Linking MS1 and MS2 signals in positive and negative modes of LC-HRMS in untargeted metabolomics using the ROIMCR approach.

Data-independent acquisition (DIA) mode in liquid chromatography (LC) high-resolution mass spectrometry (HRMS) has emerged as a powerful strategy in untargeted metabolomics for detecting a broad range of metabolites. However, the use of this approach also represents a challenge in the analysis of the large datasets generated. The regions of interest (ROI) multivariate curve resolution (MCR) approach can help in the identification and characterization of unknown metabolites in their mixtures by linking their MS1 and MS2 DIA spectral signals. In this study, it is proposed for the first time the analysis of MS1 and MS2 DIA signals in positive and negative electrospray ionization modes simultaneously to increase the coverage of possible metabolites present in biological systems. In this work, this approach has been tested for the detection and identification of the amino acids present in a standard mixture solution and in fish embryo samples. The ROIMCR analysis allowed for the identification of all amino acids present in the analyzed mixtures in both positive and negative modes. The methodology allowed for the direct linking and correspondence between the MS signals in their different acquisition modes. Overall, this approach confirmed the advantages and possibilities of performing the proposed ROIMCR simultaneous analysis of mass spectrometry signals in their differing acquisition modes in untargeted metabolomics studies.

Perfluoroalkyl Acids (PFAAs) Concentrations and Oxidative Status in Two Generations of Great Tits Inhabiting a Contamination Hotspot.

The ubiquity of perfluoroalkyl acids (PFAAs) contrasts with the limited information about their effects. We report here PFAA plasma concentrations in wild populations of great tits ( Parus major) settled at and in the vicinity of a fluorochemical plant in Antwerp (Belgium). Using two generations we obtained novel results on some poorly known issues such as differences between sexes, maternal transfer of the compounds and potential associations with the oxidative status. For five out of the 11 detected PFAAs, the concentrations were the highest ever reported in birds' plasma, which confirms that Antwerp is one of the main hotspots for PFAAs pollution. Contrary to other studies conducted in birds, we found that females presented higher mean concentrations and detection frequencies for two compounds (perfluorooctanesulfonic acid (PFOS) and perfluoroundecanoic acid (PFUnDA)) than males. Maternal transfer and the dietary intake appear to be the main route of exposure for nestlings to PFOS but not to other compounds. Finally, PFAA concentrations tended to correlate positively with protein damage in adult birds while in nestlings they positively correlated with higher activity of antioxidant enzymes (glutathione peroxidase and catalase). Experimental work is needed to confirm oxidative stress as a pathway for the pernicious effects of PFAAs.

High levels of PFOS in eggs of three bird species in the neighbourhood of a fluoro-chemical plant.

We studied perfluorooctane sulfonate (PFOS) levels in the eggs of three primarily invertivorous bird species sampled in 2006 near a fluoro-chemical plant: the great tit (Parus major), the northern lapwing (Vanellus vanellus) and the Mediterranean gull (Larus melanocephalus). Our study reported some of the highest PFOS levels ever measured in wildlife to date (i.e. up to 46182ng/g ww in lapwing eggs). A pronounced decrease in PFOS concentration in the Northern lapwing eggs with distance from the fluoro-chemical plant was found. A similar relationship was found for the great tit, with eggs being collected close to the fluoro-chemical plant having significantly higher PFOS levels than eggs collected 1700m further away. When comparing the PFOS levels in eggs for the three species, collected between 1700 and 5500m no significant differences were observed. In addition, when comparing PFOS levels in eggs between Northern lapwing and great tits closer to the plant (900-1700m) no significant differences were found neither. Despite the high levels found in great tit eggs, plasmatic biochemical biomarker responses did not appear to be affected.

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.

Assessing the Risk of Fipronil-Treated Seed Ingestion and Associated Adverse Effects in the Red-Legged Partridge.

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.

Imidacloprid-treated seed ingestion has lethal effect on adult partridges and reduces both breeding investment and offspring immunity.

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.

Experimental exposure of red-legged partridges (Alectoris rufa) to seeds coated with imidacloprid, thiram and difenoconazole.

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.

Transcriptomic effects of Perfluoralkyl acids on the adipose tissue of a songbird species at environmentally relevant concentrations.

