The impact of lead poisoning from ammunition sources on raptor populations in Europe.

Poisoning caused by ingestion of spent lead (Pb) ammunition in food items is a common cause of death of raptors. However, there has been no previous attempt to assess the impact of lead poisoning on populations of raptors throughout Europe or examine how this relates to the prevalence of hunting. We used measurements of lead concentration in the liver from over 3000 raptors of 22 species found dead or dying in the wild in 13 countries and a lead poisoning threshold of 20 ppm (dry weight) to assess the proportion of these in which lead poisoning caused or contributed to death. The prevalence of lead poisoning as a cause of death of raptors varied substantially among European countries and was positively correlated across countries with the reported number of hunters per unit area. Ten species had a non-zero proportion of individuals with concentrations exceeding the lead poisoning threshold ranging between 0.3% and 16.5%. The estimated annual conditional death rate from lead poisoning for these ten species averaged 0.44% (range 0.06-0.85%). Scavenging species feeding regularly on carcasses of game animals,tended to have a high annual probability of death from lead poisoning. So too did some predators which only sometimes scavenge, but prey on frequently hunted birds, such as gamebirds, waterfowl and pigeons, which may contain ingested or embedded lead shot. Small-bodied predators had a low annual probability of death from lead poisoning. Modelling indicated that European populations of adult raptors of the ten focal species averaged 6.0% smaller (range 0.2-14.4%) than they would be without the effects of lead poisoning. A given rate of lead poisoning mortality resulted in greater expected population reductions for species with high annual survival rate and late age at first breeding.

70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria.

We have developed an in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS). When mRNA for a nuclear-encoded yeast mitochondrial matrix protein, Put2, was translated in a wheat germ cell-free system, import into posttranslationally added yeast mitochondria was negligible. However, when a yeast PRS was added, significant import was observed. The import stimulating activity of the yeast PRS was shown to consist of at least two distinct factors. One of these is the recently purified 70-kD heat shock-related protein Ssalp/Ssa2p, two proteins that are 98% homologous. The other factor is an N-ethylmaleimide-sensitive protein(s). Both factors act synergistically.

The chloroplast import receptor is an integral membrane protein of chloroplast envelope contact sites.

A chloroplast import receptor from pea, previously identified by antiidiotypic antibodies was purified and its primary structure deduced from its cDNA sequence. The protein is a 36-kD integral membrane protein (p36) with eight potential transmembrane segments. Fab prepared from monospecific anti-p36 IgG inhibits the import of the ribulose-1,5-bisphosphate carboxylase small subunit precursor (pS) by interfering with pS binding at the chloroplast surface. Anti-p36 IgGs are able to immunoprecipitate a Triton X-100 soluble p36-pS complex, suggesting a direct interaction between p36 and pS. This immunoprecipitation was specific as it was abolished by a pS synthetic transit peptide, consistent with the transit sequence receptor function of p36. Immunoelectron microscopy localized p36 to regions of the outer chloroplast membrane that are in close contact with the inner chloroplast membrane. Comparison of the deduced sequence of pea p36 to that of other known proteins indicates a striking homology to a protein from spinach chloroplasts that was previously suggested to be the triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) (Flügge, U. I., K. Fischer, A. Gross, W. Sebald, F. Lottspeich, and C. Eckerskorn. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:39-46). However, incubation of Triton X-100 solubilized chloroplast envelope material with hydroxylapatite indicated that p36 was quantitatively absorbed, whereas previous reports have shown that phosphate translocator activity does not bind to hydroxylapatite (Flügge, U. I., and H. W. Heldt. 1981. Biochim. Biophys. Acta. 638:296-304. These data, in addition to the topology and import inhibition data presented in this report support the assignment of p36 as a receptor for chloroplast protein import, and argue against the assignment of the spinach homologue of this protein as the chloroplast phosphate translocator.

Diclofenac residues in carcasses of domestic ungulates available to vultures in India.

