Elwood F. Hill

Nineteenth Century Mercury: Hazard to Wading Birds and Cormorants of the Carson River, Nevada

Contemporary mercury interest relates to atmospheric deposition, contaminated fish stocks and exposed fish-eating wildlife. The focus is on methylmercury (MeHg) even though most contamination is of inorganic (IoHg) origin. However, IoHg is readily methylated in aquatic systems to become more hazardous to vertebrates. In response to a classic episode of historical (1859–1890) IoHg contamination, we studied fish-eating birds nesting along the lower Carson River, Nevada. Adult double-crested cormorants (Phalacrocorax auritus), snowy egrets (Egretta thula) and black-crowned night-herons (Nycticorax nycticorax) contained very high concentrations of total mercury (THg) in their livers (geo. means 134.8μg/g wet weight (ww), 43.7 and 13.5, respectively) and kidneys (69.4, 11.1 and 6.1, respectively). Apparently tolerance of these concentrations was possible due to a threshold-dependent demethylation coupled with sequestration of resultant IoHg. Demethylation and sequestration processes also appeared to have reduced the amount of MeHg redistributed to eggs. However, the relatively short time spent by adults in the contaminated area before egg laying was also a factor in lower than expected concentrations of mercury in eggs. Most eggs (100% MeHg) had concentrations below 0.80μg/g ww, the putative threshold concentration where reproductive problems may be expected; there was no conclusive evidence of mercury-related depressed hatchability. After hatching, the young birds were fed diets by their parents averaging 0.36–1.18μgMeHg/g ww through fledging. During this four to six week period, accumulated mercury concentrations in the organs of the fledglings were much lower than found in adults, but evidence was detected of toxicity to their immune (spleen, thymus, bursa), detoxicating (liver, kidneys) and nervous systems. Several indications of oxidative stress were also noted in the fledglings and were most apparent in young cormorants containing highest concentrations of mercury. This stress was evidenced by increased thiobarbituric acid-reactive substances, low activities of enzymes related to glutathione metabolism and low levels of reduced thiols, plus an increase in the ratio of oxidized to reduced glutathione. At lower concentrations of mercury, as was found in young egrets, we observed elevated activities of protective hepatic enzymes, which could help reduce oxidative stress. Immune deficiencies and neurological impairment of fledglings may affect survivability when confronted with the stresses of learning to forage and the ability to complete their first migration.

BRAIN CHOLINESTERASE ACTIVITY OF APPARENTLY NORMAL WILD BIRDS

Organophosphorous and carbamate pesticides are potent anticholinesterase substances that have killed large numbers of wild birds of various species. Cause of death is diagnosed by demonstration of depressed brain cholinesterase (ChE) activity in combination with chemical detection of anticholinesterase residue in the affected specimen. ChE depression is determined by comparison of the affected specimen to normal ChE activity for a sample of control specimens of the same species, but timely procurement of controls is not always possible. Therefore, a reference file of normal whole brain ChE activity is provided for 48 species of wild birds from North America representing 11 orders and 23 families for use as emergency substitutes in diagnosis of anticholinesterase poisoning. The ChE values, based on 83 sets of wild control specimens from across the United States, are reproducible provided the described procedures are duplicated. Overall, whole brain ChE activity varied nearly three-fold among the 48 species represented, but it was usually similar for closely related species. However, some species were statistically separable in most families and some species of the same genus differed as much as 50%.

Toxicity of anticholinesterase insecticides to birds: Technical grade versus granular formulations

The acute toxicities of 13 granular anticholinesterase insecticides were compared with their technical grade active ingredients by administering single oral doses of chemical to adult Northern bobwhites (Colinus virginianus) and evaluating resultant LD50 values and dose-response curves. Similar tests with ringed turtledoves (Streptopelia risoria) were conducted with five of the granular formulations to check for interspecific differences. The test chemicals were Amaze 15G (isofenphos), Counter 15G (terbufos), Dasanit 15G (fensulfothion), Diazinon 14G (diazinon), Di-Syston 15G (disulfoton), Dyfonate 20G (fonofos), Furadan 10G (carbofuran), Lorsban 15G (chlorpyrifos), Nemacur 15G (fenamiphos), Parathion 10G (parathion), Tattoo 10G (bendiocarb), Temik 15G (aldicarb), and Thimet 15G (phorate). Information is also presented on dose-response relations and their use in hazard assessment, granule size and hazard, response patterns, and toxic signs. The general conclusions were: (1) The organophosphates (fenamiphos and fensulfothion) and the carbamate (aldicarb) were the most toxic of the insecticides tested. (2) The granular formulation and its technical grade active ingredient were of equivalent toxicity, or the granular was significantly less toxic. (3) The dose-response curve enhances hazard assessment. (4) Ingestion of a single granule of Temik 15G was shown to be life threatening to bobwhite-sized birds, and ingestion of fewer than five granules could be lethal to sparrow-sized birds for Dasanit 15G, Diazinon 14G, Dyfonate 20G, Furadan 15G, and Nemacur 15G. (5) It is therefore suggested that the hazard associated with granular insecticides may be more dependent on which species (cf. size and feeding behavior) inhabit a treated area than on the actual application rate.

