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Anticholinesterase poisoning of birds: Field monitoring and diagnosis of acute poisoning

Organophosphorus and carbamate pesticides are cholinesterase (ChE) inhibiting chemicals that have been responsible for avian die-offs. Identification of chemicals implicated in these die-offs is difficult and sometimes conclusions are solely circumstantial. However, when marked depression (inhibition) of brain ChE activity accompanies organophosphorus or carbamate residues in body tissues or ingesta, cause-effect diagnosis is enhanced. To achieve this end, normal brain ChE activity is estimated for controls of the affected species and then die-off specimens are individually evaluated for evidence of ChE inhibition. This approach to evaluation of antiChE poisoning may also be used to monitor exposure of vertebrates to field application of organophosphorus or carbamate pesticides. Problems associated with this kind of evaluation, and the main topic of this report, include variability of brain ChE activity among species, postmortem influences of ambient conditions (storage or field) on ChE activity, and differential patterns of ChE activity when inhibited by organophosphorus or carbamate compounds. Other topics discussed are the ChE assay procedure, example case reports and interpretation, and research needed for improving the diagnostic utility of ChE activity in a field situation.

Environmental Toxicology and Chemistry↗

Diagnosis of anticholinesterase poisoning in birds: Effects of environmental temperature and underfeeding on cholinesterase activity

Brain cholinesterase (ChE) activity has been used extensively to monitor exposure to organophosphorus (OP) and carbamate (CB) insecticides in wild birds. A series of factorial experiments was conducted to assess the extent to which noncontaminant-related environmental conditions might affect brain ChE activity and thereby confound the diagnosis of OP and CB intoxication. Underfeeding (restricting intake to 50% of control for 21 d or fasting for 1–3 d) or exposure to elevated temperature (36 ± 1°C for 1 d) caused only slightreductions (10–17%)in brain AChE activity in adult male Japanese quail ( Coturnix coturnix japonica ). This degree of “reduction” in brain AChE activity is considerably less than the 50% “inhibition” criterion employed in the diagnosis of insecticide-induced mortality, but nevertheless approaches the 20% “inhibition” level used as a conservative estimate of sublethal exposure to a known insecticide application.

Environmental Toxicology and Chemistry↗

Ultrastructural changes in the hepatocytes of juvenile rainbow trout and mature brown trout exposed to copper or zinc

Morphological changes in hepatocytes of mature brown trout (Salmo trutta Linnaeus) and juvenile rainbow trout (Salmo gairdneri Richardson), accompanying chronic exposures to copper and zinc, were examined by transmission electron microscopy. At a concentration of copper not inhibitory to the final stages of gonadal development or spawning of brown trout, structural alterations included contraction of mitochondria and a tendency for nuclei to be slightly enlarged. Concentrations of copper or zinc lethal to a small fraction (10% and 4%, respectively) of a population of juvenile rainbow trout exposed for 42 d during larval and early juvenile development caused hepatocyte changes in survivors indicative of a reduction in ability to maintain intracellular water and cation balance and possible intranuclear metal sequestering. Specific structural alterations included increased vesiculation of rough endoplasmic reticulum, an increase in the abundance of electron-dense particles in the nucleus, increases in the numbers of multilaminar and globular inclusions, pooling of glycogen, increased autophagocytic activity and an increase in the number of necrotic cells. At advanced stages of toxicosis (concentrations of copper or zinc lethal to approximately 50% of the juveniles exposed for 42 d during development), loss in integrity of mitochondrial membranes, rupturing of plasma and nuclear membranes, separation of granular and fibrillar nuclear components, fragmentation of endoplasmic reticulum, and extensive autophagic vacuolization were significant features of hepatocytes of surviving juvenile rainbow trout.