Gyps vulture populations across the Indian subcontinent are declining rapidly and evidence indicates that veterinary use of the non-steroidal anti-inflammatory drug (NSAID) diclofenac is the major cause. Exposure of vultures to diclofenac is likely to arise from the consumption of livestock carcasses that have been treated shortly before death, however, detailed information regarding the prevalence and residual levels of diclofenac in carcasses available to vultures in India remains unreported. Here, we present data on diclofenac residues in 1848 liver samples taken from carcasses of dead livestock sampled at 67 sites in 12 states within India, between May 2004 and July 2005. Diclofenac residues were detected in carcasses in all states except Orisa, where only one site was sampled. The overall prevalence of detectable diclofenac (>10 microg kg(-1)) across all states was 10.1% and varied significantly among states, with up to 22.3% prevalence determined in Bihar. The geometric mean concentration of diclofenac found in samples in which the drug was detected was 352 microg kg(-1). The prevalence of carcasses containing diclofenac is similar to that previously proposed to be required to have caused the observed Gyps vulture declines in India. On the 11th of May 2006, the Drug Controller General (India) ordered the withdrawal of all licenses granted for the manufacture of diclofenac for veterinary use within India. However, if Gyps vultures are to be protected, potentially substantial existing stocks now need to be quickly and effectively removed from the Indian veterinary market.

Lead contamination and associated disease in captive and reintroduced red kites Milvus milvus in England.

Since 1989, a red kite Milvus milvus reintroduction programme has been underway in the United Kingdom, with 4-6 week old nestlings brought into captivity and held for 6-8 weeks before reintroduction. As scavengers, red kites may consume unretrieved game, and ingest shot or lead (Pb) fragments in their prey's flesh. We evaluated exposure to Pb in captive and wild red kites by taking blood samples from 125 captive young red kites prior to release, through analysing 264 pellets (regurgitated by wild birds) collected from under a roost site, and analysing Pb concentrations in livers and/or bones of 87 red kites found dead between 1995 and 2003. Lead isotope analyses of livers were also conducted in an effort to identify Pb exposure routes. Forty-six (36.8%) kites sampled prior to release had elevated blood Pb concentrations (201-3340 microg l(-1)). The source of this Pb was probably small fragments of lead ammunition in the carcasses of birds or mammals either fed to the nestlings by their parents or, more likely, subsequently whilst in captivity. Once released, kites were also exposed to lead shot in their food, and a minimum of 1.5-2.3% of regurgitated pellets contained Pb gunshot. Seven of 44 red kites found dead or that were captured sick and died within a few days had elevated (>6 mg kg(-1) dry weight [d.w.]) liver Pb concentrations, and six of these (14%) had concentrations of >15 mg kg(-1) d.w., compatible with fatal Pb poisoning. Post-mortem analyses indicated that two of these birds had died of other causes (poisoning by rodenticide and a banned agricultural pesticide); the remaining four (9%) probably died of Pb poisoning. Bone samples from 86 red kites showed a skewed distribution of Pb concentration, and 18 samples (21%) had Pb concentrations >20 mg kg(-1) d.w., indicating elevated exposure to Pb at some stage in the birds' life. Lead isotopic signatures (Pb (208/206); Pb (206/207)) in liver samples of the majority of kites were compatible with those found in lead shot extracted from regurgitated pellets. Lead isotope ratios found in the livers of kites with very low Pb concentrations were distinct from UK petrol Pb isotopic signatures, indicating that birds were exposed to little residual petrol Pb. We conclude that the primary source of Pb to which red kites are exposed is lead ammunition (shotgun pellets or rifle bullets), or fragments thereof, in their food sources; in some cases exposure appears sufficient to be fatal. We make recommendations to reduce Pb poisoning in both captive and wild red kites and other scavenging species.

Diclofenac disposition in Indian cow and goat with reference to Gyps vulture population declines.

Gyps vultures across India are declining rapidly and the NSAID diclofenac has been shown to be the major cause. Vultures scavenge livestock carcasses that have been treated with diclofenac within the days preceding death. We present data on diclofenac disposition in Indian cow and goat, and field data on the prevalence of diclofenac in carcases in the environment. In the disposition experiment, animals were treated with a single intramuscular injection of diclofenac at 1000 microg kg-1 bw. In cow, diclofenac was detectable in liver, kidney and intestine up to 71 h post-treatment; in plasma, half-life was 12.2 h. In goat, tissue residues were undetectable after 26 h. Prevalence of diclofenac in liver from 36 dead livestock collected in the field was 13.9%. Data suggest that diclofenac residues in Indian cow and goat are short-lived, but diclofenac prevalence in carcasses available to vultures may still be very high.

Widespread exposure to lead affects the body condition of free-living whooper swans Cygnus cygnus wintering in Britain.

