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Despite the importance of reptiles in agroecosystems, little is known about the effects of agricultural intensification and pesticide use on these animals. We compared antioxidant and haematological biomarkers in the wild Italian wall lizards Podarcis siculus from three olive groves representing a gradient of management intensity. Lizards from the conventional grove showed induced antioxidant defences relative to those from the organic field. However, this induction did not avoid the occurrence of oxidative stress in males from intensively managed olive groves, who showed TBARS levels 58%–133% higher than males from the other sites. Haematological responses also suggested increased stress in females from the intensively managed olive groves, with a heterophil-to-lymphocyte ratio 5.3 to 14.8-fold higher than in the other sites. The observed stress responses of lizards along the studied gradient of agricultural management suggest their potential usefulness as non-destructive biomarkers to environmental stressors associated with agricultural intensification., The present study was supported by FEDER through COMPETE – Programa Operacional Factores de Competitividade, Fundação para a Ciência e Tecnologia (PTDC/BIA-BIC/3488/2012), CESAM’s Strategic Programme (UIDB/50017/2020 + UIDP/50017/2020), the EU’s FP7 Project HERPESTI (IEF-2012-328328) and the Doctoral Fellowship of EF (SFRH/BD/88955/2012).

The ingestion of lead (Pb) shot pellets is a well-known cause of avian mortality, but exposure of birds to Pb may have other sublethal effects that can affect population sustainability. One of these effects is the alteration of the immunological status. Pb can affect most components of the avian immune system and imbalance the relationship among them. Pb exposure typically alters the ratio between T- and T-type responses mounted by different classes of T-lymphocytes, causing the depression of the T responses that are associated with cell-mediated immunity. Immunodepressing effects of Pb on birds are observed at blood levels above 50 μg dL, but developing birds show immunodepressing effects at much lower concentrations (> 10 μg dL). Impacts of Pb on the avian immune system also relate to reduced resistance to infection. We review immunotoxic effects of Pb on birds affected by shot ingestion as well as by other sources of exposure to this element., Part of the information included in this review was compiled as part of a research project funded by the Spanish Ministry of Science and Innovation (grant CGL2010-17030) and included in the PhD Thesis of NVC, who benefited from an FPI fellowship of the Spanish Ministry of Science and Innovation (ref. BES-2011-045670).

Some domestic waste disposal sites (DWDS) accumulate enormous amounts of food that humans discard. This food resource is exploited by different species whose population dynamics have as a result been altered. The effects of feeding on human waste on individual health parameters such as body condition are still unknown. Traditionally, body condition in wildlife has been assessed using morphological indices based on size and weight. Expanding the range of indices to include the analysis of indicators at the physiological level can provide a more comprehensive picture of the animals' health status. In this work, we studied how different amounts of waste consumption (assessed using stable isotope analysis) affect nestling health and body condition in an expanding population of the endangered Egyptian Vulture (Neophron percnopterus). We used morphometric and physiological approaches, including plasma biochemistry, antioxidant defences and oxidative stress biomarkers. The results of the multivariate redundancy analysis and variance partitioning showed that the proportion of diet originating from DWDS is the main factor explaining physiological indicators in the studied vulture population. Nestlings that were fed on food from DWDS had lower levels of vitamins (e.g. α-tocopherol) and carotenoids (zeaxanthin and lutein), which have important functions as antioxidant defences, immunostimulant properties and ornamental purposes. Nevertheless, nestlings that were fed on food from DWDS were better nourished and experienced fewer food shortages than individuals not fed using this resource, which had lower levels of cholesterol. Possibly due to the easy access to food, breeding pairs using DWDS could have fewer difficulties in feeding their nestlings and thus could engender more regular brood sizes than those not exploiting this type of resource. Our integrative approach using diverse components was crucial for a reliable assessment of health and body condition in Egyptian Vulture nestlings and its relationship to the consumption of food derived from human-altered environments., This study was financed by University of Barcelona (J. R., A. H. M. and H. T.), and Red Electrica de España-SA and we are grateful to Mercedes Gil. H.T was supported by a predoctoral grant from the IRBIO-University of Barcelona (APIF 2014).

Drilled seeds are an important food resource for many farmland birds but may pose a serious risk when treated with pesticides. Most compounds currently used as seed treatment in the EU have low acute toxicity but may still affect birds in a sub-chronic or chronic way, especially considering that the sowing season lasts several weeks or months, resulting in a long exposure period for birds. Tebuconazole is a triazole fungicide widely used in agriculture but its toxicity to birds remains largely unknown. Our aim was to test if a realistic scenario of exposure to tebuconazole treated seeds affected the survival and subsequent reproduction of the red-legged partridge (Alectoris rufa). We fed captive partridges with wheat seeds treated with 0%, 20% or 100% of tebuconazole application rate during 25 days in late winter (i.e. tebuconazole dietary doses were approximately 0.2 and 1.1 mg/kg bw/day). We studied treatment effects on the physiology (i.e. body weight, biochemistry, immunology, oxidative stress, coloration) and reproduction of partridges. Exposed birds did not reduce food consumption but presented reduced plasmatic concentrations of lipids (triglycerides at both exposure doses, cholesterol at high dose) and proteins (high dose). The coloration of the eye ring was also reduced in the low dose group. Exposure ended 60 days before the first egg was laid, but still affected reproductive output: hatching rate was reduced by 23% and brood size was 1.5 times smaller in the high dose group compared with controls. No significant reproductive effects were found in the low dose group. Our results point to the need to study the potential endocrine disruption mechanism of this fungicide with lagged effects on reproduction. Risk assessments for tebuconazole use as seed treatment should be revised in light of these reported effects on bird reproduction., Funding support was by CSIC (Intramural 201330E041), FEDENCA (Real Federación Española de Caza) and Oficina Nacional de la Caza with the partnership of Fundación Biodiversidad. Ana Lopez-Antia is a postdoctoral researcher of the Research Foundation - Flanders (FWO). This study contributes to the projects REGRESEEDS (Spanish Ministry of Economy and Competitiveness; CGL 2016-75278-R) and AGROPERDIZ (Junta de Comunidades de Castilla-La Mancha; SBPLY/17/180501/000245).

The ingestion of lead (Pb) ammunition is the most important exposure pathway to this metal in birds and involve negative consequences to their health. We have performed a passive monitoring of Pb poisoning in birds of prey by measuring liver (n = 727) and blood (n = 32) Pb levels in individuals of 16 species found dead or sick in Spain between 2004 and 2020. We also performed an active monitoring by measuring blood Pb levels and biomarkers of haem biosynthesis, phosphorus (P) and calcium (Ca) metabolism, oxidative stress and immune function in individuals (n = 194) of 9 species trapped alive in the field between 2016 and 2017. Passive monitoring results revealed some species with liver Pb levels associated with severe clinical poisoning (>30 μg/g d.w. of Pb): Eurasian griffon vulture (27/257, 10.5%), red kite (1/132, 0.8%), golden eagle (4/38, 10.5%), and Northern goshawk (1/8, 12.5%). The active monitoring results showed that individuals of bearded vulture (1/3, 33.3%), Eurasian griffon vulture (87/118, 73.7%), Spanish imperial eagle (1/6, 16.7%) and red kite (1/18, 5.6%) had abnormal blood Pb levels (>20 μg/dL). Blood Pb levels increased with age, and both monitoring methods showed seasonality in Pb exposure associated with a delayed effect of the hunting season. In Eurasian griffon, blood Pb concentration was associated with lower δ-ALAD activity in blood and P levels in plasma, and with higher blood lipid peroxidation and plasma carotenoid levels in agreement with other experimental and field studies in Pb-exposed birds. The study reveals that Pb poisoning is a significant cause of death and sublethal effects on haem biosynthesis, P metabolism and oxidative stress in birds of prey in Spain., This study has been funded by several contracts for the detection of poisoning events in wildlife with regional governments and the Ministry of Agriculture, Food and Environment of Spain and with the project CGL2013-40975-R of the Spanish Ministry of Science and Innovation. PRC benefited from a contract with reference PTA2017-14583-I financed by the Spanish Ministry of Science and Innovation and CSIC. MMH was supported by a Juan de la Cierva postdoctoral research contract provided by the Spanish Government (IJCI-2014-20171) and currently by the JCCM and the European Regional Development Fund (SBPLY/17/180501/000514). MEOS was supported by a contract of the Ramón y Cajal program of the Spanish Ministry of Science.

