Mónica Martínez-Haro

Effects of lead exposure on oxidative stress biomarkers and plasma biochemistry in waterbirds in the field

Medina lagoon in Andalusia has one of the highest densities of spent lead (Pb) shot in Europe. Blood samples from waterbirds were collected in 2006-2008 to measure Pb concentration (PbB), δ-aminolevulinic acid dehydratase (ALAD), oxidative stress biomarkers and plasma biochemistry. PbB above background levels (>20 μg/dl) was observed in 19% (n=59) of mallards (Anas platyrhynchos) and in all common pochards (Aythya ferina) (n=4), but common coots (Fulica atra) (n=37) and moorhens (Gallinula chloropus) (n=12) were all <20 μg/dl. ALAD ratio in mallards and coots decreased with PbB levels >6 μg/dl. In mallards, an inhibition of glutathione peroxidase (GPx) and an increased level of oxidized glutathione (oxGSH) in red blood cells (RBC) were associated with PbB levels >20 μg/dl. In coots, PbB levels were negatively related to vitamin A and carotenoid levels in plasma, and total glutathione in RBCs; and positively related with higher superoxide dismutase and GPx activities and % oxGSH in RBCs. Overall, the results indicate that previously assumed background levels of PbB for birds need to be revised.

Bioaccessibility of Pb from Ammunition in Game Meat Is Affected by Cooking Treatment

Background The presence of lead (Pb) ammunition residues in game meat has been widely documented, yet little information exists regarding the bioaccessibility of this Pb contamination. We study how cooking treatment (recipe) can affect Pb bioaccessibility in meat of animals hunted with Pb ammunition. Methodology/Principal Findings We used an in vitro gastrointestinal simulation to study bioaccessibility. The simulation was applied to meat from red-legged partridge (Alectoris rufa) hunted with Pb shot pellets and cooked using various traditional Spanish game recipes involving wine or vinegar. Total Pb concentrations in the meat were higher in samples with visible Pb ammunition by X-ray (mean±SE: 3.29±1.12 µg/g w.w.) than in samples without this evidence (1.28±0.61 µg/g). The percentage of Pb that was bioaccessible within the simulated intestine phase was far higher in meat cooked with vinegar (6.75%) and wine (4.51%) than in uncooked meat (0.7%). Risk assessment simulations using our results transformed to bioavailability and the Integrated Exposure Uptake Biokinetic model (IEUBK; US EPA) show that the use of wine instead of vinegar in cooking recipes may reduce the percentage of children that would be expected to have >10 µg/dl of Pb in blood from 2.08% to 0.26% when game meat represents 50% of the meat in diet. Conclusions/Significance Lead from ammunition in game meat is more bioaccessible after cooking, especially when using highly acidic recipes. These results are important because existing theoretical models regarding Pb uptake and subsequent risk in humans should take such factors into account.

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.

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.

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.

Pb-Al relationships in waterfowl feces discriminate between sources of Pb exposure.

Hunted mallards (n = 56) were collected in the Ebro Delta (Spain) in 2007-08 to evaluate the use of feces as a non-invasive monitoring method to study lead shot ingestion. The prevalence of Pb shot ingestion in these birds was 28.6%, and similar to that reported before a ban on Pb shot use in 2003. Lead concentrations in terminal intestinal contents were higher in mallards with Pb shot in their gizzard. Feces Pb concentrations >34 microg/g d.w were indicative of Pb shot ingestion, and background Pb levels were <12.5 microg/g d.w. To discriminate between birds ingesting soil Pb, and those ingesting Pb shot, we correlated Al and Pb levels and showed that mallards with ingested Pb shot in the gizzard stood out as outliers within the regression. Feces Pb-Al relationships can be used as a simple non-invasive tool in monitoring programs regarding Pb shot ingestion in birds.

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.