Lead poisoning, through the ingestion of spent lead gunshot, is an established cause of morbidity and mortality in waterbirds globally, but the thresholds at which blood levels begin to affect the physiology of birds in the wild are less well known. Here we determine the prevalence of lead exposure in whooper swans and, for the first time, identify the level of blood lead associated with initial reductions in body condition. Blood lead elevated above background levels (i.e. >20 µg dL(-1)) was found in 41.7% (125/300) of swans tested. Blood lead was significantly negatively associated with winter body condition when levels were ≥44 µg dL(-1) (27/260 = 10%). Our findings indicating that sub-lethal impacts of lead on body condition occur at the lower end of previously established clinical thresholds and that a relatively high proportion of individuals in this population may be affected, reaffirm the importance of reducing contamination of the environment with lead shot.

Mechanisms of mitochondrial protein import.

Mitochondria import most of their proteins from the cytosol. Precursor forms of most matrix proteins as well as some IM and IMS proteins are synthesized on cytoplasmic ribosomes with N-terminal cleavable signal sequences. Many other mitochondrial proteins including IM carrier proteins contain internal targeting sequences. Three multisubunit translocases, one in the OM and two in the IM, participate in the import process. These translocases co-operate with cytosolic chaperones, chaperone-like soluble proteins in the IMS as well as chaperones in the matrix. Insertion of carrier proteins into the IM only requires a membrane potential. On the other hand, translocation of preproteins across the IM into the matrix requires (i) a membrane potential, (ii) GTP hydrolysis, which occurs at the outer side of the IM, and (iii) ATP-dependent interactions occurring at the matrix side. Following import, the cleavable signal sequence of most preproteins is removed in one step by the MPP. In some cases, removal of the signal sequence is achieved in two steps; first by MPP and second by either mitochondrial intermediate peptidase or by IM peptidases. Imported proteins must be folded properly to perform their functions.

Arsenic levels in the soils and macrophytes of the 'Entremuros' after the Aznalcóllar mine spill.

In April 1998, a holding lagoon containing pyrite ore processing waste rich in arsenic, failed and released 5-6 million m(3) of highly polluting sludge and acidic water. Over 2700 ha of the internationally important Doñana National and Natural Parks were contaminated. The area of Natural Park to sustain the greatest impact was known as the Entremuros. This paper presents 0-5 cm soil monitoring data from the Entremuros, from sampling campaigns 6 and 18 months after the disaster; as well as macrophyte root, rhizome and stem data from samples taken 18 months after the spill. Results show a clear, decreasing, north-south arsenic soil pollution trend, both 6 and 18 months after the spill, and suggest a small reduction in total soil arsenic levels occurred over time; although a significant increase in extractable arsenic is also noted. The two macrophytes (Typha dominguensis and Scirpus maritimus) studied herein are not accumulating arsenic in stem parts, however, accumulation of arsenic on iron plaque on the roots of these plants may be occurring. Further work is recommended in order to determine the ecotoxicological significance of this process in relation to the avian food-chains of Doñana, and elsewhere.

Preparation of protein A-peroxidase monoconjugate using a heterobifunctional reagent, and its use in enzyme immunoassays.

Protein A-peroxidase monoconjugate was prepared in solution using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)-propionate. The yield of the monoconjugate was much higher than that obtained with current methods. An immunoassay method was developed in which protein A-peroxidase monoconjugate served as a universal tool. Protein A-peroxidase monoconjugate was taken up by IgG molecules immobilized on an excess of solid phase antigen. The ability of free antigen to inhibit the binding of antibody, measured as inhibition of conjugate up take, served as the basis for quantification in the assay. The method was applied to human chorionic gonadotropin (HCG) and human IgG. Optimal assay conditions were developed and it was found that as little as 1 ng of HCG/ml and 2 ng of IgG/ml could be detected. The method is of comparable sensitivity to other available immunoassay methods and gives accurate, reproducible results.

Antiallergic activity of 2-phenyl-8-azapurin-6-ones.

The synthesis and antiallergic activity in the rat passive cutaneous anaphylactic reaction of a series of 2-phenyl-8-azapurin-6-ones are described. Early in the investigation, a linear free-energy equation was established in which the activity was related to the size and hydrogen bonding capacity of the ortho substituent in the phenyl ring. This relationship was used to provide guidance and limits for subsequent work leading to 2-o-propoxyphenyl-8-azapurin-6-one which is 40 times more potent than disodium cromoglycate. It is suggested that good antiallergic activity in this series is associated with coplanarity of the phenyl group with the azapurin-6-one which would be favored by a high degree of hydrogen bonding.