Despite the importance of reptiles in agroecosystems, little is known about the effects of agricultural intensification and pesticide use on these animals. We compared antioxidant and haematological biomarkers in the wild Italian wall lizards Podarcis siculus from three olive groves representing a gradient of management intensity. Lizards from the conventional grove showed induced antioxidant defences relative to those from the organic field. However, this induction did not avoid the occurrence of oxidative stress in males from intensively managed olive groves, who showed TBARS levels 58%-133% higher than males from the other sites. Haematological responses also suggested increased stress in females from the intensively managed olive groves, with a heterophil-to-lymphocyte ratio 5.3 to 14.8-fold higher than in the other sites. The observed stress responses of lizards along the studied gradient of agricultural management suggest their potential usefulness as non-destructive biomarkers to environmental stressors associated with agricultural intensification.

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., The study was financed by the Spanish Ministry of Science through the project Regreseeds (ref CGL2016-75278-R) and contributes to Agroperdiz (project SBPLY/17/180501/000245; Junta de Comunidades de Castilla-La Mancha). All the experimental procedures were approved by the competent authority Junta de Comunidades de Castilla-La Mancha (permit reference 11-2017).

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., The study was financed but the Spanish Research Agency of the Ministry of Science (project CGL2016-75278-R) and the Junta de Comunidades de Castilla-La Mancha (project SBPLY/17/180501/000245). MEOS was supported by a contract of the Ramón y Cajal program of the Spanish Ministry of Science.

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., This work was supported by the Junta de Comunidades de Castilla-La Mancha (Project PPII-2014-028-P). JRE was supported by a Research and Innovation Strategy for Smart Specialisation (RIS3) postdoctoral contract (2017/11747) from the Junta de Comunidades de Castilla-La Mancha.

Sown seeds are a key component of many farmland birds’ diets due to natural food shortages in autumn and winter. Because these seeds are often treated with pesticides, their ingestion by birds can result in toxic effects. For risk assessment, data on treated seed toxicity should be combined with information about exposure risk for wild birds and the factors that modulate it. We characterized the exposure of red-legged partridges to pesticide-treated seeds through the analysis of digestive contents of birds shot by hunters (n = 194) in an agricultural region in central Spain. We measured the contribution of sown seeds to the partridges' diet and how it related to pesticide exposure. Moreover, we evaluated the influence of landscape composition on the intake of sown seeds and pesticides by partridges. During peak sowing time, seeds constituted half (50.7%) of the fresh biomass ingested by partridges, which consumed mostly winter cereal seeds (42.3% of biomass). Residues of seven fungicides and one insecticide (active ingredients) were detected in 33.0% of birds. The presence of pesticides in digestive contents was linked to the ingestion of cereal sown seeds. Moreover, dietary exposure of birds to pesticides was modulated by landscape characteristics, being lower in areas with heterogeneous landscapes, greater habitat mosaic and more natural vegetation. The estimated dietary intake of pesticides resulting from our field observations, in combination with experimental data on pesticide toxicity, raise concerns about the risks that pesticide-treated cereal seeds pose to granivorous bird populations. Our results highlight the importance of farming landscape composition and diversification, which should be considered as a priority in the agricultural policy to mitigate pesticide risks to farmland birds through the consumption of treated seeds., This study was funded by the projects AGROPERDIZ (SBPLY/17/180501/000245; Junta de Comunidades de Castilla-La Mancha and “Fondo Europeo de Desarrollo Regional”) and REGRESEEDS (CGL2016-75278; Spanish Ministry of Science). MEOS was supported by Ramón y Cajal grant (Spanish Ministry of Science).

We monitor pollutant accumulation and investigate associated changes at the physiological level within the population of an obligate avian scavenger, the Egyptian Vulture (Neophron percnopterus), from Catalonia (NE Spain). This population is expanding its range, presumably because of the use of human waste disposal sites as food resource. We hypothesized that habitat urbanization, presumably associated with feeding from human wastes, could influence the accumulation of persistent organic pollutants and metals. The aim of this study was to explore the relationship between accumulated pollutants and biochemical parameters in nestling blood. We used the proportion of urban surface within an 8 km radius of each nest as a proxy to study the relationship between anthropic influence and pollutant accumulation. Observed blood levels of metals, organochlorine pesticides, polychlorinated biphenyls (PCBs), per- and polyfluoroalkylated substances (PFAS) and polybrominated diphenyl ethers (PBDEs) were relatively low, as expected for nestling individuals due to short-term exposures. CB-180 and PBDEs were associated with variations in blood biochemistry parameters; hexa-BDEs appeared positively associated with activities of the enzymes aspartate aminotransferase and lactate dehydrogenase, whereas CB-180 accumulation was associated with an increased activity of creatine phosphokinase and elevated glutathione levels. Increased CB-180 levels were also related to decreased blood concentrations of calcium, cholesterol, α-tocopherol and lutein. A proportion of urban surfaces as low as 6.56% within a radius of 8 km around the nest appears related to the accumulation of CB-180, the majority of analysed PFAS and of PBDE congeners 99 and 209, and increased urbanization was also associated with decreased plasma levels of α-tocopherol and carotenoids. These associations suggest that changes in blood profiles of vitamins, carotenoids or other analytes, despite related to increased plasma levels of CB-180, would be consequence of exploitation of artificial food sources rather than of a direct effect of the pollutants., All experimental procedures described in the present study were authorized by the Service of Biodiversity and Animal Protection, Department of Territory and Sustainability, Generalitat de Catalunya (permit references SF/419-426, SF/492-500, SF/492-497, SF/174-181 and SF/091-098 for years 2012, 2013, 2014, 2015 and 2016, respectively). This study was financed by the Spanish Ministry of Science and Innovation through the research supporting funds from the Ramón y Cajal Program (ref. RYC-2015-17504 to MEOS), the University of Barcelona (JR, AHM and HTA), and Red Electrica de España-SA. HTA was supported by a predoctoral grant from the IRBIO-University of Barcelona (APIF 2014).

European populations of black-necked grebes (Podiceps nigricollis) congregate every year to moult at the salt ponds of the Odiel Marshes (SW Spain). However, the Odiel Marshes are part of one of the most metal-polluted coastal estuaries in the world, which may pose risks to wildlife. We assessed the exposure of grebes to metal pollution during the critical moulting period in the Odiel Marshes and its potential to cause adverse health effects. Levels of metals in red blood pellet (as a biomarker of exposure), plasma carotenoids, eye redness, and body condition (as biomarkers of effects) were studied. Metal content was also analyzed in the brine shrimp Artemia parthenogenetica, the most important food for grebes in this hypersaline ecosystem during the moulting period. Results showed that, in comparison to toxicity thresholds, grebes had relatively high blood levels of arsenic (As), mercury (Hg) and zinc (Zn). The high loads found in Artemia and the way blood levels vary during the moulting period indicate that shrimp consumption may be the main route of metal exposure for grebes. Plasma carotenoids and body condition showed a positive association with exposure to As, while the relationship of lutein-like carotenoids with Hg accumulation was negative at the beginning of the moulting period to become positive afterwards. Moreover, eye redness was negatively affected by As accumulation. Factors including food resource availability, seasonal fluctuations in physiological status, and interannual variations in the degree of environmental contamination should be considered in monitoring efforts when using moult migrant waterbirds as sentinel species., J. Rodríguez-Estival was supported by a Research and Innovation Strategy for Smart Specialisation (RIS3) postdoctoral contract (2017/11747) from the Junta de Comunidades de Castilla-La Mancha. The study was financially supported by Junta de Andalucía (Consejería de Innovación, Ciencia y Empresa, Project P07-CVI-02700).

