analytical methods based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Quality parameters such as compound recovery, intra and inter-day variation, linearity and limits of detection were calculated in order to validate the methods. In addition, the ionization conditions and the optimization of the most appropriate transitions permitted unequivocal identification. Once the sampling and analytical procedure was set-up, 59 target compounds were monitored in 63 samples. Pharmaceutical, followed by pesticides, OPEs and PFAS were widespread along all IBAs studied at concentrations from 0.5 to 41083 ng/L. Overall, this study highlights the need to monitor the presence of contaminants in areas of high ecological interest to contribute to pollution control and mitigation towards protection of biodiversity.

Effect of mineral supplementation on lead bioavailability and toxicity biomarkers in sheep exposed to mining pollution.

The chronic exposure of livestock to lead (Pb) pollution in historical mining areas may represent significant and unnecessary costs for farmers and primary producers, in addition to important food safety risks. Here, we evaluate the effect of mineral supplements, in the form of a commercial mineral block (MB), to reduce Pb bioavailability and toxicity in sheep through an experimental approach under real farming conditions in an abandoned mining area. Blood, fecal Pb levels, and soil ingestion, along with different blood and plasma biomarkers were studied. Experiment 1 was carried out with 3-months-old female lambs, n = 54, fenced in two contiguous MB and non-MB-supplemented plots. After 20 days of treatment, blood Pb level was lower in MB-supplemented sheep than in those that were non-MB-supplemented. Experiment 2 was carried out with 2-months-old female lambs, n = 34, fenced in a single plot and MB-supplemented during the first 20 days of experiment. After MB supplementation, blood Pb level in sheep was also reduced by almost half, falling below the threshold of subclinical intoxication, and then increased again after 20 days without MB. Experiment 3 was carried out with adult rams, n = 10, fenced in a single MB-supplemented plot during the first 20 days of experiment. In this case, blood Pb level decreased by day 40. Soil ingestion was not reduced by MB supplementation in any of the experiments. MB supplementation favored antioxidant status by increasing SOD activity and reducing GPX activity and MDA levels. In conclusion, the MB supplementation seemed to reduce Pb bioavailability by increasing its fecal excretion, but renal excretion and bone deposition may also have favored the reduction of blood Pb concentration. Mineral supplements may be a new easy-to-apply and cost-effective way to reduce livestock exposure in Pb polluted sites.

Metals and metalloids in freshwater fish from the floodplain of Tablas de Daimiel National Park, Spain.

The Tablas de Daimiel National Park (TDNP) is a floodplain ecosystem in central Spain with a potential risk of heavy metal and metalloid pollution. The objective of this study was to know the accumulation of arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb), and selenium (Se) in muscle of six species of freshwater fish from the floodplain of TDNP. We obtained muscle samples of Cyprinus carpio (n = 89); Squalius pyrenaicus (n = 16); Ameiurus melas (n = 9); Lepomis gibbosus (n = 8); Micropterus salmoides (n = 6) and Carassius auratus (n = 5). A. melas, which is a predatory species, had significantly higher Hg concentrations than omnivorous or herbivorous species (i.e. C. carpio). On the contrary, A. melas showed lower concentrations of As, Pb and Se than omnivorous species (i.e. S. pyrenaicus and L. gibbosus ). The concentration of Hg was positively associated with fish size in C. carpio and A. melas. Some individuals of C. carpio (5.7%) and S. pyrenaicus (12.5%) showed Pb muscle concentrations above the maximum residue levels established by the European legislation for human consumers. The observed muscle Se concentrations can be associated with adverse effects on fish such as blood changes, reduced growth, mortality of juveniles and reproductive failure. The accumulation of Se in this floodplain located in a seleniferous area and the contamination produced by spent Pb shot pellets used for hunting in the past are discussed as potential sources of the elevated levels of these two elements in fish from this floodplain wetland.

Egg Overspray with Herbicides and Fungicides Reduces Survival of Red-Legged Partridge Chicks.

