Jaime Rodríguez-Estival

Sub-chronic effects of nitrate in drinking water on red-legged partridge (Alectoris rufa): Oxidative stress and T-cell mediated immune function

In order to evaluate the effects of nitrates on birds, we have exposed captive red-legged partridges to nitrates concentrations of 0 (control), 100 (dwell water in farming areas) or 500 mg/l (fertirrigation level). The cellular immune response, plasma biochemistry, methemoglobin concentration (metHb), and oxidative stress biomarkers in blood and tissues were studied after two weeks of exposure. Several blood parameters such as aspartate aminotransferase, creatinine phosphokinase and lactate dehydrogenase activities and magnesium level decreased with nitrate exposure, whereas alkaline phosphatase activity and creatinine level increased. The oxidant effect of nitrates was evidenced by the increase in blood metHb, accompanied by the lipid peroxidation of red blood cells, the increased levels of oxidized glutathione (GSH) in liver, and the generation of oxidative DNA damage in plasma lymphocytes. GSH in erythrocytes was negatively correlated with blood metHb. The cellular immune function was slightly lower at partridges exposed to nitrates. These results suggest that adverse effects of nitrates on birds occur at concentrations potentially present in the field.

Blood Pb and δ-ALAD inhibition in cattle and sheep from a Pb-polluted mining area

The effects of Pb pollution on cattle and sheep raised in an ancient mining area were studied through the use of blood Pb (PbB) levels and δ-aminolevulinic acid dehydratase (δ-ALAD) activity. Lead levels in livestock blood from the mining area (n=110) were significantly elevated when compared to the controls (n=79). In 91.4% of cattle (n=58) and 13.5% of sheep (n=52) sampled in the mining area, PbB levels corresponded to subclinical exposure (6-35 μg/dl). Two young cattle (<2 years) from the mining area (n=5) had PbB levels indicative of clinical poisoning (>35 μg/dl). Elevated PbB was also accompanied by δ-ALAD activity inhibition in blood, which confirms that measurable effects of Pb poisoning were taking place. Observed PbB levels suggest that a potential risk to human consumers of beef from the Pb polluted areas may also exist, as has been shown previously for game meat from the same mining area.

Interactions between endogenous and dietary antioxidants against Pb-induced oxidative stress in wild ungulates from a Pb polluted mining area

Certain physiologic disorders, attributed to lead (Pb) exposure are related to the generation of oxidative stress. Organisms rely on a complex antioxidant system, composed of endogenously produced compounds such as glutathione (GSH), superoxide dismutase (SOD) and GSH peroxidase (GPX); and dietary antioxidants such as vitamins A and E. The aim of this work was to study the interactions between both groups of antioxidants and to evaluate their role in fighting Pb-induced oxidative stress in wild ungulates living in a Pb mining area. We studied red deer (n=54) and wild boar (n=60) from mining and control sites. Liver Pb, copper (Cu) and selenium (Se) levels were measured in liver and bone. Levels of GSH, oxidized GSH (GSSG), lipid peroxidation (TBARS), α-tocopherol, free retinol and retinyl esters, and the activities of SOD and GPX were measured in liver. Wild boar and red deer from the mining sites had higher bone (geometric means: 7.36 vs. 1.23μg/g dw for boar; and 0.46 vs. 0.11μg/g dw for deer) and liver Pb levels (0.81 vs. 0.24μg/g dw for boar; and 0.35 vs. 0.11μg/g dw for deer) than the controls. Pb exposure in deer was associated with lower GSH, α-tocopherol and retinyl esters, and higher free retinol and TBARS. A similar effect on vitamin A balance was observed in wild boar, but this was accompanied with an increase in GSH and α-tocopherol. GPX activity and Se levels were higher in wild boar (mean in controls: 670IU/mg protein and 1.3μg/g dw, respectively) than in red deer (150IU/mg protein and 0.3μg/g). These differences may indicate that red deer is more sensitive to Pb-induced oxidative stress than wild boar. Both endogenous and nutritional antioxidants may be negatively affected by Pb exposure, and their interactions are essential to fight against Pb-mediated oxidative damage.

Mercury exposure and mechanism of response in large game using the Almaden 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.

Levels of organochlorine pesticides and polychlorinated biphenyls in the critically endangered Iberian lynx and other sympatric carnivores in Spain.