Among reptiles, freshwater turtle species have high potential for metal accumulation because of their long lifespan or their aquatic and terrestrial habits. In order to monitor metal bioaccumulation, determine potential toxic effects, and investigate tools for non-invasive metal sampling in reptiles, we studied lead (Pb) and mercury (Hg) accumulation in Mediterranean pond turtles (Mauremys leprosa) inhabiting two former mining areas, one of them with high environmental concentrations of Pb (Sierra Madrona-Alcudia Valley district) and the other one with high environmental concentrations of Hg (Almadén district). Individuals from the Pb mining area showed mean blood concentrations (i.e. 5.59 μg Pb/g dry weight, d.w.) that were higher than those measured in other populations. Blood Hg concentrations were highest (8.83 μg Hg/g d.w.) in the site close to the former Hg mines, whereas blood Hg concentrations in terrapins from another site of Almadén district, located ∼28 km downstream, were not different from locations at the non-mining area. Animals from the Pb-contaminated site showed evidence of oxidative stress, whereas those from the Hg-contaminated site showed increased activity of the antioxidant enzyme glutathione peroxidase, as well as reduced circulating levels of the main endogenous antioxidant peptide, glutathione. Concentrations measured in feces and carapace scutes were useful indicators to monitor blood concentrations of Pb, but not of Hg. Our results provide evidence of the usefulness of freshwater turtles as sentinels of chronic metal pollution, and validate non-invasive tools to advance Pb monitoring in reptiles., Permits for animal collection and manipulation were provided by the Environment (references DGPFEN/SEN/avp_16_024 and 178/17) and Livestock (reference 678714) departments of the Junta de Comunidades de Castilla-La Mancha. This study was co-funded by the University of Castilla-La Mancha and the Diputación de Ciudad Real through the project reference DIPUCR16 to MEOS, and the Junta de Comunidades de Castilla-La Mancha (PPII-2014-028). MMH was supported by a Juan de la Cierva postdoctoral research contract from the Government of Spain (IJCI-2014-20171) and currently by the Junta de Comunidades de Castilla-La Mancha and the European Regional Development Fund (SBPLY/17/180501/000514).

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 ± 4.05 and 33.73 ± 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 ± 2.13%) and Pb (1.68 ± 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., J. Rodríguez-Estival was supported by a Research and Innovation Strategy for Smart Specialisation (RIS3) postdoctoral contract (2017/11747) from the Junta de Comunidades de Castilla-La Mancha. The study was financially supported by the Junta de Comunidades de Castilla-La Mancha (Project PPII-2014-028-P).

The ingestion of spent lead (Pb) from ammunition is a known cause of mortality in waterfowl, but little is known about sublethal effects produced by Pb poisoning on birds, especially in wild populations. The authors studied potential sublethal effects associated with Pb exposure in mallards (Anas platyrhynchos) from the Ebro delta (northeastern Spain) after a ban on Pb ammunition. They analyzed the relationships between blood Pb levels and oxidative stress, immune response, and carotenoid-based coloration, which are known to be influenced by oxidative stress. Levels of Pb were reduced by half from 6 yr to 9 yr after the ban. Lipid peroxidation was positively related to Pb levels in females. The δ-aminolevulinic acid dehydratase activity was suppressed by Pb exposure and negatively associated with the activity of antioxidant enzymes. Carotenoid levels were positively associated with blood Pb concentration in both sexes, and males with higher Pb levels presented a less intense coloration in legs and beak. Levels of Pb were positively related to hemolytic activity of circulating immune system components and negatively related to lysozyme levels. In summary, Pb exposure was associated in a gender-specific way with increased oxidative stress, consequences on color expression, and impaired constitutive immunity. In females, antioxidants seemed to be allocated mostly in reproduction rather than in self-maintenance, whereas males seemed to better maintain oxidative balance to the detriment of coloration., Funding was provided by the Spanish Ministry of Science and Innovation (grants CGL2007-62797 and CGL2010-17030), and FPI fellowship (BES-2011-045670, to N. Vallverdu-Coll), and the Juan de la Cierva program (to M. Ortiz-Santaliestra).

Despite the efforts of the European Commission to implement measures that offset the detrimental effects of agricultural intensification, farmland bird populations continue to decline. Pesticide use has been pointed out as a major cause of decline, with growing concern about those agro-chemicals that act as endocrine disruptors. We report here on the effects of flutriafol, a ubiquitous systemic fungicide used for cereal seed treatment, on the physiology and reproduction of a declining gamebird. Captive red-legged partridges (Alectoris rufa; n = 11–13 pairs per treatment) were fed wheat treated with 0%, 20% or 100% of the flutriafol application rate during 25 days in late winter. We studied treatment effects on the reproductive performance, carotenoid-based coloration and cellular immune responsiveness of adult partridges, and their relationship with changes in oxidative stress biomarkers and plasma biochemistry. We also studied the effect of parental exposure on egg antioxidant content and on the survival, growth and cellular immune response of offspring. Exposed partridges experienced physiological effects (reduced levels of cholesterol and triglycerides), phenotypical effects (a reduction in the carotenoid-based pigmentation of their eye rings), and most importantly, severe adverse effects on reproduction: a reduced clutch size and fertile egg ratio, and an overall offspring production reduced by more than 50%. No effects on body condition or cellular immune response of either exposed adult or their surviving offspring were observed. These results, together with previous data on field exposure in wild partridges, demonstrate that seed treatment with flutriafol represents a risk for granivorous birds; they also highlight a need to improve the current regulation system used for foreseeing and preventing negative impacts of Plant Protection Products on wildlife. The ingestion of flutriafol treated seeds reduces partridge's reproductive output and represents a threat for granivorous bird populations., This work was supported by CSIC (Intramural 201330E041), FEDENCA (Real Federación Española de Caza) and Oficina Nacional de la Caza with the partnership of Fundación Biodiversidad. Ana Lopez Antia is a postdoctoral researcher of the Research Foundation – Flanders (FWO). This study contributes to the project REGRESEEDS supported by the Spanish Ministry of Economy and Competitiveness (ref. CGL2016-75278-R).

P.J. Van den Brink et al., The United Nations Sustainability Development Goals (SDGs) have been established to end poverty, protect the planet and ensure prosperity for all. Delivery of the SDGs will require a healthy and productive environment. An understanding of the impacts of chemicals, which can negatively impact environmental health, is therefore essential to the delivery of the SDGs. However, current research on and regulation of chemicals in the environment tends to take a simplistic view and does not account for the complexity of the real world, which inhibits the way we manage chemicals. There is therefore an urgent need for a step-change in the way we study and communicate h the impacts and control of chemicals in the natural environment. To do this requires the major research questions to be identified so that resources are focused on questions that really matter. In this paper, we present the findings of a horizon scanning exercise to identify research priorities of the European environmental science community around chemicals in the environment. Using the key questions approach, we identified 22 questions of priority. These questions covered: overarching questions around which chemicals we should be most concerned about and where, impacts of global megatrends, protection goals and sustainability of chemicals; the development and parameterisation of assessment and management frameworks; and mechanisms to maximise the impact of the research. The research questions identified in this paper provide a first-step in the path forward for the research, regulatory and business communities to better assess and manage chemicals in the natural environment., We would like to thank the financial support provided by Baylor University, Tony and Donna Robert, the University of York, and the US Environmental Protection Agency.

