MEASUREMENT OF PERSISTENT ORGANIC POLLUTANTS, PERFLUORINATED COMPOUNDS, AND TOXIC METALS IN THE BLOOD OF HUMBOLDT PENGUINS (SPHENISCUS HUMBOLDTI) AT PUNTA SAN JUAN, PERU USING DRIED BLOOD SPOTS.

The Humboldt penguin (Spheniscus humboldti) population at the Punta San Juan Marine Protected Area in Peru is considered critical to the long-term sustainability of this endangered species in Peru. Exposure of the rookery to environmental toxicants is a mounting concern because of regional growth of industries and human populations. Whole blood samples were collected from 30 free-ranging penguins in 2011 as part of a broader population health monitoring program. Dried blood spots (DBS) containing 50 µl of blood were prepared and analyzed to assess exposure to five groups of environmental contaminants. Concentrations of elements arsenic, cadmium, iron, lead, mercury, selenium, and thallium were analyzed using inductively coupled plasma mass spectrometry. Persistent organic pollutant concentrations were measured using gas chromatography-tandem mass spectrometry to analyze organochlorine pesticides (OCP; p,p'-DDT, p,p'-DDE, ß-hexachlorocyclohexane, t-nonachlor, and oxychlordane), polychlorinated biphenyls (congeners 138 and 153), and polybrominated flame retardants (polybrominated biphenyl-153 and polybrominated diphenyl ether congeners 47 and 99). Per- and polyfluoroalkyl substances, including perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid were measured using liquid chromatography-tandem mass spectrometry. Results revealed low levels of exposure to these selected contaminants, at levels not considered to be of concern for wildlife health. DBS methodology was considered effective in a field-based setting for quantification of whole blood concentrations of environmental contaminants in penguins.

Acute maduramicin toxicosis in pregnant gilts.

Ionophores are used as feed additives for the control of coccidiosis and growth promotion in farm animals. Reports of maduramicin toxicosis in farm animals are scarce. The present work describes an acute maduramicin toxicosis affecting 22 pregnant gilts, 2 pregnant sows and 2 boars, resulting in a total mortality of 65% within 2days. The clinical and histopathological findings observed shared similar characteristics to acute ionophore toxicosis in pigs, being characterized by severe myodegeneration in skeletal muscle and degenerative changes in the myocardium. Important clinical pathology indices found were elevated levels of CPK and ALT. In contrast to the pregnant gilts, the two pregnant sows completely recovered after 1month and farrowed 2months after the intoxication event healthy piglets. The lack of effect of maduramicin on the fetuses might be indicative of poor placental penetration of maduramicin. Moreover, the present work reports for the first time maduramicin levels in livers (0.5mg/kg) of gilts exposed to lethal concentrations of maduramicin (18.5mg/kg) in the feed. As the average feed intake of the gilts was estimated to be 3.5kg feed/day, the mean maduramicin intake leading to the observed high mortality rate was 0.4mg/kg body weight/day.

New validated multiresidue analysis of six 4-hydroxy-coumarin anticoagulant rodenticides in hen eggs.

Anticoagulant rodenticides are frequently a cause of poisoning of domestic animals, wildlife, and human beings. A toxicosis in 6,000 laying hens caused by the malicious addition of unknown amounts of coumatetralyl bait as well as the insecticides aldicarb, methomyl, and imidacloprid in the drinking water, was investigated in the current study. In order to determine a possible carryover of coumatetralyl into eggs, a rapid and reliable analytical method was developed and fully validated for the simultaneous detection of 6 anticoagulant rodenticides (warfarin, coumatetralyl, coumachlor, bromadiolone, difenacoum, and brodifacoum) in yolk and albumen using high-performance liquid chromatography (HPLC) with fluorescence detection. The method developed was reproducible, sensitive, accurate, and linear within the range of 0.01-1 mg/kg, which is the concentration range of bromadiolone and warfarin found in yolk in previously reported studies. The coefficient of variations of within and between days was 1.0-8.5% for yolk and 0.6-3.8% for albumen, while recoveries from spiked albumen and yolk samples were all in the range of 79-99% and 51-95%, respectively. Limits of detection in yolk were 0.01 mg/kg for warfarin and 0.003 mg/kg for the remaining compounds; in albumen, the limit of detection was 0.003 mg/kg for warfarin, coumatetralyl, and coumachlor, and 0.0015 mg/kg for difenacoum and brodifacoum. The application of the validated method revealed the presence of coumatetralyl in the yolk only at levels of 0.0057 mg/kg and 0.0052 mg/kg on the second and fourth day of the poisoning. In conclusion, the HPLC method demonstrated suitability for application in official analysis of anticoagulants in hen eggs.

Diagnostic analysis of veterinary dried blood spots for toxic heavy metals exposure.

Dried blood spots (DBS) on filter paper have been used in human medicine since the 1960s, predominantly for screening in-borne metabolic disorders and more recently, for toxicology. Despite its 50-year existence, this technology has not been adopted by veterinarians for routine diagnoses and research. We have validated a novel DBS analytical procedure for the routine measurement of toxic heavy metals using 50 µL of whole blood on a single DBS by inductively coupled plasma mass spectrometry (ICP-MS). Targeted heavy metals are arsenic, cadmium, mercury, lead, selenium and thallium. The limits of quantitation (LOQ) on DBS are: arsenic 1.7 µg/L, cadmium 4.0 µg/L, mercury 13.7 µg/L, lead 13.3 µg/L, selenium 6.3 µg/L and thallium 1.5 µg/L. These LOQs suffice for routine diagnoses of heavy metal intoxication in domesticated and wildlife species as well as for basic, applied and epidemiological studies. The technique is ideal for population studies involving investigations of wildlife exposure to heavy metals and other environmental pollutants. The small blood volume involved (50 µL) makes it feasible to study small animals (birds, reptiles, amphibians and small mammals) that were previously excluded, or difficult to study due to the relatively large sample volumes required by current gold standard blood collection techniques.

