Clinical lead toxicosis occurred one month after the captive vultures were returned to their aviary following old minium paint removal. Although ten birds had been exposed to severe lead contamination, only one died. The vultures were poisoned with lead(II) ions in the paint dust and chips that were sanded off the steel aviary construction and contaminated the soil to such an extent that its 10cm-deep surface layer contained as much as 25.85 mg/g of lead. Vultures are scavengers feeding on meat on the bone and entire carcasses or parts thereof supplied on the ground. Therefore, feeding on soiled carrion from the ground was the direct way of lead exposure for these captive vultures. Generally, kidney and liver concentrations of lead above 8-10 μg/g on a fresh weight basis are diagnostic for lead toxicosis in animals . Liver, kidney and blood lead concentrations in the male Egyptian Vulture that died were consistent with acute intoxication and levels causing mortality . Likewise, laboratory analyses confirmed severe liver and kidney damage and anaemia . The present case is similar to an outbreak of lead toxicosis in captive Houbara Bustards (Chlamydotis undulata macqueenii) on a private farm in the United Arab Emirates where the birds ingested large flakes of green paint peeling off the metal poles supporting the aviary mesh . Fortunately, the blood lead concentration was approximately 2.5 times lower in the cage-mate female Egyptian Vulture and decreased with D-penicillamine and later CaNa2EDTA treatment.
Background blood lead concentrations in Pb-non-exposed control Cinereous Vultures were in agreement with those measured in other birds [17, 20, 33, 34]. Regarding the development of blood lead levels in Cinereous Vultures surviving the lead toxicosis, it is important to realise that initially high sub-lethal values decreased even without therapy within the first month after lead ingestion, probably due to excretion and deposition in bones  and that the decrease of blood lead levels following CaNa2EDTA administration was not as substantial as might be expected (cf. Figure 2).
Interestingly, it was possible to examine the temporal development of the intoxication and evaluate both adverse effects of lead and effects of chelation therapy on selected blood parameters in the group of surviving Cinereous Vultures.
Calcium levels are reported not to change in lead-exposed falcons, while phosphorus rises . Unlike Wernery et al. , this study found phosphorus levels equal and lower in pre-treatment and post-treatment Cinereous Vultures, respectively, with the exception of the extreme of phosphorus rise in the male Egyptian Vulture who died. The current data also document a considerable decrease of iron both in pre-treatment and post-treatment birds. This iron decrease in lead-exposed birds is related to the development of anaemia, whereas it is the effect of CaNa2EDTA inducing urinary excretion of iron in the post-treatment birds . Importantly, iron deficiency is known to increase susceptibility to lead poisoning in infants and children .
Chelation therapy effectively improved haematocrit in Pb-exposed vultures in the present study. This parameter reflects the extent and efficiency of oxygen-carrying capacity of blood and thus the bird’s ability for physical performance and is often used as an indicator of condition in birds . Blood collection is considered non-destructive sampling for the lead toxicosis diagnostics and clinical state of health evaluation ; however, stress of handling can result in death in anaemic birds.
There were no significant differences in total protein levels of control healthy Pb-non-exposed birds and pre- and post-treatment vultures, and the values were within published normal ranges of the Cinereous Vulture .
The significant increase of alkaline phosphatase activity in the post-treatment group of vultures may be explained by collagen degradation and induction of bone metabolism after CaNa2EDTA therapy . While the activity of this enzyme is a measure of condition in nestlings and growing birds , it may be hypothesised that chelation therapy-induced bone resorption may rather result in poor performance.
The significant increase of creatine kinase activity in lead-exposed vultures well corresponds with published data  and a return to normal of this enzyme activity provides information on the efficacy of chelation therapy. Unfortunately, normal plasma chemistry values of enzymes were published in concentration units  and do not allow for a comparison with activities of the current study.
Lead impairs general health, growth, immune, reproductive, and many other physiological functions in birds, the biosynthesis of haemoglobin inclusive . Some of its adverse effects are associated with the oxidative damage of lipids, proteins, and DNA . The current results on lipid peroxidation (TBARS) and the ferric reducing ability of plasma (FRAP assay) demonstrate oxidative stress in lead-exposed vultures (cf. Table 1). While the FRAP values returned to normal following therapy with CaNa2EDTA, TBARS slightly increased in comparison with pre-treatment birds. Data on the increase of TBARS correspond with published findings that demonstrate enhanced lipid peroxidation in the liver of rats treated with CaNa2EDTA .