ACUTE ORAL TOXICITY OF SODIUM CYANIDE IN BIRDS

Sensitivities of six avian species, black vulture (Coragyps atratus), American kestrel (Falco sparverius), Japanese quail (Coturnix japonica), domestic chicken (Gallus domesticus), eastern screech-owl (Otus asio), and European starling (Sturnus vulgaris), to acute poisoning by sodium cyanide (NaCN) were compared by single dose LD50s. Three species, domestic chickens, black vultures, and turkey vultures (Cathartes aura), were dosed with NaCN to determine cyanide residues in those that died and also in survivors, in addition to postmortem fate. Three flesh-eating species (black vulture, American kestrel, and eastern screech-owl; LD50s 4.0–8.6 mg/kg) were more sensitive to NaCN than three species (Japanese quail, domestic chicken, and European starling; LD50s 9.4–21 mg/kg) that fed predominantly on plant material. Elevated concentrations of cyanide were found in the blood of birds that died of cyanide poisoning; however, concentrations in birds that died overlapped those in survivors. Blood was superior to liver as the tissue of choice for detecting cyanide exposure. No gross pathological changes related to dosing were observed at necropsy.

Case histories of bald eagles and other raptors killed by organophosphorus insecticides topically applied to livestock

Since 1982 when secondary poisoning of a red-tailed hawk (Buteo jamaicensis) was documented following the recommended use of famphur applied topically to cattle, the Patuxent Wildlife Research Center has tested dead birds of prey for poisoning by famphur and other pouron organophosphorus (OP) insecticides. Brain cholinesterase (ChE) activity was first determined, then if ChE was depressed ≥50%, stomach and/or crop contents were evaluated for anti-ChE compounds. This report presents the circumstances surrounding the OP-caused deaths of eight bald eagles (Haliaeetus leucocephalus), two red-tailed hawks, and one great horned owl (Bubo virginianus) between March 1984 and March 1985. OP poisoning of raptors by pour-on insecticides in the United States is widespread, but its magnitude is unknown.

Seasonal variation in diagnostic enzymes and biochemical constituents of captive northern bobwhites and passerines

1. A variety of biochemical measurements were taken periodically in captive northern bobwhite (Colinus virginianus L.), European starlings (Sturnus vulgaris L.), red-winged blackbirds (Agelaius phoeniceus L.) and common grackles (Quiscalus quiscula L.) to determine whether baseline values remain sufficiently stable throughout the year for general clinical use in the absence of concurrent control specimens. 2. Variables included whole blood hematocrit and hemoglobin, plasma lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, creatine kinase, butyrylcholinesterase, alkaline phosphatase, glucose, albumin, total protein, creatinine, urea nitrogen, uric acid, cholesterol, and triglycerides, and brain acetylcholinesterase. Butyryl- and acetylcholinesterase were included because of their specific uses in toxicology. 3. Significant seasonal differences were detected for each of the variables except brain acetylcholinesterase in at least one of the species. Significant species differences were detected during at least one season for all of the variables measured. 4. All species were maintained outdoors, but only northern bobwhites came into reproductive condition and showed sex-differences in the clinical variables during their normal breeding season. 5. It was concluded that reference values for the 18 clinical variables measured could be calculated from our data for adult specimens of the species studied, and that results for one species cannot be extrapolated with certainty to any other species. 6. Estimated normal bounds for each of the 18 variables measured by commonly used clinical procedures are presented for reproductively quiescent northern bobwhites, European starlings, red-winged blackbirds, and common grackles.