Perfluoralkyl acids (PFAS) have been regarded as global pollutants for at least twenty years, with potentially negative physiological effects on multiple vertebrate species including humans. Here we analyze the effects of the administration of environmentally-relevant levels of PFAS on caged canaries (Serinus canaria) by using a combination of physiological, immunological, and transcriptomic analyses. This constitutes a completely new approach to understand the toxicity pathway of PFAS in birds. While we observed no effects on physiological and immunological parameters (e.g, body weight, fat index, cell-mediated immunity), the transcriptome of the pectoral fatty tissue showed changes compatible with the known effects of PFAS as obesogens in other vertebrates, particularly in mammals. First, transcripts related to the immunological response were affected (mainly enriched), including several key signaling pathways. Second, we found a repression of genes related to the peroxisome response and fatty acid metabolism. We interpret these results as indicative of the potential hazard of environmental concentrations of PFAS on the fat metabolism and the immunological system of birds, while exemplifying the ability of transcriptomic analyses of detecting early physiological responses to toxicants. As the potentially affected functions are essential for the survival of the animals during, for example, migration, our results underline the need for tight control of the exposure of natural populations of birds to these substances.

Understanding PFAAs exposure in a generalist seabird species breeding in the vicinity of a fluorochemical plant: Influence of maternal transfer and diet.

Perfluoroalkyl acids (PFAAs) are a focus of scientific and regulatory attention nowadays. However, PFAAs dynamics in the environment and the factors that determine wildlife exposure are still not well understood. In this study we examined PFAAs exposure in chicks of a generalist seabird species, the lesser black-backed gull (Larus fuscus), breeding 49 km away of a PFAAs hotspot (a fluorochemical plant in Antwerp, Belgium). In order to study the pathways of PFAAs exposure, we measured how chicks' PFAAs burden varied with age, sex, and body condition. In addition, we related PFAA concentrations to chicks' diet using stable isotope signatures. For this purpose, we studied plasma PFAA concentrations in 1-week and 4-week-old gull chicks. Only 4 (PFOS, PFOA, PFDA and PFNA) out of the 13 target PFAA compounds were detected. Measured concentrations of PFOS and PFOA were generally high compared to other seabird species but were highly variable between individuals. Furthermore, our results suggest that maternal transfer plays a significant role in determining chicks' PFAAs burden, and that there are variable sources of exposure for PFOS and PFOA during post-hatching development. The association between PFOS and specific stable isotopes (i.e. δ15N and δ13C) suggests a higher exposure to PFOS in birds with a predominantly marine diet. We also found that males' condition was positively associated with PFOS plasmatic concentration, probably due to the indirect effect of being fed a high quality (marine) diet which appears PFOS rich. Yet, exact exposure source(s) for PFOA remain(s) unclear. Given that PFOS concentrations measured in some chicks surpassed the toxicity reference value calculated for top avian predators, continued monitoring of exposure and health of this gull population, and other wildlife populations inhabiting the area, is highly recommended.

Birds feeding on tebuconazole treated seeds have reduced breeding output.

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.

Limited reproductive impairment in a passerine bird species exposed along a perfluoroalkyl acid (PFAA) pollution gradient.

Although bird eggs have been used in biomonitoring studies on perfluoroalkyl acids (PFAAs), effects of environmental concentrations on reproduction remain largely unknown in wild birds. In the present study we examined the associations between the concentrations of 4 perfluoroalkyl sulfonic acids (PFSAs) and 11 perfluoroalkyl carboxylic acids (PFCAs) in the eggs of great tits (Parus major), collected along a distance gradient from a pollution source, and multiple reproductive parameters (including the start of egg laying, clutch size, hatching success, fledging success and total breeding success) along with egg shell thickness and body condition of the nestlings. The PFAA concentrations measured at the plant site were among the highest ever reported in wild bird eggs. PFAA concentrations decreased sharply with increasing distance (0-11 km) from the plant, but remained relatively elevated in the adjacent sites. PFAAs were grouped into principal components (PCs) to prevent collinearity. High concentrations of PFOS, PFDS, PFDoDA, PFTrDA and PFTeDA (grouped as PC1) were associated with a reduced hatching success of nests where at least one egg hatched, thinner egg shells and increased survival of the hatched chicks. High concentrations of PFDA (PC2) were associated with a reduced hatching success, especially in nests where no eggs hatched, an earlier start of egg laying and a reduction of total breeding success, mainly caused by the failure in hatching. Although the major manufacturer of PFAAs phased out the production of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and related products in 2002, concentrations appear to have increased since previous measurements. Surprisingly, despite the very high concentrations close to the fluorochemical plant, there was no clear evidence for reproductive impairment as the observed associations between PFAA concentrations and reproductive parameters were rather limited compared to previous studies in songbirds. These findings also suggest potential differences in sensitivity between species. CAPSULE: Despite the very high PFAA concentrations at the perfluorochemical hotspot, correlations with reproductive parameters were limited.

Variation in PFAA concentrations and egg parameters throughout the egg-laying sequence in a free-living songbird (the great tit, Parus major): Implications for biomonitoring studies.