A new series of cardioselective adrenergic beta-receptor blocking compounds. 1-(2-Acyl-4-acylaminophenoxy)-3-isopropylaminopropan-2-ols.

A series of 1-(2-acyl-4-acylaminophenoxy)-3-isopropylaminopropan-2-ols has been synthesized and examined for beta-receptor blocking and antiarrhythmic activity. Several of these compounds are more than 20 times as active in blocking cardiac beta-receptors than vascular beta-receptors when given intravenously to anesthetized cats. The activities have been correlated quantitatively with partition and steric substitution constants. The observed relationships are consistent with a tentative proposal that the vascular receptor is situated in a more lipophilic environment than the cardiac receptor so that there is a differential transport effect between the two types of receptor.

Magnetic excitations in a weak-stripe-domain structure: A 2D dynamic micromagnetic approach

The high-frequency susceptibility spectra of ferromagnetic films supporting a weak-stripe-domain structure are computed using a 2D dynamic micromagnetic model that we have developed. The existence of multiple resonances resulting from the excitation of surface and volume modes is predicted. The main features of spectra (number of resonances, resonance frequencies, intensities, and linewidths) strongly depend on the equilibrium spin configuration and on the rf exciting field orientation. These theoretical results are successfully compared with zero-field microwave permeability measurements.

[125I]neurokinin A labels pharmacologically distinct populations of NK2 binding sites in hamster and rabbit urinary bladder.

The pharmacological profile of NK2 binding sites has been characterised in homogenates of rabbit urinary bladder and compared with that present in homogenates of hamster bladder. In both species, [125I]neurokinin A-specific binding to urinary bladder membranes was displaced by neurokinin A and the NK2 agonist [beta-Ala8]neurokinin A-(4-10) whilst the NK1 ligands [Sar9,Met(O2)11]substance P and (+/-)-CP-96,345, and the NK3 agonist, senktide, were only weak displacers or ineffective. At rabbit NK2 sites, the rank order of affinity of NK2 receptor-selective antagonists was; MEN 10,376 > MEN 10,207 > L-659,877 >> R 396. In contrast, the rank order of displacement of [125I]neurokinin A-specific binding to hamster bladder membranes was: L-659,877 > R 396 > MEN 10,376 > MEN 10,207. These data demonstrate that [125I]neurokinin A binds to pharmacologically distinct NK2 binding sites in hamster and rabbit urinary bladder.

Lead shot ingestion by waterbirds in the Camargue, france: an investigation of levels and interspecific differences.

Between January 1988 and February 1989, gizzards were collected from 1155 birds from the groups; Anatidae, Charadriidae, Scolopacidae and Rallidae, shot in the Camargue, and examined for the presence of ingested lead shot. Shot was found in nine out of ten Anatidae species, three of 15 Scolopacidae species and all of three Rallidae species. Twenty-seven per cent of Anatidae sampled had ingested shot, 13.8% had one shot present and 6.3% had more than five. Shot ingestion levels ranged from 3-60% of birds examined according to species. The incidence of shot ingestion did not appear to vary seasonally. In Anatidae, a strong interspecific relationship was found between the grit size ingested and susceptibility to ingest shot (r = 0.94). In Scolopacidae, shot ingestion may have been related to grit size ingested, feeding method and bill length. The levels of shot ingestion presented are for most species higher than have been reported elsewhere in the world. This may result from the absence of grit and presence of high shot densities in Camargue marshes.

Haematological parameters as predictors of blood lead and indicators of lead poisoning in the black duck (Anas rubripes).

The validity of various haematological parameters as indicators of blood lead concentration and lead poisoning in the black duck (Anas rubripes) is discussed. Elevated (> 40 microg dl(-1)) blood lead (PbB) levels were recorded in 41% of the 229 wild birds examined. No correlation was found between PbB concentration and haematocrit. Zinc protoporphyrin (ZPP) was positively correlated with PbB. Haemoglobin, delta-aminolevulinic acid dehydratase (ALA-d) activity and non-activated/activated ALA-d ratios were negatively correlated with PbB. The minimum PbB levels at which these parameters were affected varied greatly. ZPP and haemoglobin were relatively insensitive to PbB concentrations; whilst ALA-d activity and ALA-d ratio were influenced by PbB concentrations of < 5 microg dl(-1). Log(10) ALA-d ratio was more strongly correlated with PbB (r = -0.953) than log(10) ALA-d activity (r = -0.914) and the predictive validity (% false -ve and +ve predictions) of the ratio method was higher. The high individual variability in ALA-d activities at low PbB levels was greatly reduced by the use of the ALA-d ratio method. The ALA-d ratio method proved the most efficient for screening black duck for lead poisoning in the field.