Within the environmental risk assessment conducted for pesticide registration in the European Union (EU), avian reproductive toxicity is characterized after exposing adults. However, eggs of ground-nesting species can be exposed when pesticide applications occur during laying or incubation. We simulated environmentally realistic exposure of red-legged partridge (Alectoris rufa) eggs to an herbicide (2,4-D) and a fungicide (tebuconazole) applied to winter cereal crops during the breeding season of most farmland birds. We analyzed the effects on hatching success, offspring survival, and physiology. Exposure by overspray led to greater pesticide accumulation in the eggshell or content than exposure through contact with treated soil (3.1-13.7 times higher, depending on the pesticide and target sample). Egg overspray with tebuconazole significantly increased chick mortality, which was 26% higher than that of controls. 2,4-D caused a similar but a close to significant increase (chick mortality 24% higher than controls). Exposure to either pesticide through contact with treated soils did not affect chick survival but altered some biochemical parameters posthatching. Our experiment shows that egg spraying with pesticides should be considered as a relevant exposure scenario in risk assessment procedures, given its potential to affect the reproductive success of ground-nesting farmland birds.

Assessment of ecotoxicological risks to river otters from ingestion of invasive red swamp crayfish in metal contaminated areas: Use of feces to estimate dietary exposure.

The invasive red swamp crayfish (Procambarus clarkii) has become a major food resource for Eurasian otters (Lutra lutra) in the Iberian Peninsula. Crayfish accumulate large amounts of metals, and hence otters could be at risk of exposure and intoxication through crayfish consumption. We conducted a food safety risk assessment for otters inhabiting two historical mining areas in central Spain affected by lead (Pb) and mercury (Hg) pollution. Estimated daily intakes (EDI) of Pb and Hg were non-invasively calculated from the proportion of crayfish remains and metal levels in otter feces. We considered that the abdominal muscle and the carcass of crayfish differ significantly in relative weight, total metal content and bioavailability of metals to reduce the uncertainty of risk characterization. Fecal concentrations of Hg and Pb in the polluted areas were 1.878 and 6.554 µg/g d. w., respectively (13-fold and 7-fold higher compared to a non-polluted area). EDI of Hg and Pb in the polluted areas were 66.02 and 78.26 µg/kg-day, respectively (14- and 8-fold higher than in the reference area). EDI from the Hg area were above minimum levels susceptible to cause neurotoxicity in mustelids, and 6.3% were above levels susceptible to cause histopathological lessions. In the Pb area, 16.7% of EDI were consistent with levels causing reproductive effects. Metal exposure through crayfish consumption might prevent or slow the recovey of otters in these polluted environments, thus this factor should be considered in management strategies aimed to protect otter populations.

Multi-level analysis of exposure to triazole fungicides through treated seed ingestion in the red-legged partridge.

Triazole fungicides are the most widely used products to treat cereal seeds. Granivorous birds, such as red-legged partridges (Alectoris rufa), which consume seeds left on the surface of fields after sowing, have a high risk of exposure. As triazole fungicides can affect sterol synthesis, we tested the hypothesis that treated seed consumption could alter the synthesis of sex hormones and reduce the reproductive capacity of partridges. We exposed adult partridges to seeds treated with four different formulations containing triazoles as active ingredients (flutriafol, prothioconazole, tebuconazole, and a mixture of the latter two) simulating a field exposure during the late autumn sowing season. All treatments produced biochemical changes and an overexpression of genes encoding for enzymes involved in the biosynthesis of sterols and steroid hormones, such as PMVK, ABCA1, MVD, PSCK9, DHCR7 and HSD17B7. Plasma levels of oestradiol were reduced in partridges exposed to tebuconazole. We also monitored reproduction 3 months after exposure (laying date, egg fertilization and hatching rates). We observed a 14-day delay in the laying onset of partridges that had been exposed to flutriafol as compared to controls. These results show that the consumption of seeds treated with triazole fungicides has the potential to affect granivorous bird reproduction. We recommend the evaluation of lagged reproductive effects as part of the protocols of environmental risk assessment of pesticides in wild birds in light of the effects resulting from the exposure to triazole-treated seeds.

Maternal food supplementation and perceived predation risk modify egg composition and eggshell traits but not offspring condition.

Mothers may vary resource allocation to eggs and embryos, which may affect offspring fitness and prepare them for future environmental conditions. The effects of food availability and predation risk on reproduction have been extensively studied, yet their simultaneous impacts on reproductive investment and offspring early life conditions are still unclear. We experimentally manipulated these key environmental elements using a 2×2 full factorial design in wild, free-living pied flycatchers (Ficedula hypoleuca), and measured egg composition, eggshell traits and offspring condition. Eggs laid in food-supplemented nests had larger yolks and thicker shells independently of predation risk, while eggs laid in nests exposed to predator cues had lower levels of immunoglobulins, independent of food supplementation. In nests without predator cues, shell biliverdin content was higher in eggs laid in food-supplemented nests. Incubation was 1 day shorter in food-supplemented nests and shorter incubation periods were associated with higher hatching success, but there were no direct effects of maternal treatment on hatching success. To investigate the impact of maternal treatment (via egg composition) on the offspring, we performed full brood cross-fostering after hatching to unmanipulated nests. Maternal treatment did not significantly affect body mass and immunoglobulin levels of offspring. Our results suggest that although prenatal maternal cues affected egg composition, these egg-mediated effects may not have detectable consequences for offspring growth or immune capacity. Unpredictable environmental stressors may thus affect parental investment in the eggs, but parental care may level off costs and benefits of differential maternal egg allocation.