Environmental Toxicology and Chemistry↗

Year and age effects on residues of dieldrin and heptachlor in dead gray bats, Franklin County, Missouri— 1976, 1977, and 1978

Dead gray bats ( Myotis grisescens ) containing lethal concentrations of dieldrin were found beneath a maternity roost in Bat Cave Nos. 2 and 3, Franklin County, Missouri, in 1976, 1977 and 1978. Whereas residues of dieldrin, DDE [1,1-dichloro-2,2-bis( p -chlorophenyl)ethylene] and PCB polychlorinated biphenyls in bats appeared not to change significantly over the 3 years, residues of heptachlor-related chemicals increased in 1977 to potentially dangerous concentrations and remained elevated in 1978. Lethal brain levels of dieldrin in adult bats (geometric mean = 12.1 μg/g), compared with juvenile bats (geometric mean = 6.5 μg/g), indicated that juveniles are nearly twice as sensitive. The estimated population of gray bats (as maximum number of nonflying young) at Bat Cave Nos. 2 and 3 in 1976 and 1978 was 1,800 bats, but in 1979 no bats were present. Dieldrin, perhaps in conjunction with heptachlor, may have caused the decline and disappearance of this colony. However, dieldrin was banned in 1974 and Missouri's authorization to use heptachlor on corn expired in 1981. Furthermore, three organophosphate insecticides (chlorpyrifos, dyfonate and mocap) are to be substituted. We hope that the gray bats remaining in this area of Missouri survive the residues of dieldrin and heptachlor still in their food chains and prove to be unaffected by the new organophosphates.

Environmental Toxicology and Chemistry↗

Altered avoidance behavior of young black ducks fed cadmium

Pairs of adult black ducks ( Anas rubripes ) were fed a diet containing 0, 4 or 40 ppm cadmium as cadmium chloride. One-week-old ducklings that had been fed the same dietary concentrations of cadmium as had their parents were tested for avoidance of a fright stimulus. Ducklings fed 4 ppm cadmium ran significantly farther from the stimulus than did controls or ducklings fed 40 ppm cadmium. Such an alteration in behavior could have harmful effects on wild birds.

Environmental Toxicology and Chemistry↗

Acute toxicity of diazinon is similar for eight stocks of bobwhite

Nine-week-old bobwhite ( Colinus virginianus ) from eight different game farms were tested for their sensitivity to an acute oral exposure of technical-grade diazinon (phosphorothioic acid O,O -diethyl- O -[6-methyl-2-(l-methylethyl) -4-pyrimidinyl]ester). Extraneous variables associated with interlaboratory differences in husbandry were eliminated by incubating eggs and rearing chicks to test age for all stocks simultaneously in the same facilities at the Patuxent Wildlife Research Center. Under this single set of conditions, the responses of the eight stocks of bobwhite to diazinon were statistically inseparable, with LD 50 values varying from 13 mg/kg (95% confidence interval, 8–21 mg/kg) to 17 mg/kg (95% confidence interval, 11–25 mg/kg). The pooled LD 50 for the eight stocks was 14.7 mg/kg (95% confidence interval, 13.1–16.5 mg/kg).

Environmental Toxicology and Chemistry↗

Effects of freshwater exposure to arsenic trioxide on the parr-smolt transformation of coho salmon ( Oncorhynchus kisutch )

The effects of chronic (6 months) exposure to arsenic trioxide in fresh water on the Parr-smolt transformation of coho salmon ( Oncorhynchus kisutch ) were evaluated. Exposure to 300 μg As/L (as As 2 O 3 ) appeared to delay the onset of the normal increase in plasma thyroxine concentration and cause a transient reduction of gill Na + ,K + -ATPase activity. Fish exposed to 300 μg As/L also migrated to the sea less successfully than did nonexposed smolts, but there were no effects on the survival and growth of smolts held in 28‰ salt water for 6 months.