The total antioxidant capacity of blood can be measured using the ferric reducing antioxidant power assay as a clinical marker of oxidative stress. The value of this parameter depends on non-enzymatic antioxidants including ascorbic and uric acids, bilirubin, vitamin E, α-tocopherol and albumin and is linearly related to their molar concentrations. Uric acid contributes around 60% to the plasma value . Uric acid, formed in the liver and eliminated by kidneys, represents the primary route of excretion of nitrogenous waste in birds .
Changes in the oxidation-reduction system of glutathione have also been demonstrated in the lead-exposed Cinereous Vultures both prior to and after CaNa2EDTA therapy. This phenomenon can be associated with the altered redox status and other damaged biochemical pathways due to the presence of lead(II) ions. Moreover, interactions of reduced glutathione with CaNa2EDTA can be considered due to the high reactivity of the –SH moiety of this abundant peptide. Considering the fact that over 90% of the total glutathione pool in healthy organisms is represented by its reduced form and an increased oxidised-to-reduced ratio is indicative of oxidative stress, both pre- and post-treatment birds were able to handle this issue and modulate the antioxidant response. A different response to lead exposure was reported from waterbirds  in which the level of oxidised glutathione in red blood cells increased in association with Pb levels >20 μg/dl.
On the other hand, inducible metallothioneins are the first detectable signs of exposure to heavy metals at the cellular level and may be used as biomarkers because there are probably constant levels of metallothioneins in non-stressed cells [42–45]. Metallothioneins binding various metal ions play a key role in reducing toxic effects of heavy metals in the organism .
Many environmental stressors can decrease immunocompetence in birds. Immunosuppressive effects, demonstrated even at low doses , can result in mortality because of other factors such as, for example, Aspergillus infection [5, 47]. Combinations of several stressors, though sub-lethal when considered as a single exposure, are known to enhance avian toxicity [20, 22].
Gross and microscopic lesions in the liver and kidney of the male Egyptian Vulture were consistent with lead toxicity . The liver is important for the metabolism and excretion of lead in bile. Lead is characteristic for its strong affinity for the mitochondrial membrane and impairment of respiratory and phosphorylative abilities . Importantly, there are common molecular mechanisms, such as oxidative stress and mitochondrial impairment, involved in the adverse effects of some toxins [40, 50, 51], as well as in the immune response .
Although all Cinereous Vultures survived a month after exposure and blood lead levels were decreasing, chelation therapy to reduce the risk of morbidity and mortality is considered in birds of prey such as California condors with blood lead levels ≥0.3-0.45 μg/g [17, 53]. Such high levels of lead were present at the time of intoxication/diagnosis and in vultures prior to therapy. Treatment should continue until blood lead levels are below 0.2 μg/g . As the blood lead concentrations were just around this threshold in the post-treatment birds (i.e. 0.254 ± 0.097 μg/g), the chelation therapy should probably have been more aggressive. What makes the decision to treat or not to treat the birds difficult is the lag between blood collection and availability of results of laboratory analyses for lead. It is imperative to minimize stressful stimuli to anaemic birds, therefore, one should both collect blood and administer chelation agents at one occasion.
It is also necessary to report some adverse effects such as inappetence and regurgitation after three days of D-penicillamine therapy in the Egyptian Vulture female. Side-effects of D-penicillamine are known to include gastrointestinal disturbances that may affect up to 30% of patients . Importantly, vultures dosed for 5 days in the present study as well as birds treated with CaNa2EDTA for as many as 23 consecutive days, did not demonstrate any deleterious effects . While D-penicillamine was administered per os, CaNa2EDTA is a parenteral agent chelated with Ca2+ and thus selective for divalent cations with greater affinity for EDTA, which it will exchange for Ca2+.