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: <0.01 ng mL(-1), liver: 0.4ngg(-1) wet weight, fat: 87 ng g(-1)lipid weight) than other carnivores with more anthropic and opportunistic foraging behavior, such as the red fox (Vulpes vulpes; plasma: 1.11 ng mL(-1), liver: 459 ng g(-1), fat: 1984 ng g(-1)), or with diets including reptiles at higher proportion, such as the Egyptian mongoose (Herpestes ichneumon; plasma: 7.15 ng mL(-1), liver: 216 ng g(-1), fat: 540 ng g(-1)), or the common genet (Genetta genetta; liver: 466 ng g(-1), fat: 3854 ng g(-1)). Chlorinated pesticides showed interspecific variations similar to PCBs. Organochlorine levels have declined since the 80s in carnivores from Doñana National Park, but PCB levels are still of concern in Eurasian otters (Lutra lutra; liver: 3873-5426 ng g(-1)) from the industrialized region of Madrid.

Plasma levels of pollutants are much higher in loggerhead turtle populations from the Adriatic Sea than in those from open waters (Eastern Atlantic Ocean)

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. 1.12ng/ml; ∑OCPs, 1.63 vs. 0.19ng/ml; ∑PAHs, 13.39 vs. 4.91ng/ml; p<0.001 in all cases). This is the first time that levels of PAHs are reported in the Adriatic loggerheads. With respect to inorganic contaminants, although the differences were not as great, the Adriatic turtles appear to have higher levels of some of the most toxic elements such as mercury (5.74 vs. 7.59μg/ml, p<0.01). The results of this study confirm that the concentrations are larger in turtles from the Mediterranean, probably related to the high degree of anthropogenic pressure in this basin, and thus they are more likely to suffer adverse effects related to contaminants.

Pollutant accumulation patterns in nestlings of an avian top predator: biochemical and metabolic effects

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 Bonellis 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 Bonellis 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 nestlings 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.

Lead (Pb) in sheep exposed to mining pollution: implications for animal and human health.

Livestock from the ancient mining area of Sierra Madrona and Alcudia Valley (Spain) is exposed to elevated levels of lead (Pb), as previous studies based on blood monitoring have revealed. Here we have studied blood, liver and muscle Pb levels in sheep in order to know if Pb exposure could represent a risk for human consumers of the meat and offal of these animals. A cross-sectional study was conducted with ≥4 years old (adults) ewes from the mining area (n=46) and a control area (n=21). Blood samples were taken before the sacrifice at the slaughterhouse, and liver and muscle samples were taken thereafter. At the same time, 2-3 year old rams (subadults, n=17) were blood sampled in the mining area. Blood, liver and muscle Pb levels were higher in the mining than in the control area. Blood Pb concentration in the mining area (n= 44, mean: 6.7μg/dl in ewes and 10.9μg/dl in rams) was above background levels (>6μg/dl) in 73.3 percent of animals. Liver Pb concentration in 68 percent of sheep from the mining area (n=32, mean: 6.16μg/g dry weight, d.w.) exceeded the minimum level associated with toxic exposure (5µg/g d.w.) and 87.5 percent of liver samples were above European Union Maximum Residue Levels (MRL) established for offal destined for human consumption (0.5µg/g w.w.~1.4µg/g d.w.). On the contrary, none of the muscle samples in ewes exceeded the EU MRL (0.1µg/g w.w.~0.34µg/g d.w.) established for meat, which may be related to the decline of blood Pb levels with age observed in the present study. These results suggest a potential health effect for sheep exposed to Pb pollution in this area and implications for food safety, but further research with lamb meat may be necessary to refine the risk assessment for human consumers.

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.

Chronic effects of lead (Pb) on bone properties in red deer and wild boar: Relationship with vitamins A and D3

Here we study the occurrence of abnormalities on bone tissue composition and turnover mechanisms through the Pb-mediated disruption of vitamins A and D in wild ungulates living in a lead (Pb)-polluted mining area. Red deer (Cervus elaphus) and wild boar (Sus scrofa) from the mining area had significantly higher liver and bone Pb levels than controls, which were associated with the depletion of liver retinyl esters and the corresponding increase of free retinol levels both in deer and boar from the mining area. Pb-exposed adult deer had lower carbonate content in bone mineral than controls, which was associated with the increased free retinol percentage. In wild boar, the degree of bone mineralization was also positively associated with higher burdens of retinyl esters. These results suggest that Pb-associated changes in bone composition and mineralization is likely influenced by the depletion of vitamin A in wildlife exposed to environmental Pb pollution.