Environmental Toxicology and Chemistry↗

Evaluation of the potential hazard to barn owls of talon (brodifacoum bait) used to control rats and house mice

During 1980, a radiotelemetry study on barn owls ( Tyto alba ) was conducted to evaluate the secondary hazards of the anticoagulant rodenticide TALON® (containing 0.005% brodifacoum) when it was used for controlling rats ( Rattus spp.) and house mice ( Mus musculus ) on farms. Thirty‐five active nests were located in about 1,100 km 2 (430 mi 2 ) in southwestern New Jersey. Starting on June 9, 1980, radio transmitters with tail clips were attached to 26 adult nesting barn owls and 8 fledged young of the year. In one 20‐km 2 (about 8 mi 2 ) area, which was the most intensively searched, 12 active barn owl nests were located. Of all 35 active nests, 66% were in trees, 11% in barns, 8.5% in silos, 8.5% in chimneys and 6% in miscellaneous locations. Analysis of regurgitated pellets showed that meadow voles ( Microtus pennsylvanicus ) were the most common prey at all sites. Although Norway rats ( Rattus norvegicus ) and house mice occurred in some pellet collections, they made up only a small part of the diet (3.9% rats and 2% mice) of barn owls in the study area. Baiting at farms with TALON was begun on July 24, and ended on September 29, 1980. TALON was not observed to cause or implicated in barn owl mortality during the study. However, trace residues (< 0.05 ppm, the limit of determination) of brodifacoum were found in one electrocuted barn owl. Young barn owls were fledged from at least eight sites where poisoned rodents were available on the farms for at least part of the nesting and feeding period (including pre‐ and postfledging). Furthermore, radiotelemetry confirmed that at least 9, and possibly 12, of the radio‐equipped owls were present on sites posttreatment, when census evaluations showed commensal rodents were present and consuming TALON bait.

New Jersey↗

Organochlorine residues in eggs of black-crowned night herons from Colorado and Wyoming

Eggs of black-crowned night-herons Nycticorax nycticorax (L.) were collected for analysis from seven nesting sites in Colorado and Wyoming in 1979. One egg was taken per nest from as many as 20 nests per site during early incubation. The nests were marked and revisited after hatching, but before fledging, to record the number of live young. DDE was detected in all of the collected eggs (total 147) at a mean concentration of 3.1 ppm, fresh basis (residue means are geometric). Mean DDE at the seven sites varied from 1.8 to 7.6 ppm. Polychlorinated biphenyls (PCBs) were detected in 81 eggs (mean 1.0 ppm); the highest mean at any site was 2.2 ppm. Twelve other organochlorines were each detected in 1 to 30 eggs, usually at a concentration of > 1.0 ppm. Mean total organochlorines in the eggs from the seven sites ranged from 2.0 to 10.1 ppm and the median number of compounds per egg ranged from one to five. Eggshell thickness was negatively correlated (p > 0.001, r = – 0.585) with DDE levels in the 147 eggs. Average shell thickness (0.258 ± 0.030 mm) was 8.8% lower than the average thickness (0.283 ± 0.016mm) of 40 pre-DDT eggs from this region. The nesting sites with the highest DDE and total organochlorine residues in the eggs had the thinnest shells, produced the fewest young and had more nonviable eggs and dead young. At four of the seven sites, the average number of live young per nest was > 2.0, the minimum long-term mean required for population maintenance. The source of the contaminants found in the heron eggs in this study was not determined.

Environmental Toxicology and Chemistry↗

Field and laboratory evaluation of the influence of copper-diquat on apple snails in southern Florida