As there are no veterinary chelators, the use of both human drugs containing D-penicillamine and CaNa2EDTA was extra-label. In light of this, no standard protocol is available for birds and, for example, California condors are considered in need of chelation therapy based on a threshold level recommended for lead-poisoned children . Importantly, one must bear in mind the possible harmful effects of chelation therapy. Nephrotoxicity of CaNa2EDTA can further deteriorate the renal function impaired by exposure to lead . While heavy metals including lead are well known embryo/foetal toxicants, the teratogenic potential of chelating agents has also been demonstrated to be due to depletion of essential trace elements such as copper or zinc induced by chelation . Parenteral administration of CaNa2EDTA can be painful, it may result in muscle damage at the injection site  and hamper early release of birds from captive facilities. For example, semiannual recapture of California condors for health checkups indicated the need of clinical intervention to avert morbidity and mortality in about 20% of birds . However, extensive stays in captivity associated with chelation therapy can probably result in behavioural and reproductive problems due to disruption of pair bonds and inability to maintain nesting territories . Chelating agents reduce body stores of lead. On the other hand, the reduction of blood lead levels may result in the mobilisation of skeletal stores of lead associated with redistribution of lead concentrations and worsening signs of toxicity. Multiple treatments may, therefore, be indicated by follow-up tests . As shown by the present pre- and post-treatment data in lead-poisoned vultures, the benefits and risks of chelation therapy should carefully be considered in individual birds because only some blood parameters normalised after CaNa2EDTA administration (i.e., haematocrit, creatine kinase and ferric reducing ability of plasma) and others worsened. Nevertheless, it is these authors’ opinion that chronic sublethal effects of lead exposure associated even with lower blood lead levels outweigh the adverse action of chelation therapy in decision-making how to manage these cases because there is no apparent threshold below which adverse effects of lead do not occur . Apart from measures to decrease the lead burden, supportive therapy is often necessary in poisoned birds. Taken together, these results warrant supplying lead-intoxicated and CaNa2EDTA-treated birds with antioxidants, iron and calcium to counteract the adverse effects both of lead exposure and chelation therapy [54, 56].
There is a diversity of environmental pollutants that can impair avian reproduction either through the action on adult birds and developing embryos and foetuses, or both [61–63]. Lead, affecting multiple physiological functions in birds , belongs to such toxins. Its accumulation can exert reproductive effects in breeding birds and detrimental effects may include changes in egg size, eggshell thickness, hatchability and fledging success . The reproductive success was found to be negatively correlated with maternal Pb . Female birds are known to eliminate heavy metals in their eggs . While blood lead levels reflect recent exposure, egg contamination can be due to recent or chronic exposure [8, 9] and result both from maternal blood and lead mobilisation from organs and tissues during egg formation . The clutch of the Cinereous Vulture typically includes only a single egg of approximately 237g . The Cinereous Vulture female in this study laid a normal sized egg. Regarding the small clutch size and the total lead content estimate of 1.18mg, this excretion route cannot represent an effective way of depuration in this species. However, as shown by the lead concentration in the foetal liver consistent with levels causing mortality in birds , it can pose a threat for the developing avian egg. Incubation lasts from 52 to 55 days in the Cinereous Vulture and, despite the high Pb burden in the egg, the development ceased not earlier than on day 40. In agreement with published data , lead concentrations were higher in the contents compared to the eggshell. Reproduction during subsequent breeding seasons can also be at risk from lead mobilised from bone deposits because birds that have survived acute exposure may have relatively high bone Pb. Interestingly, the femur Pb concentration found in the Cinereous Vulture male 14 months after exposure was lower than that indicative of excessive exposure and observed in birds that have died of Pb poisoning [8, 9]. This bird’s lead burden was probably further decreased by chelation therapy.
Bone lead concentrations in long-lived species of birds reflect life time exposure and increase with age . Although this bioaccumulation is less-readily related to pathophysiological mechanisms and mortality when compared with soft tissues and blood levels , sub-lethal effects can result in bone composition alteration such as lower degree of mineralisation and higher bone fragility .
The global populations of both avian scavengers are declining and their conservation status is near threatened and endangered in the Cinereous Vulture (Aegypius monachus) and Egyptian Vulture (Neophron percnopterus), respectively . Lead exposure of captive birds with its lifelong health consequences and reproduction impairment can hamper the success of conservation breeding and make birds unavailable for reintroductions [8, 67]. In view of this, the accident was a serious management failure. As the danger of lead is notoriously known, the decision not to remediate the soil prior to restocking birds in the aviary is hard to understand.