Amphibians and reptiles are the two most endangered groups of vertebrates. Environmental pollution by pesticides is recognised as one of the major factors threatening populations of these groups. However, the effects of pesticides on amphibians and reptiles have been studied for few substances, which is partly related to the fact that these animals are not included in the mandatory toxicity testing conducted as part of environmental risk assessments of pesticides. Whether risks of pesticides to amphibians and reptiles are addressed by surrogate taxa used in risk assessment is currently under debate. In order to develop a scientifically sound and robust risk assessment scheme, information needs to be gathered to examine whether fish, birds and mammals are valid surrogates for amphibians and reptiles. We updated a systematic review of scientific literature that was recently published compiling toxicity data on amphibians and reptiles. The outcome of this review was analysed with the purposes to (1) compare endpoints from amphibians and reptiles with the available information from fish, birds and mammals, and (2) develop species sensitivity distributions (SSDs) for those substances tested in at least six amphibian species (no substances were found tested in at least six reptile species) to identify a candidate amphibian model species to be used as surrogate in risk assessment. A positive correlation was found between toxicity recorded on fish and amphibians, the former revealing, in general, to be more sensitive than the latter to waterborne pollutants. In the terrestrial environment, although birds and mammals were more sensitive than amphibians and reptiles to at least 60% of tested substances, just a few weak significant correlations were observed. As a general rule, homoeothermic vertebrates are not good surrogates for reptiles and terrestrial amphibians in pesticide risk assessment. However, some chemical-dependent trends were detected, with pyrethroids and organochlorine insecticides being more toxic to amphibians or reptiles than to birds or mammals. These trends could ultimately help in decisions about protection provided by surrogate taxa for specific groups of substances, and also to determine when risk assessment of pesticides needs to pay special consideration to amphibians and reptiles. The outcome of this review reflects that there is still much information needed to reduce uncertainties and extract relevant conclusions on the overall protection of amphibians and reptiles by surrogate vertebrates., This study was self-funded, but most of the data used to conduct the meta-analysis presented here were generated by a previous study funded by the European Food and Safety Authority through the outscored procurement OC/EFSA/PRAS/2015/01.

Pesticide research traditionally has focused on compounds with high acute toxicity or persistence, but the adverse sublethal effects of pesticides with different properties also may have important consequences on exposed wildlife. The authors studied the effects of thiram, a fungicide used for seed coating with known effects as endocrine disruptor. Red-legged partridges (Alectoris rufa; n = 15 pairs per treatment group) were fed wheat treated with 0%, 20%, or 100% of the thiram application rate used in autumn (25 d) and late winter (10 d) to mimic cereal sowing periods. The authors studied the effects on reproductive performance, carotenoid-based ornamentation and cellular immune responsiveness of adult partridges, and their relationship with changes in oxidative stress biomarkers and plasma biochemistry. The authors also studied the effect of parental exposure on egg antioxidant content and on the survival, growth, and cellular immune response of offspring. Exposure to thiram-coated seeds delayed egg laying, reduced clutch size, and affected egg size and eggshell thickness. Partridges exposed to the 20% thiram dose exhibited reduced egg fertility and brood size (55% and 28% of controls, respectively). Chick survival was unaffected by parental exposure to treated seeds, but adverse effects on their growth rate and cellular immune response were apparent. These effects on reproduction and immune function may have important demographic consequences on farmland bird populations. Environ Toxicol Chem 2015;34:1320–1329. © 2015 SETAC

Temporary ponds, where many amphibians from temperate regions breed, show an annual cycle with a maximum water volume in spring followed by a progressive desiccation throughout late spring and summer. This desiccation leads to a decrease in dissolved oxygen and an increase in nitrogen levels, which can additionally increase because of anthropogenic sources such as chemical fertilizers. We analyzed the toxicity posed by environmentally relevant levels of a common nitrogenous fertilizer, ammonium nitrate, at different conditions of oxygen availability to Bufo calamita tadpoles, which typically develop in ephemeral ponds. Ammonium nitrate (90.3 mg N–NO3NH4/l) and hypoxic conditions (initial dissolved oxygen 4.53 ± 0.40 mg/l) caused significant lethal effects after 7 and 12 days of exposure, respectively. At the end of experiment (16 days), mortality rates were 32.5 % in individuals exposed to the fertilizer and 15 % in those growing under hypoxic conditions. When both stressors were combined, they showed an additive effect on tadpole survival. Malformations, such as oedemas and spinal curvatures, and locomotory abnormalities, were detected after 12 days of experiment in >90 % of individuals exposed to 45.2 mg N–NO3NH4/l under hypoxic conditions, whereas none of these stressors by separate related to abnormality rates >35 %. Delayed development was also observed in tadpoles exposed to ammonium nitrate with hypoxia affecting developmental rate only after 12 days of exposure. The results are discussed in terms of potential mechanisms linking negative effects of both factors as well as in terms of potential alterations of the ecological plasticity that often allows amphibians to survive in unpredictable environments.

Avian embryos are exposed to yolk androgens that are incorporated into the egg by the ovulating female. These steroids can affect several aspects of embryo development, often resulting in increases in overall size or the speed of growth of different traits. However, several studies suggest that they also entail immune costs to the offspring. In this study, we explored whether variation in yolk androgen concentration affected several measures of the constitutive and cell-mediated immune axes in the spotless starling (Sturnus unicolor). Using a within-brood design, we injected different doses of androgens (testosterone and androstenedione) into the eggs. Our study showed that experimentally increased yolk androgens led to sex-specific immunosuppression in both the innate and adaptive axes of the immune system. Both cell-mediated immune response (CMI) and lysozyme activity decreased with increasing androgen levels injected into the egg in the case of male nestlings, whereas there were no effects on females. The effects that we found were always linear: no quadratic or threshold patterns were detected. We found no effects of the experimental treatment in hemolysis or agglutination capacity, but these measures were negatively correlated with CMI, suggesting negative correlation among different branches of the immune system. Blood (trypanosomes and hemosporidians) and intestinal (coccidia) parasites were not affected by the experimental increase of yolk androgen levels. Our results show that in our study species yolk androgens induce immunosuppression in some axes of the male nestling immune system. Further studies should analyze the proximate causes for these contrasting effects in different axes of the immune system and the reason for the differential impact on males and females., This study was financed by project grants CGL2008–03501 and CGL2011–26318 to D.G. (Ministerio de Ciencia e Innovación [MICINN]). J.M. was supported by an FPI (Formación de Personal Investigador) grant (BES-2009–021383) from MICINN. L.P.-R. was supported by a postdoctoral grant from MICINN through the Juan de la Cierva Subprogram (JCI-2008–2059), followed by a postdoctoral contract from the Spanish Ministry of Economy and Competitiveness through the Severo Ochoa Program for Centres of Excellence in Research, Development, and Innovation (SEV-2012–0262). M.E.O.-S. was supported by a postdoctoral grant from MICINN through the Juan de la Cierva Subprogram (JCI-2009–03865), followed by a Marie Curie Intra-European Fellowship of the European Union’s 7th Framework Program (2012–328328).

Pesticide coated seeds are known to be potentially toxic for birds, but the risk of poisoning will depend on how likely the individuals are to consume them. To refine the risk assessment of coated seed consumption by birds we studied the consumption and avoidance of seeds treated with imidacloprid, thiram, maneb or rhodamine B under different scenarios of food unpredictability (diversity or changes in food sources). In a first set of experiments, we examined during four days the amount of ingested food by red-legged partridges (. Alectoris rufa) when offered untreated seeds, treated seeds or both. In the latter case, we also assessed the effect of a daily interchange in the position of feeders containing treated and untreated food. A second experiment, conducted with imidacloprid only, consisted of offering, during 27. h, fixed overall amounts of treated and untreated food, equally distributed in a different number of feeders per pen (1, 2, 4 or 8 feeders of each type of food) in order to diversify food sources. All the tested pesticide-treated seeds were avoided in two-choice experiments, and imidacloprid and thiram were also avoided in one-choice experiments. We found that imidacloprid treated seeds were avoided, probably as a consequence of a conditioned aversion effect due to the post-ingestion distress. However, under a diversification of two-choice food sources with multiple feeders, imidacloprid-treated seeds were ingested by partridges at increasing amounts that can produce sublethal effects or even death. Thiram treated seeds were also initially avoided in one-choice experiment, but probably mediated by a sensory repellence that progressively decreased with time. Our results reveal that the risk of pesticide exposure in birds may increase by unpredictability of food resources or prolonged availability of coated seeds, so pesticide registration for seed coating should consider worst-case scenarios to avoid negative impacts on farmland birds., Study financed by FEDENCA (Real Federación Española de Caza) and Oficina Nacional de Caza with the partnership of Fundación Biodiversidad.

