A heavy burden: Metal exposure across the land-ocean continuum in an adaptable carnivore.

Urbanisation and associated anthropogenic activities release large quantities of toxic metals and metalloids into the environment, where they may bioaccumulate and threaten both wildlife and human health. In highly transformed landscapes, terrestrial carnivores may be at increased risk of exposure through biomagnification. We quantified metallic element and metalloid exposure in blood of caracals (Caracal caracal), an adaptable felid inhabiting the rapidly urbanising, coastal metropole of Cape Town, South Africa. Using redundancy analysis and mixed-effect models, we explored the influence of demography, landscape use, and diet on the concentration of 11 metals and metalloids. Although species-specific toxic thresholds are lacking, arsenic (As) and chromium (Cr) were present at potentially sublethal levels in several individuals. Increased use of human-transformed landscapes, particularly urban areas, roads, and vineyards, was significantly associated with increased exposure to aluminium (Al), cobalt (Co) and lead (Pb). Foraging closer to the coast and within aquatic food webs was associated with increased levels of mercury (Hg), selenium (Se) and arsenic, where regular predation on seabirds and waterbirds likely facilitates transfer of metals from aquatic to terrestrial food webs. Further, several elements were linked to lower haemoglobin levels (chromium, mercury, manganese, and zinc) and elevated levels of infection-fighting cells (mercury and selenium). Our results highlight the importance of anthropogenic activities as major environmental sources of metal contamination in terrestrial wildlife, including exposure across the land-ocean continuum. These findings contribute towards the growing evidence suggesting cities are particularly toxic areas for wildlife. Co-exposure to a suite of metal pollutants may threaten the long-term health and persistence of Cape Town's caracal population in unexpected ways, particularly when interacting with additional known pollutant and pathogen exposure. The caracal is a valuable sentinel for assessing metal exposure and can be used in pollution monitoring programmes to mitigate exposure and promote biodiversity conservation in human-dominated landscapes.

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

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

In vitro assessment of mineral blocks as a cost-effective measure to reduce oral bioavailability of lead (Pb) in livestock.

Soil contamination in former mining districts is a persistent problem resulting from the historic lack of legal requirements as regards land restoration after mine closures. Much of this polluted land is currently being used worldwide for livestock and big game production, with the consequent health risks for the animals exposed and the subsequent threats to food safety. Soil remediation and restoration may be unfeasible or difficult to accomplish in the short term when pollution affects large territories and other alternatives must, therefore, be explored in order to reduce the probability of grazing animals being exposed to this contamination. In this paper, we study the use of mineral blocks (MBs) as a potential alternative by which to reduce the oral bioavailability of lead (Pb) in polluted soils by means of a simplified in vitro assay simulating gastrointestinal pH conditions. Experiments were carried out with twelve commercial MBs of different compositions in order to identify the most useful to be tested in further in vivo bioavailability studies. The results showed that one of them reduced the bioaccessibility of Pb from polluted soil by 88.2% and 75.9% under gastric and intestinal conditions, respectively, when compared with assays containing only polluted soil without MBs. The MB in question had the highest phosphorus content (7%) and one of the highest calcium contents (10%) of all those tested. Furthermore, negative correlations were detected between the content of calcium and phosphorus in the MBs and the percentage of bioaccessible Pb under gastric conditions, and between phosphorus and bioaccessible Pb under intestinal conditions. The use of MBs with a high phosphorus and calcium content should consequently be tested in vivo as a cost-effective (€ 0.6-1.5/sheep/month) tool by which to reduce the bioavailability of Pb for extensive grazing livestock reared in contaminated areas.

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

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

Specific carotenoid pigments in the diet and a bit of oxidative stress in the recipe for producing red carotenoid-based signals.