Effect of Game Management on Wild Red-Legged Partridge Abundance

The reduction of game and fish populations has increased investment in management practices. Hunting and fishing managers use several tools to maximize harvest. Managers need to know the impact their management has on wild populations. This issue is especially important to improve management efficacy and biodiversity conservation. We used questionnaires and field bird surveys in 48 hunting estates to assess whether red-legged partridge Alectoris rufa young/adult ratio and summer abundance were related to the intensity of management (provision of supplementary food and water, predator control and releases of farm-bred partridges), harvest intensity or habitat in Central Spain. We hypothesized that partridge abundance would be higher where management practices were applied more intensively. Variation in young/adult ratio among estates was best explained by habitat, year and some management practices. Density of feeders and water points had a positive relationship with this ratio, while the density of partridges released and magpies controlled were negatively related to it. The variables with greatest relative importance were feeders, releases and year. Variations in post-breeding red-legged partridge abundance among estates were best explained by habitat, year, the same management variables that influenced young/adult ratio, and harvest intensity. Harvest intensity was negatively related to partridge abundance. The other management variables had the same type of relationship with abundance as with young/adult ratio, except magpie control. Variables with greatest relative importance were habitat, feeders, water points, releases and harvest intensity. Our study suggests that management had an overall important effect on post-breeding partridge abundance. However, this effect varied among tools, as some had the desired effect (increase in partridge abundance), whereas others did not or even had a negative relationship (such as release of farm-reared birds) and can be thus considered inefficient or even detrimental. We advise reconsidering their use from both ecological and economical points of view.

Identifying Sources of Pb Exposure in Waterbirds and Effects on Porphyrin Metabolism Using Noninvasive Fecal Sampling

Waterbird feces (mainly mallard Anas platyrhynchos and coot Fulica atra) were collected from four wetlands in Southern Spain in the field or during capture (n = 558 and n = 59, respectively) to study lead (Pb) shot ingestion. Lead and aluminum (Al) concentrations along with Pb isotope signatures were used to identify sources of Pb exposure. The profile and concentrations of porphyrins and biliverdin in feces were used as biomarkers of toxicological effects. Feces with Pb concentrations ≥ 34 μg/g d.w. showed higher Pb/Al ratios, together with lower (206)Pb/(207)Pb and (208)Pb/(207)Pb ratios, and higher (208)Pb/(206)Pb ratios, than feces with <34 μg/g d.w. Isotope signatures and Pb/Al ratios together indicated that Pb shot ingestion was the likely cause of the high Pb levels in some samples, whereas sediment ingestion was linked to lower/background levels. Coproporphyrin I and protoporphyrin IX were also higher in feces with Pb ≥ 34 μg/g d.w., indicating measurable disruption in heme synthesis. Noninvasive fecal sampling permits study of the degree and source of Pb exposure and physiological effects, with low-effort and minimal disturbance to waterbirds.

Avian digestive tract simulation to study the effect of grit geochemistry and food on Pb shot bioaccessibility.

Lead shot dissolution was investigated in a dynamic in vitro simulated avian gizzard-intestine system. The method allows simulated digestive fluid to pass (at intervals) from a gizzardlike environment to an intestine-based one, and then considers the dissolution of Pb shot (0-3 pellets) in the presence of differing grit geochemistries (siliceous and calcareous) and variable amounts of food (0-4 g of partially milled wheat seed). Dissolved Pb levels in simulated gizzards were consistently higher in the presence of siliceous, than with calcareous, grit. This was also seen in simulated intestines, except when less food was used (0-1 g), when Pb levels in solution were higher in calcareous systems. The Pb concentrations in gizzard and intestine solutions increased directly with the number of Pb shot used. In all treatments Pb levels in intestine liquids were lower than in gizzard liquids. Calcareous grit simulations maintained 2.5-34 times more Ca in solution than those that used siliceous grit. Dietary supplementation with calcareous grit may reduce Pb bioaccessibility of ingested Pb shot in birds by reducing gizzard acidity, by enhancing Pb precipitation (as Pb-carbonate), and by promoting higher dissolved Ca levels in the intestine, which may then compete with Pb for intestinal absorption.