Cyanide and migratory birds at gold mines in Nevada, USA

Since the mid-1980s, cyanide in heap leach solutions and mill tailings ponds at gold mines in Nevada has killed a large but incompletely documented number of wildlife (>9,500 individuals, primarily migratory birds). This field investigation documents the availability of cyanide at a variety of ‘typical’ Nevada gold mines during 1990 and 1991, describes wildlife reactions to cyanide solutions, and discusses procedures for eliminating wildlife loss from cyanide poisoning. Substantial progress has been made to reduce wildlife loss. About half of the mill tailings ponds (some up to 150 ha) in Nevada have been chemically treated to reduce cyanide concentrations (the number needing treatment is uncertain) and many of the smaller heap leach solution ponds and channels are now covered with netting to exclude birds and most mammals. The discovery of a cyanide gradient in mill tailings ponds (concentration usually 2–3 times higher at the inflow point than at reclaim point) provides new insight into wildlife responses (mortality) observed in different portions of the ponds. Finding dead birds on the tops of ore heaps and associated with solution puddling is a new problem, but management procedures for eliminating this source of mortality are available. A safe threshold concentration of cyanide to eliminate wildlife loss could not be determined from the field data and initial laboratory studies. New analytical methods may be required to assess further the wildlife hazard of cyanide in mining solutions.

Divergent effects of postmortem ambient temperature on organophosphorus- and carbamate-inhibited brain cholinesterase activity in birds

Abstract Diagnosis of wildlife mortality from organophosphorus and carbamate pesticides is made by demonstration of critical depression (e.g., >50%) of whole brain cholinesterase (ChE) activity and detection of anti-ChE pesticide residue. In the field, this 50% criterion is usually far exceeded for organophosphorus poisonings, while ChE activity may be near normal for carbamate poisonings. Because dead wildlife may remain in the field for variable durations before collection, the temporal effect of moderate ambient temperatures on uninhibited and inhibited brain ChE was evaluated. Adult Japanese quail ( Coturnix japonica ) were dosed with 0, 1, 3, or 9 mg of carbofuran or dicrotophos per kg body weight, survivors were euthanized after 1 hr, and all specimens were randomized to 18, 25, or 32°C for 0, 1, 2, or 3 days. Toxic response was dose dependent for all variables measured. Carbofuran evoked rapid response at all dosages and death within 30 min or the subject seemed to recover within 60 min, while response to dicrotophos was not pronounced for about 30 min but then persisted through time of euthanasia. Postmortem reactivation was demonstrated for carbamylated ChE while activity of phosphorylated ChE continued to decrease. Rapid inhibition and spontaneous reactivation of carbamylated enzyme explained both toxicity and postmortem changes for carbofuran treatments. Phosphorylation of ChE is a comparatively slow and essentially irreversible reaction that explains both delayed response to dicrotophos and continued depression of postmortem enzyme. Control whole brain ChE activity was resilient in intact birds exposed to above freezing temperatures to 32°C for 1 day or to 25°C for 3 days.

Down-regulation of muscarinic receptors and the m3 subtype in white-footed mice by dietary exposure to parathion

The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs after dietary exposure for 14 d to sublethal concentrations of parathion in a field rodent species, and that significant though incomplete recovery in AChE and mAChRs occurs in 7 d following termination of exposure.

SEX AND STORAGE AFFECT CHOLINESTERASE ACTIVITY IN BLOOD PLASMA OF JAPANESE QUAIL

Blood plasma cholinesterase (ChE) activity is a sensitive indicator of exposure to organophosphorus and carbamate insecticides. Effects of sex and storage of samples were studied as sources of variability by treating breeding Japanese quail (Coturnix japonica) with 3 mg of dicrotophos or carbofuran per kg of body weight and comparing blood plasma ChE activities for samples collected at 1 hr postdosage and assayed fresh, after 1 and 2 days of refrigeration (4 C), and after 1, 7 and 28 days of freezing(−25 C). ChE activity of fresh control plasma was 34% (P < 0.01) higher in males than females. Male ChE activity remained essentially unchanged during storage while female ChE activity increased (P < 0.05) gradually over time under both storage conditions. In contrast, when plasma ChE activity was inhibited by either antiChE, male plasma ChE activity was depressed further than female ChE (P < 0.01) and remained constant during storage while female ChE activity continued to decrease (P < 0.05). These divergent effects of exposure to antiChE compounds and sample storage indicate extreme care should be exercised in use of blood plasma for evaluation of antiChE exposure in wild birds.