Colorful ornaments have been the focus of sexual selection studies since the work of Darwin. Yellow to red coloration is often produced by carotenoid pigments. Different hypotheses have been formulated to explain the evolution of these traits as signals of individual quality. Many of these hypotheses involve the existence of a signal production cost. The carotenoids necessary for signaling can only be obtained from food. In this line, carotenoid-based signals could reveal an individual's capacity to find sufficient dietary pigments. However, the ingested carotenoids are often yellow and became transformed by the organism to produce pigments of more intense color (red ketocarotenoids). Biotransformation should involve oxidation reactions, although the exact mechanism is poorly known. We tested the hypothesis that carotenoid biotransformation could be costly because a certain level of oxidative stress is required to correctly perform the conversion. The carotenoid-based signals could thus reveal the efficiency of the owner in successfully managing this challenge. In a bird with ketocarotenoid-based ornaments (the red-legged partridge; Alectoris rufa), the availability of different carotenoids in the diet (i.e. astaxanthin, zeaxanthin and lutein) and oxidative stress were manipulated. The carotenoid composition was analyzed and quantified in the ornaments, blood, liver and fat. A number of oxidative stress biomarkers were also measured in the same tissues. First, we found that color and pigment levels in the ornaments depended on food levels of those carotenoids used as substrates in biotransformation. Second, we found that birds exposed to mild levels of a free radical generator (diquat) developed redder bills and deposited higher amounts of ketocarotenoids (astaxanthin) in ornaments. Moreover, the same diquat-exposed birds also showed a weaker resistance to hemolysis when their erythrocytes were exposed to free radicals, with females also enduring higher oxidative damage in plasma lipids. Thus, higher color production would be linked to higher oxidative stress, supporting the biotransformation hypothesis. The recent discovery of an avian oxygenase enzyme involved in converting yellow to red carotenoids may support our results. Nonetheless, the effect could also depend on the abundance of specific substrate carotenoids in the diet. Birds fed with proportionally higher levels of zeaxanthin showed the reddest ornaments with the highest astaxanthin concentrations. Moreover, these birds tended to show the strongest diquat-mediated effect. Therefore, in the evolution of carotenoid-based sexual signals, a biotransformation cost derived from maintaining a well-adjusted redox machinery could coexist with a cost linked to carotenoid acquisition and allocation (i.e. a resource allocation trade-off).

Effects of invasive fish and quality of water and sediment on macrophytes biomass, and their consequences for the waterbird community of a Mediterranean floodplain.

Floodplains are among the most threatened ecosystems world-wide because of multiple stressors, i.e., invasive species, pollution and aquifer overexploitation; the Tablas de Daimiel National Park (Spain) is a clear example of these kinds of impact. This work aims to test whether invasive fish and/or the water and sediment quality are significant drivers of the decline of stonewort (Chara spp.) meadows in the Tablas de Daimiel, investigating how this could explain changes observed in the waterbird community. Bird surveys performed monthly between June 2010 and April 2014 have shown that herbivorous species like the red-crested pochard (Netta rufina) reached historical records between September 2010 and June 2011, but have decreased since then. Piscivorous waterbirds like the great cormorant (Phalacrocorax carbo) and herons increased in population after 2011, however. These changes may be due to the decline of Chara spp. meadows, connected to overexploitation by herbivores, or to changes in water and sediment quality. To test this hypothesis, we studied the growth of Chara spp. biomass in ten sites of the Tablas de Daimiel, where experimental exclosures were set up to exclude herbivory by birds, and bioturbation and herbivory by fish. Our results have shown that the absence of Chara spp. in the Tablas de Daimiel is mostly explained by presence of invasive fish (i.e. common carp). Moreover, the physicochemical characteristics of the water (lower values of conductivity and higher values of inorganic carbon and organic nitrogen), as well as of the sediment (lower values of inorganic and organic phosphorus), favour the increase of Chara spp., in the absence of the fish effect. These results led the National Park managers to begin the control of invasive fish as an urgent measure to assure the ecological conservation of this Mediterranean wetland.

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.

Mercury exposure and mechanism of response in large game using the Almadén mercury mining area (Spain) as a case study.

Mercury (Hg) accumulation, transfer, defense mechanism and adverse effects were studied in red deer (Cervus elaphus) and wild boar (Sus scrofa) from the Almadén mining district (Spain), the largest (285,000 t of Hg) and the oldest (more than 2000 years) Hg mine/refining operation site in the world. Red deer (n=168) and wild boar (n=58) liver, kidney, bones (metacarpus), testis and muscle were analyzed for total Hg and selenium (Se) within a range of distances to the Almadén mining district. The highest Hg concentrations were found in kidney (0.092 and 0.103 µg/g d.w. for red deer and wild boar, respectively) followed by the levels in liver (0.013 and 0.023 µg/g d.w. for red deer and wild boar, respectively). A significant correlation (r=-0.609, p=0.007) was found between Hg concentrations and distance to the Almadén Hg mining district. However, both red deer and wild boar closest to the mining area still showed mercury concentrations well below the concentration associated with clinical signs of Hg poisoning. Highest Se concentrations were found in kidney (2.60 and 6.08 µg/g in red deer and wild boar, respectively) and testis (2.20 µg/g in red deer). For selenium, differences between red deer and wild boar were statistically significant (p<0.05) in all tissues, concentrations being higher in wild boar than in red deer. In the diagnosis of Se deficiency, the vast majority of the examined red deer livers were deficient. A significant correlation was found between Hg and Se in kidney (r=0.386, p>0.001 for red deer and r=0.567, p=0.005 for wild boar). Liver GSSG concentrations were negatively correlated to total mercury and Hg:Se molar ratio in male deer, which could indicate a hormetic response to Hg exposure. Moreover, a positive association was found between the antioxidant element Se and antioxidant vitamins in red deer tissues.