Lead concentrations in birds of prey in Britain.

This paper reports on lead (Pb) concentrations in the livers of 424 individuals of 16 raptor species found dead and sent for analysis to the Institute of Terrestrial Ecology, Monkswood, from the early 1980s to the early 1990s. Elevated Pb concentrations in liver (>20 ppm dry wt), within the range associated with Pb poisoning mortality in raptors, were recorded in one peregrine (4% of species sample) and one buzzard (2% of species sample). These birds are likely to have ingested lead gunshot in the flesh of their prey. Another one each of these species had liver Pb concentrations of 15-20 ppm dry wt, reflecting unusually high absorption of Pb. No individuals of any other species had >15 ppm dry wt liver Pb, although some had 6-15 ppm. The source of Pb in these birds was unknown, but it could have resulted from high Pb concentrations in prey items, including some containing lead shot. Median liver Pb concentrations were generally very low (ranging from <0.07 to 1.61 ppm dry wt for species with sample sizes exceeding 10). In sparrowhawks, for which a large sample was available, liver Pb concentrations in all but one individual were low (<2.6 ppm dry wt). Pb concentrations in juveniles were significantly lower than in adults, and were lowest in recently fledged birds.

Exposure to trace elements of flamingos living in a biosphere reserve, the Camargue (France).

Recent research has shown that some adult flamingos Phoenicopterus roseus collected from the Camargue Biosphere Reserve had tissue metal concentrations considerably higher than the average for this species. As the range of these birds is large, the origin of the contamination was unknown. Blood and feather samples from nestling flamingos were therefore analysed for trace metals to determine the presence and origin of local contamination. A comparison of elemental concentration in the feathers of nestling and adult flamingos revealed higher concentrations of Cd, Cu, Pb and Se in adults, and higher concentrations of Zn in juveniles. This was attributed to increased exposure to atmospheric pollutants of adults, and either differences in Zn requirements and metabolism between adults and juveniles, or a local contamination of the juveniles' food supply by Zn. Concentrations of Zn in serum were also very elevated compared with human standards. Cd, Cu, Zn and Se concentrations were analysed in outer (O) and inner (I) barbs (i.e. barbs that are, respectively, exposed or not exposed to external deposits) of greater coverts of nestling flamingos. A comparison of elemental concentrations in these two feather components indicates a local atmospheric contamination by Cd, Cu and Pb.

The Doñana ecological disaster: contamination of a world heritage estuarine marsh ecosystem with acidified pyrite mine waste.

One of the most important bird breeding and over wintering sites in the west of Europe, the Coto Doñana, was severely impacted by the release of 5 million cubic meters of acid waste from the processing of pyrite ore. The waste entered ecologically sensitive areas of the park (including breeding areas for internationally endangered bird species) causing sustained pH decreases from pH 8.5 to 4.5 and resulting in massive metal contamination of the impacted ecosystem. The contaminating sludge waste contained arsenic at 0.6%, lead at 1.2% and zinc at 0.8% on a dry weight basis. The acid conditions facilitated the solubilization of these metals, leading to water concentrations lethal for aquatic wildlife. The accident caused considerable fish and invertebrate kills and has severe consequences for the protected bird species dependent on the impacted habitats and adjacent areas.

Mortality of globally threatened Sarus cranes Grus antigon from monocrotophos poisoning in India.

Globally threatened Sarus cranes Grus antigon are resident at Keoladeo National Park World Heritage site and the surrounding area near Bharatpur, Western Rajasthan, India. In winter, they are joined by c. 200 wintering common cranes Grus grus. On 23 November 2000, 15 Sarus cranes and three common cranes were found dead in a field adjacent to the park, where wheat seed had been sown the previous day. Chemical analyses of seed samples from the field and the cranes' alimentary tract contents identified residues of the organophosphate insecticide monocrotophos. Monocrotophos concentrations of 0.8 and 1.8 ppm were found in wheat samples, and 0.2-0.74 ppm (x=0.33) in the alimentary tract contents of five of the seven cranes examined. No other organophosphate or organochlorine pesticides were detected. We conclude that the cranes died from monocrotophos poisoning after eating treated seed. We discuss similar incidents of avian mortality, and recommend that measures be taken to reduce these.