Food safety risk assessment of metal pollution in crayfish from two historical mining areas: Accounting for bioavailability and cooking extractability.

Here we characterize the bioaccumulation of mercury (Hg) and lead (Pb) in red swamp crayfish (Procambarus clarkii) from two river courses in Central Spain that are impacted by historical Hg and Pb mining activities, respectively. We estimate the absolute oral bioavailability of metals in crayfish tissues by means of in vitro bioaccessibility simulations, and assess whether their consumption may imply a health risk for humans by estimating target hazard quotients and safe consumption rates. We also study the effect of cooking crayfish on the mobilization of the metal body burden in the context of the traditional Spanish cuisine. The results showed that crayfish from the mining districts accumulated a high level of Hg and Pb pollution in both the tail muscle and the carcass. The in vitro bioaccessibility of Hg and Pb in the edible part was 27.86 ±â€¯4.05 and 33.73 ±â€¯5.91%, respectively. Absolute bioavailability was estimated to be 38.31 for Hg, and 20.21 (adults) and 67.35% (children) for Pb. Risk indices indicated that, even after adjusting for bioavailability, it is not safe to consume crayfish from the mining-impacted rivers because of their high levels of Hg and Pb. Using the carcass as a condiment for flavouring should also be avoided. The cooking procedure extracted relatively small amounts of the total Hg (8.92 ±â€¯2.13%) and Pb (1.68 ±â€¯0.29%) body burden. Further research that will support human and ecological risk assessment, along with the implementation of advisory measures for the local population as regards crayfish consumption, are recommended.

Dietary canthaxanthin reduces xanthophyll uptake and red coloration in adult red-legged partridges.

Carotenoids give color to conspicuous animal signals that are often the product of sexual selection. Knowledge of the mechanisms involved in carotenoid-based signaling is critical to understanding how these traits evolve. However, these mechanisms remain only partially understood. Carotenoids are usually viewed as scarce dietary antioxidants whose allocation to ornaments may trade off against health. This trade-off would ensure its reliability as a signal of individual quality. In the case of red (keto)carotenoids, the literature suggests that some species may show constraints in their uptake. Canthaxanthin is one of the most common ketocarotenoids in red ornaments of animals. It is often commercially used as a dietary supplement to obtain redder birds (e.g. poultry). We increased the dietary canthaxanthin levels in captive red-legged partridges (Alectoris rufa). This species shows red non-feathered parts mostly pigmented by another common ketocarotenoid: astaxanthin. We studied the impact on the uptake of carotenoids and vitamins and, finally, on coloration. We also tested the potential protective effect of canthaxanthin when exposing birds to a free radical generator (diquat). Canthaxanthin did not apparently protect birds from oxidative stress, but interfered with the absorption of yellow carotenoids (lutein and zeaxanthin). Zeaxanthin is a precursor of astaxanthin in enzymatic pathways, and their levels in tissues and eggs were lower in canthaxanthin-supplied birds. This led to lower astaxanthin levels in ornaments and paler coloration. As far as we know, this is the first report of a carotenoid supplementation decreasing animal coloration. The results have implications for understanding carotenoid-based signaling evolution, but also for improving husbandry/experimental procedures.

Mapping the spatio-temporal risk of lead exposure in apex species for more effective mitigation.

Effective mitigation of the risks posed by environmental contaminants for ecosystem integrity and human health requires knowing their sources and spatio-temporal distribution. We analysed the exposure to lead (Pb) in griffon vulture Gyps fulvus-an apex species valuable as biomonitoring sentinel. We determined vultures' lead exposure and its main sources by combining isotope signatures and modelling analyses of 691 bird blood samples collected over 5 years. We made yearlong spatially explicit predictions of the species risk of lead exposure. Our results highlight elevated lead exposure of griffon vultures (i.e. 44.9% of the studied population, approximately 15% of the European, showed lead blood levels more than 200 ng ml(-1)) partly owing to environmental lead (e.g. geological sources). These exposures to environmental lead of geological sources increased in those vultures exposed to point sources (e.g. lead-based ammunition). These spatial models and pollutant risk maps are powerful tools that identify areas of wildlife exposure to potentially harmful sources of lead that could affect ecosystem and human health.