Pollutants and disease are factors implicated in amphibian population declines, and it is hypothesized that these factors exert a synergistic adverse effect, which is mediated by pollutant-induced immunosuppression. Polybrominated diphenyl ethers (PBDEs) are ubiquitous pollutants that can exert immunotoxicity, making them of interest to test effects on amphibian immune function. We orally exposed Lithobates (Rana) pipiens tadpoles to environmentally realistic levels (0-634 ng/g wet diet) of a pentabromodiphenyl ether mixture (DE-71) from as soon as they became free-swimming through metamorphic climax. To assess adaptive immune response in juvenile frogs, we used an enzyme-linked immunosorbent assay to measure specific IgY production following immunization with keyhole limpet hemocyanin (KLH). Specific KLH antibody response was significantly decreased in juvenile frogs that had been exposed to PBDEs as tadpoles. When assessing innate immune responses, we found significantly different neutrophil counts among treatments; however, phagocytic activity of neutrophils was not significantly different. Secretion of antimicrobial skin peptides (AMPs) nonsignificantly decreased with increasing PBDE concentrations, and no significant effect of PBDE treatment was observed on efficacy of AMPs to inhibit chytrid fungus (Batrachochytrium dendrobatidis) growth. Our findings demonstrate that environmentally realistic concentrations of PBDEs are able to alter immune function in frogs; however, further research is needed to determine how these alterations impact disease susceptibility in L. pipiens.

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 ¿g ml¿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 ¿g g¿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., This study was part of NewRaptor (project ID 230465/F20) funded by the Norwegian Research Council and the Norwegian University of Science and Technology (NTNU). We acknowledge the following people for their help in obtaining the samples: Nathalie Briels and Sina Thu Randulff from NTNU and Gjøran Stenberg from Nord University (Norway), Mario León-Ortega, M. Paz Aldeguer, Pedro María Mojica and José Enrique Martínez from the University of Murcia (Spain).

In this paper we determined the levels of 63 environmental contaminants, including organic (PCBs, organochlorine pesticides, and PAHs) and inorganic (As, Cd, Cu, Pb, Hg and Zn) compounds in the blood of loggerhead turtles (Caretta caretta) from two comparable populations that inhabit distinct geographic areas: the Adriatic Sea (Mediterranean basin) and the Canary Islands (Eastern Atlantic Ocean). All animals were sampled at the end of a period of rehabilitation in centers of wildlife recovery, before being released back into the wild, so they can be considered to be in good health condition. The dual purpose of this paper is to provide reliable data on the current levels of contamination of this species in these geographic areas, and secondly to compare the results of both populations, as it has been reported that marine biota inhabiting the Mediterranean basin is exposed to much higher pollution levels than that which inhabit in other areas of the planet. According to our results it is found that current levels of contamination by organic compounds are considerably higher in Adriatic turtles than in the Atlantic ones (∑. PCBs, 28.45 vs. 1.12. ng/ml; ∑. OCPs, 1.63 vs. 0.19. ng/ml; ∑. PAHs, 13.39 vs. 4.91. ng/ml; p

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., This study was funded by the Spanish Ministry of Education and Science (grant CGL2010-17030). N.V.-C. was supported by an FPI scholarship funded by the Spanish Ministry of Economy and Competitiveness (BES-2011-045670).

Due to reductions in winter food resources, newly sown cereal seeds have become a key component of many bird species' diets, but these seeds are often treated with pesticides that may cause toxic effects. To complete an appropriate risk assessment, data on treated seed toxicity need to be combined with information about the risk of exposure of birds in the field and the factors that modulate such exposure. We studied the abundance of pesticide-treated seeds available for birds in the field, the pesticides and their concentrations in treated seeds, and the bird species observed in the field that were feeding on these pesticide-treated seeds. The exposure of red-legged partridge to treated winter cereal seeds was characterized through the analysis of crop and gizzard contents of hunted individuals (n = 189). Moreover, we measured the contribution of cereal seeds in the autumn–winter diet of partridges in order to assess the potential risk of exposure to pesticide-treated seeds. Density of treated seeds on the soil surface after sowing (11·3 ± 1·2 seeds m in the centre of field and 43·4 ± 5·5 seeds m in the headlands) was enough to provide, in an area between 6 and 50 m, doses of six active ingredients above those indicating acute (i.e. a dose capable of killing 50% of individuals of a sensitive species) and / or chronic (no observed effect level) toxicity. Up to 30 bird species were observed consuming treated cereal seeds in recently sown fields. Corn bunting was identified as an appropriate focal passerine species for the risk assessment of pesticide-treated seeds. We found that treated seeds were an important route of pesticide ingestion for red-legged partridge; pesticide residues (six fungicides and two insecticides) were found in 32·3% of crops and gizzards. Cereal seeds represented more than half (53·4 ± 4·3%) of total biomass consumed by partridges from October to February. Synthesis and applications. The field exposure data combined with previous studies about the toxicity to partridges of using pesticide-treated seeds point to an unacceptable risk of this practice to farmland birds. Our results suggest that the prophylactic use of pesticide-coated seeds should be avoided, with the approval of this treatment considered on a case-by-case basis and accompanied with specific measures to minimize risks of adverse effects on avian communities., This study was financed by the Spanish Ministry of Agriculture and Environment (Project OAPN 755/2012), CSIC (Intramural 201330E041),FEDENCA (Real Federación Española de Caza) and Oficina Nacional de la Caza with the partnership of Fundación Biodiversidad.

Animal body condition refers to the health and physiological state of individuals, and multiple parameters have been proposed to quantify this key concept. Food intake is one of the main determinants of individual body condition and much debate has been generated on how diet relates to body condition. We investigated this relationship in free-living Bonelli’s eagle (Aquila fasciata) nestlings sampled at two geographically distant populations in Spain. Nestlings’ main prey consumption was estimated by isotopic analyses. A multi-biomarker approach, including morphometric and blood biochemical measures (i.e. hematocrit, plasma biochemistry and oxidative stress biomarkers), enabled us to integrate all the body condition measures taken. A greater consumption of a preferred prey [i.e. the European rabbit (Oryctolagus cuniculus)] improved nestling body condition, as indicated by lower levels of cholesterol in plasma, greater activity of enzymes mediating in protein catabolism, higher levels of tocopherol and glutathione, and less glutathione peroxidase activity, which also suggested lower degree of oxidative stress. On the other hand, increased diet diversity was positively correlated with higher levels of oxidized glutathione, which suggests that these nestlings had poorer body condition than those with a higher frequency of preferred prey consumption. Several factors other than diet [i.e. altitude of nesting areas, nestling sex and age, sampling time (before or after midday) and recent food ingestion] had an effect on certain body condition measures. Our study reveals a measurable effect of diet on a predator’s body condition and demonstrates the importance of considering the potential influence of multiple intrinsic and extrinsic factors when assessing animal body condition., This study was funded by projects CGL2007-64805 and CGL2010-17056 from the “Ministerio de Ciencia e Innovación, Gobierno de España”, the “Àrea d’Espais Naturals de la Diputació de Barcelona” and Miquel Torres S.A. J. Resano-Mayor was supported by a predoctoral grant from the “Departamento de Educación, Gobierno de Navarra; Plan de Formación y de I+D 2008–2009”.

Chytridiomycosis, the emerging disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) is responsible for declines and extirpations of amphibian populations worldwide. Environmental covariates modify the host-Bd interaction and thus affect the ongoing spread of the pathogen. One such covariate may be the intensity of ultraviolet B (UV-B) radiation. In a field experiment conducted in Laguna Grande de Penalara (central Spain), a mountainous region where the presence of Bd has been documented since 1997, we analyzed the potential effect of environmental UV-B (daily maximum 2.5-3.9 W/m(2) ) on the susceptibility of larvae of the common toad (Bufo bufo) to Bd. The proportion of infected individuals increased as tadpoles developed. The prevalence of Bd was significantly lower in tadpoles exposed to environmental UV-B intensities (2.94%) than in tadpoles not exposed to the radiation (9.72%). This finding mirrors that seen for a second amphibian species, the European midwife toad (Alytes obstetricans), for which conditional prevalence (i.e., prevalence of infection conditioned on the probability of a site being infected) across the Iberian Peninsula was inversely correlated with the intensity of UV-B.