Identification of carotenoid pigments and their fatty acid esters in an avian integument combining HPLC-DAD and LC-MS analyses.

Yellow-orange-red ornaments present in the integuments (feathers, bare parts) of birds are often produced by carotenoid pigments and may serve to signal the quality of the bearer. Although carotenoid esterification in tissues is a common phenomenon, most of the work on avian carotenoids has been focused on the identification of free forms or have been done after sample saponification. Here we determined free and esterified carotenoid composition in a bird species with red ornaments: the red-legged partridge (Alectoris rufa). Carotenoids from leg integument were extracted and processed by TLC to separate three major carotenoid groups (free form, mono- and diesters with fatty acids), whereas saponified extracts gave only free forms of carotenoids. TLC fractions were then analyzed by HPLC-DAD with C18 phase column for a preliminary identification of carotenoid groups. The final characterization of free carotenoids and its esters with fatty acids was performed with direct extracts analyzed by LC-MS and LC-MS/MS with a C30 phase, always with a system coupled to DAD. The main carotenoid (λ(max) 478 nm and [M+H](+) at m/z 597.2) was identified as astaxanthin by comparison with standards. A second carotenoid (λ(max) between 440 and 480 nm and [M+H](+) at m/z 581.3) was not identified among any of the commercially available carotenoid standards, although it could correspond to pectenolone according to its fragmentation pattern. Both the unidentified carotenoid and astaxanthin formed monoesters with fatty acids, but only astaxanthin was in its diesterified form. Monoesters were mainly formed with palmitic, stearic, oleic and linoleic acids. Complementary analyses of fatty acid composition in partridge integument by GC-MS revealed high amounts of these and other fatty acids, such as myristic, arachidic and docosanoic acids. The combination of HPLC-DAD and LC-MS/MS spectra was especially useful to identify the carotenoids present in the esterified forms and the probable masses of the fatty acids included in them, respectively.

Heavy metal exposure in large game from a lead mining area: effects on oxidative stress and fatty acid composition in liver.

The Pb mining area of the valley of Alcudia and the Sierra Madrona mountains (S. Spain) has been exploited intermittently for over 2100 years, since Roman occupation and up until the late 1900s. Red deer (n=168) and wild boar (n=58) liver and bone (metacarpus) were analyzed for Pb, Zn, Cu, Cd, As and Se. Lipid peroxidation, total and oxidized glutathione (GSH), superoxide dismutase (SOD), GSH peroxidase (GPX) and fatty acid composition were studied in liver of red deer. The concentrations of Pb in liver and bone of red deer and wild boar were higher in the mining area than in the control area, and higher in the wild boar than red deer, but well below the level associated with clinical signs of Pb poisoning. Liver levels of Cu, Cd and Se were also higher in red deer from the mining area. Red deer from the mining area had 39% less total GSH than in the control area. The percentage of docosahexaenoic acid in liver of red deer from the mining area was 16% lower than in the control area.

Metal levels in the bones and livers of globally threatened marbled teal and white-headed duck from El Hondo, Spain.

El Hondo is a key area for marbled teal and white-headed duck. We present Pb, Cu, Zn, Se, and As data for bone and liver in birds found dead between 1996 and 2001. Several metals were higher in adult white-headed ducks than in marbled teal. They were higher in female than in male white-headed ducks, and did not differ with sex in marbled teal, but did by age. Lead in liver of adults was influenced by Pb shot ingestion, which was detected in 21% of marbled teal and in 71% of white-headed duck. No marbled teal had liver levels indicative of Pb poisoning, while 86% of white-headed ducks did. Selenium, Zn, and Cu were elevated in 13%, 7%, and 39% of birds, respectively. Whilst Pb shot poses the greatest threat to these species, further work should assess exposure via plants, invertebrates, water, and sediments for other metals, and investigate possible sub-lethal effects.