Effects of Lead Exposure on Sperm Quality and Reproductive Success in an Avian Model.

Lead (Pb) poisoning via ingestion of shot pellets is a frequent cause of death in wild birds and also has a wide range of subclinical effects. Here we report on the sublethal effects Pb exposure has on the breeding performance of red-legged partridges (Alectoris rufa). We studied the effects of Pb exposure on sperm quality, reproductive success, egg properties, laying performance, antioxidant levels, and carotenoid-based coloration. Birds were exposed by oral gavage to one or three No. 6 Pb shot pellets (2.8 mm in diameter, mean mass ± SD: 109 ± 7.97 mg). We show that exposure to three pellets (330 mg) reduced the hatching rate of females and decreased the acrosome integrity and sperm motility of males. In addition, females exposed to 1 pellet (110 mg) produced heavier eggs and chicks, whereas males exposed to 1 pellet presented an increase in sperm vigor. Sperm viability, concentration, progressiveness or fecundation rate were not affected by Pb treatment. Pb exposure increased circulating antioxidant levels in males, whereas the percentage of carotenoid-pigmented eye-ring area decreased in exposed females. Several sperm parameters showed positive relationships with coloration and antioxidant levels, suggesting that males displaying redder ornaments may be more capable of protecting sperm from oxidative stress in the event of sublethal Pb exposure.

Risk assessment of lead poisoning and pesticide exposure in the declining population of red-breasted goose (Branta ruficollis) wintering in Eastern Europe.

The red-breasted goose Branta ruficollis is a globally threatened species (IUCN Vulnerable) and the only European goose species currently in decline. Working on the wintering grounds on the Black Sea Coast, we address two potential causes of decline of this species for the first time: lead poisoning, and contamination from pesticides. We quantified the densities of spent Pb shot in three wetlands used by the geese in north-east Bulgaria, and analysed the Pb concentration in the faeces of red-breasted geese and the more abundant greater white-fronted geese Anser albifrons, using Al concentration as an indicator of soil ingestion. Pb shot densities in sediments were low, and we found no evidence for Pb shot ingestion in red-breasted geese. On the other hand, we found that the geese were feeding on wheat whose seeds were treated with four fungicides: thiram, tebuconazole, difenoconazole and fludioxonil, and the two first were even detected in geese faecal samples. Using data on the daily food intake, we estimated the exposure levels of the geese to these fungicides, both by measuring the concentrations remaining on seeds and by estimating the amount used to coat the seeds at the time of sowing. We found that the exposure rates estimated during the sowing period for both geese species can exceed the recognized hazardous doses for thiram, and to a lesser extent for tebuconazole, which indicates that some pesticides may be playing a previously overlooked role in the decline of red-breasted geese.

Sublethal Pb exposure produces season-dependent effects on immune response, oxidative balance and investment in carotenoid-based coloration in red-legged partridges.

Ingestion of lead (Pb) shot pellets constitutes the main cause of Pb poisoning in avifauna. We studied the effects of sublethal Pb exposure on immunity, carotenoid-based coloration, oxidative stress and trade-offs among these types of responses during spring and autumn in red-legged partridges (Alectoris rufa). We evaluated constitutive immunity testing lysozyme and natural antibody levels, and blood bactericidal and phagocytic activities. We studied induced immunity by testing PHA and humoral responses. We analyzed fecal parasite and bacterial abundance and oxidative stress biomarkers. Pb exposure in spring reduced natural antibody levels, whereas in autumn, it reduced lysozyme levels and increased phagocytic activity. Pb exposure increased PHA response in both seasons, and decreased T-independent humoral response in autumn. Pb exposure also increased noncoliform and decreased coliform Gram-negative gut bacteria. In spring, Pb exposure decreased antioxidant levels and increased coloration in males, whereas in autumn, it increased retinol levels but reduced coloration in both genders. Our results suggest that in spring, Pb-exposed females used antioxidants to cope with oxidative stress at the expense of coloration, whereas Pb-exposed males increased coloration, which may reflect an increased breeding investment. In autumn, both genders prioritized oxidative balance maintenance at the expense of coloration.