Landscape connectivity is a very recurrent theme in landscape ecology as it is considered pivotal for the long term conservation of any organism’s populations. Nevertheless, this complex concept is still surrounded by uncertainty and confusion, largely due to the separation between structural and functional connectivity. Amphibians are the most threatened vertebrates around the globe, in Europe mostly due to habitat alteration, and to their particular life cycle. Pond breeding amphibians are considered to be organised in metapopulations, enhancing the importance of landscape connectivity in this group of animals. We sampled the amphibian species present in two pond groups in Central Western Spain. We applied the graph theory framework to these two pond networks in order to determine the importance of each pond for the entire network connectivity. We related the pond importance for connectivity with the species richness present in each pond. We tested if connectivity (partially) determined the presence of the amphibian species sampled using logistic regression. The results show that the structural connectivity of the pond network impacts on the amphibian species richness pattern and that the importance of the pond for the connectivity of the network is an important factor for the presence of some species. Our results, hence, attest the importance of (structural) landscape connectivity determining the pattern of amphibian (functional) colonization in discrete ponds.

A wide variety of agricultural chemicals with potential to affect amphibian health are released into the environment daily. Most of these chemicals are xenobiotic compounds that are highly toxic to embryos, tadpoles, and terrestrial stages. Other substances that occur in pristine environments at harmless concentrations, such as inorganic nitrogenous compounds, may reach potentially toxic levels as a consequence of certain human activities, including the application of fertilizers. Most of the studies that analyze the effects of inorganic nitrogen on amphibian embryos are conducted with anurans, whereas little information exists regarding urodeles. Embryos of newts and salamanders usually exhibit longer times to hatch than frogs and toads. A longer hatching time results in a longer exposure of embryos to diffuse environmental pollution and therefore a higher risk of suffering toxic effects during the embryonic stage. We demonstrate that ammonium nitrate, a widely used nitrogenous fertilizer, at concentrations used in areas of high agricultural intensity, affects embryonic development of the Iberian newt (Lissotriton boscai). Although ammonium nitrate did not have significant lethal effects, it reduced the growth rate of exposed embryos, which were 9.6% smaller than controls. Hatching synchrony remained similar across treatments, and hatching date was not affected by ammonium nitrate, indicating that the effect on growth was not time- dependent. Researchers have demonstrated fitness costs in smaller than average tadpoles, suggesting that ammonium nitrate exposure could have long-term negative consequences for the Iberian newt.

Many amphibians are known to suffer embryonic die-offs as a consequence of Saprolegnia infections; however, little is known about the action mechanisms of Saprolegnia and the host-pathogen relationships. In this study, we have isolated and characterized the species of Saprolegnia responsible for infections of embryos of natterjack toad (Bufo calamita) and Western spadefoot toad (Pelobates cultripes) in mountainous areas of Central Spain. We also assessed the influence of the developmental stage within the embryonic period on the susceptibility to the Saprolegnia species identified. Only one strain of Saprolegnia was isolated from B. calamita and identified as S. diclina. For P. cultripes, both S. diclina and S. ferax were identified. Healthy embryos of both amphibian species suffered increased mortality rates when exposed to the Saprolegnia strains isolated from individuals of the same population. Embryonic developmental stage was crucial in determining the sensitivity of embryos to Saprolegnia infection. The mortalities of P. cultripes and B. calamita embryos exposed at Gosner stages 15 (rotation) and 19 (heart beating) were almost total 72 h after challenge with Saprolegnia, while those exposed at stage 12 (late gastrula) showed no significant effects at that time. This is the first study to demonstrate the role of embryonic development on the sensitivity of amphibians to Saprolegnia., Funding was provided by the Spanish Ministry of Education and Science (refs. CGL2005-03727 and Flora Micologica Iberica VI, CGL2006-12732-C02-01), and by the Diputacion de Avila (Inst. Gran Duque de Alba).

This study examined the effects of arsenic exposure and the biotransformation capacity of developing Iberian green frogs (Rana perezi) with the main aim of finding valid biomarkers to monitor pollution and toxicological problems. Total arsenic concentration and arsenic species distribution were determined using liquid chromatography coupled with inductively coupled plasma-mass spectrometry (LC-ICP-MS). The level of accumulation of arsenic and its species distribution differed depending on the metamorphosis stage and level of exposure. Tetramethylarsonium cation was the major compound detected and continuously increased in concentration during frog development. Results suggest that rather low As accumulation is obtained and that Rana perezi metabolised arsenic during embryonic and larval development.

Fipronil is an insecticide commonly used in agriculture, but there are growing concerns over its environmental impacts (e.g., harmful effects on pollinators). Fipronil-treated seed ingestion might threaten granivorous farmland birds, in particular, Gallinaceous birds that are particularly sensitive to this insecticide. We report here on exposure risk and effects in a game bird of high socioeconomic importance, the red-legged partridge (Alectoris rufa). We fed captive birds with untreated maize (controls) or with a mixture of untreated–treated maize (ratio 80:20; exposed birds) during 10 days at the beginning of the breeding period (n = 12 pairs in each group). We first show that exposed partridges did not reject treated seeds but reduced food intake and lost body condition. We further studied the effects of treated seed ingestion on adult survival, oxidative balance, plasma biochemistry, carotenoid-based coloration, cellular immune response, steroid hormone levels, and reproduction. Fipronil exposure altered blood biochemistry and sexual hormone levels and reduced cellular immune response, antioxidant levels, and carotenoid-based coloration. Exposed pairs also had reduced egg fecundation rate and produced eggs with fewer antioxidants and offspring that had reduced cellular immune response. These negative effects on adult partridges, their reproductive performance, and offspring quality highlight that fipronil-treated seed ingestion is a significant threat to wild birds., The present study was financed by FEDENCA (Real Federación Española de Caza) and Oficina Nacional de Caza with the partnership of Fundacion Biodiversidad.

The ingestion of imidacloprid treated seeds by farmland birds may result in exposure to toxic amounts of this insecticide. Here we report on the effects that the exposure to the recommended application rate and to 20% of that rate may produce on birds feeding on treated seeds. Experimental exposure to imidacloprid treated seeds was performed on red-legged partridges (Alectoris rufa) (n=15 pairs per treatment group: control, 20% or 100% of the recommended application rate) during two periods that corresponded to the autumn (duration of exposure: 25 days) and late winter (10 days) cereal sowing times in Spanish farmlands. We studied effects on the survival, body condition, oxidative stress biomarkers, plasma biochemistry, carotenoid-based coloration, T-cell mediated immune response and reproduction of exposed adult partridges, and on the survival and T-cell immune response of their chicks. The high dose (recommended application rate) killed all partridges, with mortality occurring faster in females than in males. The low dose (20% the recommended application rate) had no effect on mortality, but reduced levels of plasma biochemistry parameters (glucose, magnesium and lactate dehydrogenase), increased blood superoxide dismutase activity, produced changes in carotenoid-based integument coloration, reduced the clutch size, delayed the first egg lay date, increased egg yolk vitamins and carotenoids and depressed T-cell immune response of chicks. Moreover, the analysis of the livers of dead partridges revealed an accumulation of imidacloprid during exposure time. Despite the moratorium on the use of neonicotinoids in the European Union, birds may still be at high risk of poisoning by these pesticides through direct sources of exposure to coated seeds in autumn and winter., This study financed by FEDENCA (Real Federación Española de Caza) and Oficina Nacional de Caza (Project number: UCTR100161) with the partnership of Fundación Biodiversidad.

Lead (Pb) poisoning has caused significant mortality in waterfowl populations worldwide. In spite of having been banned since 2003, prevalence of Pb shot ingestion in mallards (Anas platyrhynchos) from the Ebro delta was still 15.5% in 2011-12. We collected mallard eggs from this area to study the effects of maternally transferred Pb on eggshell properties and on immune response and oxidative balance of ducklings. Eggshell Pb levels were positively correlated with Pb levels in the blood of ducklings. Ducklings with blood Pb levels above 180 ng mL-1 showed reduced body mass and died during the first week post hatching. Blood Pb levels positively correlated with humoral immune response, endogenous antioxidants and oxidative stress biomarkers, and negatively correlated with cellular immune response. Pb shot ingestion in birds can result in maternal transfer to the offspring that can affect their developing immune system and reduce their survival in early life stages., The founding of this study was provided by the Spanish Ministry of Science and Innovation (grants CGL2007-62797 and CGL2010-17030). NVC benefits from an FPI fellowship (BES-2011-045670), MEOS was contracted under Juan de la Cierva program and MMH benefits from a Marie Curie Fellowship (PIEF-GA-2011-299747) within the 7th Framework Programme (FP7 2007–2013) of the European Commission.