Over the past decades, there has been growing scientific attention and public concern towards perfluoroalkyl acids (PFAAs), due to their widespread presence in the environment and associations with adverse effects on various organisms. Bird eggs have often been used as less-invasive biomonitoring tools for toxicological risk assessments of persistent organic pollutants, including some PFAAs. Hereby, it is typically assumed that one random egg is representative for the PFAA concentrations of the whole clutch. However, variation of PFAA concentrations within clutches due to laying sequence influences can have important implications for the egg collection strategy and may impede interpretations of the quantified concentrations. Therefore, the main objective of this paper was to study variation patterns and possible laying sequence associations with PFAA concentrations in eggs of the great tit (Parus major). Eight whole clutches (4-8 eggs) were collected at a location in the Antwerp region, situated about 11 km from a known PFAA point source. The ∑ PFAA concentrations ranged from 8.9 to 75.1 ng g-1 ww. PFOS concentrations ranged from 6.7 to 55.1 ng g-1 ww and this compound was the dominant contributor to the total PFAA profile (74%), followed by PFDoA (7%), PFOA (7%), PFDA (5%), PFTrA (4%) and PFNA (3%). The within-clutch variation (70.7%) of the ∑ PFAA concentrations was much larger than the among-clutch variation (29.3%) and concentrations decreased significantly for some PFAA compounds throughout the laying sequence. Nevertheless, PFAA concentrations were positively and significantly correlated between some egg pairs within the same clutch, especially between egg 1 and egg 3. For future PFAA biomonitoring studies, we recommend to consistently collect the same egg along the laying sequence, preferably the first or third egg if maximizing egg exposure metrics is the main objective.

Brood size is reduced by half in birds feeding on flutriafol-treated seeds below the recommended application rate.

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.

Perfluoroalkylated acids in the eggs of great tits (Parus major) near a fluorochemical plant in Flanders, Belgium.

Perfluoroalkyl acids (PFAAs) are highly persistent substances which have been detected in wildlife around the world, including birds. Although bird eggs have often been used to determine and monitor PFAAs levels in the marine environment, this has rarely been done in the terrestrial environment. In the present study we examined the concentrations and composition profile of 12 PFAAs (4 perfluoroalkyl sulfonic acids (PFSAs) and 8 perfluoroalkyl carboxylic acids (PFCAs) in the eggs of great tits (Parus major) collected at a fluorochemical plant and in three other areas, representing a gradient in distance from the pollution source (from 1 to 70 km), in Antwerp, Belgium. The PFSA concentrations measured at the site of the fluorochemical plant were among the highest ever reported in eggs with median concentrations of 10380 ng/g (extrapolated), 99.3 ng/g and 47.7 ng/g for PFOS, PFHxS and PFDS respectively. Furthermore, the median concentration of 19.8 ng/g for PFOA was also among the highest ever reported in bird eggs. Although these concentrations decreased sharply with distance from the fluorochemical plant, levels found in the adjacent sites were still high compared to what has been reported in literature. Moreover, based on what is known in literature, it is likely that these concentrations may cause toxicological effects. PFOS was the dominant contributor to the PFSA and PFAAs (63.4-97.6%) profile at each site, whereas for PFCAs this was PFOA at the plant site and the nearest locations (41.0-52.8%) but PFDoA (37.7%) at the farthest location. Although there is some evidence that PFAAs concentrations close to the plant site are decreasing in comparison with earlier measurements, which may be due to the phase out of PFOS, more research is necessary to understand the extent of the toxicological effects in the vicinity of this PFAAs hotspot.

Lead exposure reduces carotenoid-based coloration and constitutive immunity in wild mallards.

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. Environ Toxicol Chem 2016;35:1516-1525. © 2015 SETAC.

Altered immune response in mallard ducklings exposed to lead through maternal transfer in the wild.

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.

Adverse effects of thiram-treated seed ingestion on the reproductive performance and the offspring immune function of the red-legged partridge.

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.

Experimental approaches to test pesticide-treated seed avoidance by birds under a simulated diversification of food sources.

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.

Reducing Pb poisoning in birds and Pb exposure in game meat consumers: the dual benefit of effective Pb shot regulation.

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 <2% over the following three hunting seasons after ban reinforcement. Pb shot ingestion in mallards decreased from a pre-ban value of 30.2% to 15.5% in the post-ban period. Liver Pb levels were predominantly defined by the presence of ingested shot, whereas muscle levels were defined by the presence of both ingested and embedded shot. Only 2.5% of mallard muscle tissue had Pb levels above European Union regulations for meat (0.1µg/g wet weight) in the 2008-09 season, when Pb shot ingestion prevalence was also at a minimum (5.1%). Effective restrictions in Pb ammunition use have a dual benefit since this reduces Pb exposure for game meat consumers due to embedded ammunition as well as reducing Pb poisoning in waterbirds.