Carry-over of aflatoxin B1 to aflatoxin M1 in high yielding Israeli cows in mid- and late-lactation.

The potent hepatotoxin and carcinogen aflatoxin B1 (AFB1) is a common mycotoxin contaminant of grains used in animal feeds. Aflatoxin M1 (AFM1) is the major metabolite of AFB1 in mammals, being partially excreted into milk, and is a possible human carcinogen. The maximum permitted concentration of AFM1 in cows' milk is 0.05 µg/kg in Israel and the European Union. Since milk yield and the carry-over of AFB1 in the feed to AFM1 in the milk are highly correlated, it was considered important to determine the AFM1 carry-over in Israeli-Holstein dairy cows, distinguished by world record high milk production. Twelve such cows were used to determine AFM1 carry-over following daily oral administration of feed containing ~86 µg AFB1 for 7 days. The mean carry-over rate at steady-state (Days 3-7) was 5.8% and 2.5% in mid-lactation and late-lactation groups, respectively. The carry-over appears to increase exponentially with milk yield and could be described by the equation: carry-over% = 0.5154 e(0.0521 × milk yield), with r(2) = 0.6224. If these data truly reflect the carry-over in the national Israeli dairy herd, the maximum level of AFB1 in feed should not exceed 1.4 µg/kg, a value 3.6 times lower than the maximum residue level currently applied in Israel.

Examination of Eurasian griffon vultures (Gyps fulvus fulvus) in Israel for exposure to environmental toxicants using dried blood spots.

The griffon vulture (Gyps fulvus) is one of seven species of Old World Gyps vultures found over a wide range from the Iberian peninsula in the west through the Balkans, Turkey, and the Middle East to India in the east. The population of the griffon vultures in Israel has suffered a dramatic decrease, and in recent years productivity has been severely reduced. In this study, whole-blood samples taken from 25 apparently healthy griffon vultures at various stages of maturity were examined to investigate whether the vultures are being excessively exposed to environmental contaminants that might deleteriously affect their reproduction. Five groups of environmental contaminants, comprising toxic elements, organochlorine pesticides, polychlorinated biphenyls, polybrominated diphenyl ethers, and perfluorinated compounds, were monitored in dried blood spots. Results of the analyses showed low levels of exposure of griffon vultures to environmental contaminants compared with the sparse data available on griffon vultures and other diurnal raptors in other countries.

A database of avian blood spot examinations for exposure of wild birds to environmental toxicants: the DABSE biomonitoring project.

DABSE, a database for avian blood spot examination for exposure to toxicants, is a new biomonitoring project in wild birds that has the goal of providing reference values of harmful agents, as measured in whole blood stored as dried blood spots. Once these "normal" values have been established, the diagnosis of environmental contaminant-mediated ill-health (such as manifestations of sickness, increased mortality, a reduction in population, poor breeding success, abnormal behavior) in an individual bird or in a population could be facilitated by comparing exposure values in the investigation to reference values of the same species in the database. One might then identify the cause and pave the way for a mitigating response. The toxicant component of DABSE is being examined at the low ng ml(-1) level in 200 µl of whole blood. As these analyses are invariably very costly, an effort has been made to lower these costs and so enable more testing by quantitating representative compounds as markers for the whole group. These markers are invariably found in birds' blood at the highest concentration of all the constituents in that group. The toxicant groups comprise:- (a) elements-arsenic, cadmium, lead, mercury and selenium; (b) organochlorine pesticides, markers being p,p'-DDT, p,p'-DDE, ß-HCH, HCB and oxychlordane; (c) polychlorinated biphenyls (PCBs), the marker being congener 153; (d) polybrominated diphenyl esters (PBDEs), the marker being congener 47; (e) perfluorinated compounds (PFCs), the markers being perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA). DABSE will be expanded to determine exposure to infectious diseases and perhaps acute toxicoses.

Alpha-chloralose poisoning in dogs and cats: a retrospective study of 33 canine and 13 feline confirmed cases.

Alpha-chloralose (AC) is an anaesthetic compound also used as a rodenticide, and has dose-dependent central nervous system mixed effects of excitation and depression. The objectives of this study were to detail the clinical and clinicopathological characteristics, as well as the treatment and prognosis, of AC toxicosis in dogs and cats. Medical records were retrospectively reviewed for AC poisoning between the years 1989 and 2004, and 33 dogs and 13 cats were included in the study. The most common clinical signs were seizures, muscle tremor, hyperaesthesia, hypothermia, salivation, myosis, stupor, coma and ataxia. Coma was more common, while salivation and ataxia were less common in cats compared to dogs. Although hypothermia was very common, especially in cats (90.9%), hyperthermia was frequently observed in dogs (21%). Treatment in all patients was supportive and symptomatic, and the most commonly used anticonvulsants were diazepam and barbiturates; however, severe unresponsive seizures in three dogs had to be controlled with inhalant gas anaesthesia. The hospitalisation period was 1-3 days, and the overall mortality rate was 6.5%. Alpha-chloralose poisoning seems to have a favourable prognosis in dogs and cats.