The exposure to persistent pollutants such as organochlorine compounds (OCs) or metals has been associated with declines in top predator populations, which can accumulate high amounts of these pollutants from their prey. However, understanding how variation in OC and metal accumulation in wild species affects their biochemical and physiological responses is a big challenge, especially for endangered predators like the Bonelli's eagle (Aquila fasciata). This bird of prey is an interesting study model because the differences in diet composition among populations and territories can account for important pollutant uptake variations. We compared OC and metal accumulation in blood of Bonelli's eagle nestlings from three populations across Spain as a function of origin, age class (nestlings vs. adults), sex and number of siblings per nest, and related accumulation patterns to responses indicative of body condition, biochemistry and antioxidant status. Nestlings from Catalonia, the most industrialized area, showed the highest concentrations of PCBs and arsenic, and the lowest concentrations of zinc. The two former substances, together with DDTs, exerted an overall influence on nestling's physiology. PCBs and arsenic were associated with reduced retinol levels, pointing to oxidative damage in exposed individuals, which was also consistent with the low zinc levels in individuals from the polluted region. Increased plasma DDT levels were related to reduced body condition and lower levels of triglycerides. Mercury accumulation in Castile and Leon was higher in nestlings that were alone in the nest than in nestlings that shared it with a sibling; this suggests an increased mercury uptake from secondary prey in territories where preferred prey (i.e. rabbits) are scarce, which are also the territories where productivity is reduced. Overall, the results reveal a spatial variation in pollutant accumulation patterns and associated physiological effects, and suggest the major role that territory quality may have in such patterns., Study financed by the Spanish Ministry of Economy and Competitiveness (project ref. CGL2010-17056 to JR and Juan de la Cierva contract to MEOS) and Navarra Government (PhD fellowship of the I + D Plan 2008–2009 to JRM).

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., This study was funded by the Spanish Ministry of Education and Science (grant CGL2010-17030). Nuria Vallverdu-Coll was supported by an FPI scholarship funded by the Spanish Ministry of Economy and Competitiveness (BES-2011-045670).

We exposed larval southern leopard frogs (Rana sphenocephala) to lead-contaminated sediments to determine the lethal and sublethal effects of this metal. Tadpoles were laboratory-raised from early free-swimming stage through metamorphosis at lead concentrations of 45, 75, 180, 540, 2360, 3940, 5520, and 7580 mg/kg dry weight in sediment. Corresponding pore water lead concentrations were 123, 227, 589, 1833, 8121, 13,579, 19,038, and 24,427 microg/L. Tadpoles exposed to lead concentrations in sediment of 3940 mg/kg or higher died within 2 to 5 days of exposure. At lower concentrations, mortality through metamorphosis ranged from 3.5% at 45 mg/kg lead to 37% at 2360 mg/kg lead in sediment. The LC50 value for lead in sediment was 3728 mg/kg (95% CI = 1315 to 72,847 mg/kg), which corresponded to 12,539 microg/L lead in pore water (95% CI = 4000 to 35,200 microg/L). Early growth and development were depressed at 2,360 mg/kg lead in sediment (8100 microg/L in pore water) but differences were not evident by the time of metamorphosis. The most obvious effect of lead was its pronounced influence on skeletal development. Whereas tadpoles at 45 mg/kg lead in sediment did not display permanent abnormalities, skeletal malformations increased in frequency and severity at all higher lead concentrations. By 2360 mg/kg, 100% of surviving metamorphs displayed severe spinal problems, reduced femur and humerus lengths, deformed digits, and other bone malformations. Lead concentrations in tissues correlated positively with sediment and pore water concentrations.

The use of lead (Pb) ammunition in the form of shot pellets has been identified as a Pb exposure risk in wildlife and their human consumers. We explore the hypothesis that Pb shot ban enforcement reduces the risk of avian Pb poisoning as well as Pb exposure in game meat consumers. We assessed compliance with a partial ban on Pb shot commencing in 2003 by examination of 937 waterbirds harvested by hunters between 2007 and 2012 in the Ebro delta (Spain). Prevalence of Pb shot ingestion was determined, as were Pb concentrations in liver and muscle tissue to evaluate the potential for Pb exposure in game meat consumers. Hunted birds with only embedded Pb shot (no steel) declined from 26.9% in 2007-08 to, This study was supported by the Spanish Ministry of Science and Innovation (grants CGL2007-62797 and CGL2010-17030). NVC benefits from an FPI Fellowship (BES-2011-045670), MAT and MEOS were contracted under the Juan de la Cierva program and MMH benefits from a Marie Curie Fellowship (PIEF-GA-2011-299747).

Pesticide coated seeds are commonly used in agriculture, and may be an important source of food for some birds in times of scarcity, as well as a route of pesticide ingestion. We tested the lethal and sub-lethal effects of treated seed ingestion by the red-legged partridge (Alectoris rufa), a game bird of high socio-economic value in Spain. One year-old partridges (n = 42 pairs) were fed for 10 days in spring (prior to breeding) with wheat treated with difenoconazole (fungicide), thiram (fungicide) or imidacloprid (insecticide), using two doses for each pesticide (the one recommended, and its double to represent potential cases of abuse of pesticides). We investigated the direct and indirect effects on the body condition, physiology, immunology, coloration and subsequent reproduction of exposed partridges. For the latter, eggs were collected, measured and incubated and the growth and survival of chicks were monitored. Thiram and imidacloprid at high exposure doses produced mortalities of 41.6 and 58.3 %, respectively. The first death was observed at day 3 for imidacloprid and at day 7 for thiram. Both doses of the three pesticides caused sublethal effects, such as altered biochemical parameters, oxidative stress and reduced carotenoid-based coloration. The high exposure doses of imidacloprid and thiram also produced a decrease in cellular immune response measured by the phytohemagglutinin test in males. Bearing in mind the limitation of the small number of surviving pairs in some treatments, we found that the three pesticides reduced the size of eggs and imidacloprid and difenoconazole also reduced the fertilization rate. In addition, both thiram and imidacloprid reduced chick survival. These experiments highlight that the toxicity of pesticide-treated seeds is a factor to consider in the decline of birds in agricultural environments., Study financed by FEDENCA (Real Federación Española de Caza) and Oficina Nacional de la Caza with the partnership of Fundación Biodiversidad.

Ortiz-Santaliestra, Manuel E. et al., [EN]: atrachochytrium dendrobatidis(Bd), the fungus responsible for chytridiomycosis, grows on the keratinized parts of the body (i.e., the entire skin of juveniles and adults, and the oral disk of tadpoles). The fungus affects epidermal structures, generating an imbalance of the osmotic equilibrium through the skin that ultimately leads to death. Therefore, larvae are likely to be much more resistant compared with juveniles and adults, which may suffer high mortality. However, nothing is known about how susceptibility varies during the juvenile life stages (from the end of metamorphosis to sexual maturity). The coexistence of tolerant hosts (either tolerant species or unaffected developmental stages) with susceptible hosts is a major reason why chytridiomycosis has become an epidemic disease. The aim of this study was to analyze the interspecific and developmental variation in susceptibility to Bd in juveniles of three North American anurans. Leopard Frog (Lithobates pipiens) incurred no lethal effects at 104 Bd zoospores ml−1, regardless of the age at which they were exposed to the fungus. On the contrary, Bd infection was more severe for newly metamorphosed juveniles of American toads (Anaxyrus americanus) than for 4-week-old juveniles. When exposed to 104 Bd zoospores ml−1, newly metamorphosed juveniles of this species experienced mortality rates above 70%, whereas 4-week-old juveniles had mortality rates below 30%. Variations in structural characteristics of the skin or the antifungal efficiency of skin peptides are proposed as potential reasons to explain the observed developmental differences in susceptibility to Bd., [ES]: Batrachochytrium dendrobatidis (Bd), el hongo responsable de la quitridiomicosis, crece sobre las partes queratinizadas del cuerpo (i.e. la piel de juveniles y adultos y el disco oral de las larvas). El hongo afecta a las estructuras epidérmicas generando una alteración del equilibrio osmótico en la piel que termina produciendo la muerte. De este modo, las larvas suelen ser mucho más resistentes que los juveniles o los adultos, los cuales pueden sufrir elevadas mortalidades. Sin embargo, se desconoce cómo varía la susceptibilidad a lo largo de la etapa juvenil (desde el final de la metamorfosis hasta la adquisición de la madurez sexual). La coexistencia de hospedadores tolerantes (ya sean especies tolerantes o estadios de desarrollo que no se vean afectados) con hospedadores susceptibles es una de las principales razones por las que la quitridiomicosis se ha convertido en una epidemia. El objeto de este estudio fue analizar las variaciones interespecíficas y a lo largo del desarrollo en la susceptibilidad de tres especies de anuros de Norteamérica a Bd. Los juveniles de rana leopardo (Lithobates pipiens) no sufrieron efectos letales a 104 zoosporas de Bd mL−1, independientemente de la edad a la que fueron expuestos al hongo. Por el contrario, la infección por Bd resultó más severa para individuos recién metamorfoseados de sapo americano (Anaxyrus americanus) que para juveniles con cuatro semanas de vida. Al exponerlos a 104 zoosporas de Bd mL−1, los juveniles recién metamorfoseados experimentaron tasas de mortalidad por encima del 70%, mientras que los juveniles con cuatro semanas de edad sufrieron tasas de mortalidad inferiores al 30%. Las variaciones en las características estructurales de la piel y/o en la eficacia antifúngica de los péptidos cutáneos se proponen como razones potenciales para explicar las diferencias en la susceptibilidad a Bd observadas a lo largo del desarrollo., This work was funded by the University of Wisconsin Sea Grant Institute under grants from the National Sea Grant College Program, National Oceanic and Atmospheric Administration, U.S. Department of Commerce, and from the State of Wisconsin. Federal grant number NA16RG2257, project number R/EH-2.

Lead (Pb) is a highly toxic metal that can induce oxidative stress and affect the immune system by modifying the expression of immunomodulator-related genes. The aim of the present study was to investigate the association between Pb exposure and the transcriptional profiles of some cytokines, as well as the relationship between Pb exposure and changes in oxidative stress biomarkers observed in the spleen of wild ungulates exposed to mining pollution. Red deer and wild boar from the mining area studied had higher spleen, liver, and bone Pb levels than controls, indicating a chronic exposure to Pb pollution. Such exposure caused a depletion of spleen glutathione levels in both species and disrupted the activity of antioxidant enzymes, suggesting the generation of oxidative stress conditions. Deer from the mining area also showed an induced T-helper (Th)–dependent immune response toward the Th2 pathway, whereas boar from the mining area showed a cytokine profile suggesting an inclination of the immune response toward the Th1 pathway. These results indicate that environmental exposure to Pb may alter immune responses in wild ungulates exposed to mining pollution. However, evidence of direct relationships between Pb-mediated oxidative stress and the changes detected in immune responses were not found. Further research is needed to evaluate the immunotoxic potential of Pb pollution, also considering the prevalence of chronic infectious diseases in wildlife in environments affected by mining activities., J. Rodríguez-Estival had a Junta de Comunidades de Castilla-La Mancha predoctoral grant. The Department of Education and Science of the Junta de Comunidades de Castilla-La Mancha funded this study (projects PCC-05-004-2 and PCI08-0096-1295).

Temporary ponds where many amphibians breed experience a gradual desiccation that leads to growing larval densities, which can reduce tadpole survival rates and increase cannibalistic interactions among carnivorous salamander larvae. Concentrations of many agrochemicals, including nitrogenous fertilizers, can also increase as the water volume decreases. We analyzed the effects of ammonium nitrate fertilizer at two larval densities on growth and survival of Rana dalmatina tadpoles, and on cannibalistic behaviors of Salamandra salamandra larvae. We observed synergistic lethal effects until day 17 of exposure, when ammonium nitrate-related mortality of tadpoles was five times greater at high than at low density. The fertilizer impaired tadpole growth. This effect was stronger at low density than at high density. In the absence of ammonium nitrate, the incidence of cannibalism was higher at high than at low larval density. However, fertilizer exposure removed this density-dependent effect, which, under field conditions, would negate some potentially advantageous effects of cannibalism such as reduced intraspecific competition and the stimulation of defensive behaviors., MEOS is supported by the Juan de la Cierva Program of the Spanish Ministerio de Ciencia e Innovación.

Polybrominated diphenyl ethers (PBDEs) are bioaccumulative, persistent organic pollutants used as flame retardants in consumer goods. Concentrations of PBDEs in North American wildlife have been increasing for decades and been shown to have estrogenic effects on sexual development. No studies, however, have examined the effects of PBDEs on the sexual development of North American frogs at ecologically relevant concentrations. This study examined the effects of five dietary concentrations of DE-71 (0, 1.1, 6.1, 71.4, and 634 ng ΣPBDEs/g diet), a technical PBDE mixture, on the gonadal development of the northern leopard frog, Rana pipiens. Tadpoles were exposed chronically from the time they became free-swimming until metamorphosis. Frogs were killed either at metamorphic climax or 10 weeks after completing metamorphosis, processed for histology, and examined for alterations in sexual development. The experimental group exposed to PBDEs at 1.1 ng/g had a significantly larger proportion of females compared with the expected 50:50 sex ratio. At 10 weeks post-metamorphosis, male frogs exposed to 6.1 and 71.4 ng/g had significantly smaller testes, but all other measure of gonadal development tested showed no effects. No intersex or increased incidence of gonadal abnormality were detected. These findings indicate that PBDEs may disrupt sexual differentiation in frogs at low, environmentally relevant concentrations., Funding was received from the University of Wisconsin Sea Grant Institute (grant NA16RG2257, project R/EH-2) and the Holstrom Environmental Scholarship through the University of Wisconsin-Madison (awarded to N. Schmidt).

Accumulation of organochlorine compounds is well studied in aquatic food chains whereas little information is available from terrestrial food chains. This study presents data of organochlorine levels in tissue and plasma samples of 15 critically endangered Iberian lynx (Lynx pardinus) and other 55 wild carnivores belonging to five species from three natural areas of Spain (Doñana National Park, Sierra Morena and Lozoya River) and explores their relationship with species diet. The Iberian lynx, with a diet based on the consumption of rabbit, had lower PCB levels (geometric means, plasma, This is a contribution to the “Programa de Actuaciones para la Conservación del Lince en Andalucía II”, Consejería de Medio Ambiente, Junta de Andalucía (CMA). J. Millán holds a “Ramón y Cajal” contract and M.E. Ortiz-Santaliestra holds a “Juan de la Cierva” contract, both awarded by the Ministerio de Ciencia e Innovación and the European Social Fund. J. Rodríguez-Estival is granted by the Junta the Comunidades de Castilla-La Mancha.

Chytridiomycosis, the emerging disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) is responsible for declines and extirpations of amphibian populations worldwide. Environmental covariates modify the host-Bd interaction and thus affect the ongoing spread of the pathogen. One such covariate may be the intensity of ultraviolet B (UV-B) radiation. In a field experiment conducted in Laguna Grande de Peñalara (central Spain), a mountainous region where the presence of Bd has been documented since 1997, we analyzed the potential effect of environmental UV-B (daily maximum 2.5-3.9 W/m(2) ) on the susceptibility of larvae of the common toad (Bufo bufo) to Bd. The proportion of infected individuals increased as tadpoles developed. The prevalence of Bd was significantly lower in tadpoles exposed to environmental UV-B intensities (2.94%) than in tadpoles not exposed to the radiation (9.72%). This finding mirrors that seen for a second amphibian species, the European midwife toad (Alytes obstetricans), for which conditional prevalence (i.e., prevalence of infection conditioned on the probability of a site being infected) across the Iberian Peninsula was inversely correlated with the intensity of UV-B.