The recent decline of apple snail ( Pomacea paludosa ) populations in canals surrounding Loxahatchee National Wildlife Refuge in southern Florida coincided with the use of copper-diquat for the control of the aquatic weed hydrilla ( Hydrilla verticillata ). Field and laboratory studies were designed to assess the effects of copper-diquat on apple snails, which are the primary food of the endangered snail kite Rostrhamus sociabilis (formerly known as the Everglade kite). Acute toxicities (96-h LC 50 values) of Cutrine-Plus and Komeen (chelated formulations of copper) to immature apple snails were 22 and 24 μg/L, respectively. Diquat was toxic at a concentration of 1,800 μg/L and did not increase the toxicity of copper when the chemicals were used in combination. Evaluation of field samples indicated that copper concentrations were higher in detritus than in water, plants and mud, and that there was a gradient of copper concentration from the canal to the interior, the highest residues being in samples from the canal. Copper associated with detritus (up to 150 μg/g) had no effect on growth or survival of apple snails in field cage and tank studies. Also, field applications of copper-diquat to hydrilla had no effect on survival of caged adult and immature snails. Copper from field applications was rapidly taken out of solution by plants and organic material in the water and subsequently incorporated into the bottom detritus. Although the effects of repeated applications of copper-diquat and high body burdens of copper (accumulated during exposure to herbicidal treatment) on survival and reproduction of apple snails are not known, the information available indicates that treatment of hydrilla with copper-diquat was probably not responsible for the decline in the apple snail population. Application at recommended rates should pose no threat to these snails in the organically rich waters of southern Florida.

Environmental Toxicology and Chemistry↗

Subchronic mercury exposure in coturnix and a method of hazard evaluation

The sublethal toxicity of inorganic (HgCl 2 ) and organic (CH 3 HgCl) mercury chloride was studied in coturnix ( Coturnix japonica ) by feeding them mercuric compounds (CH 3 HgCl at concentrations of 0.125,0.5,2 and 8 ppm; HgCl 2 at 0.5, 2, 8 and 32 ppm) in ad libitum diets from hatching to adulthood. Differences of response to the mercurials were compared on the basis of selected indicator enzymes and plasma chemistries. Comparisons of response to equivalent concentrations of the two mercurials and dose-response relationships were made at 1,3,5,7 and 9 weeks. Changes of activity were detected for brain acetylcholinesterase (AChE) and the plasma enzymes aspartate aminotransferase (ASAT), butyrylcholinesterase (BChE), lactate dehydrogenase (LDH) and ornithine carbamoyl transferase (OCT). Changes of ASAT, LDH and OCT were then quantified by probit analysis and the mercurials were compared through their median effective concentrations (EC 50 ). This quantal procedure was based on the establishment of normal control values for each enzyme and then classifying mercury-treated outliers (more than ± 2 SD) as respondents. The EC 50 values at 9 weeks for ASAT, LDH and OCT, respectively, were 9, 3 and 63 ppm for HgCl 2 , and 5, 1 and 4 ppm for CH 3 HgCl. These results provided the basis for two hazard indices that were calculated by dividing the EC 50 into the oral LD 50 and the 5-d dietary LC 50 . Mercury also had contradictory effects on gonadal maturation in both sexes.

Environmental Toxicology and Chemistry↗

Toxicity of abate® 4E (temephos) in mallard ducklings and the influence of cold

Diets mixed to contain 0, 0.1, 1.0, 10 and 100 ppm temephos (determined chemically to contain less than 0.5, less than 0.5, 0.89, 6.0 and 59 ppm temephos, respectively) in an Abate® 4E formulation, were fed to mallard (Anas platyrhynchos) ducklings for 7 d. During this period, half of the ducklings in each dietary treatment group were housed in a heated brooder (39 to 41 °C) and half were housed in an unheated brooder (10 to 18°C). Mortality in all dietary groups in the unheated brooder was higher than in the heated brooder. High temephos-related mortality occurred in the 100 ppm group in the unheated brooder but not in any other diet-temperature groups. Ingestion of the 100 ppm temephos diet inhibited plasma Cholinesterase (ChE) activity and elevated plasma corticosterone concentration and creatine phosphokinase activity, but other selected plasma chemistries were not affected in a dose-related manner. Brain ChE activity was depressed only in the 100 ppm dietary groups; maximum inhibition of brain ChE activity was 48%. These findings suggest that diets containing up to 10 ppm temephos do not directly affect duckling survival during the first week of life and that the toxicity of 100 ppm temephos is markedly enhanced by cold.

Environmental Toxicology and Chemistry↗

Adaptation of Selenastrum capricornutum (Chlorophyceae) to copper

Selenastrum capricornutum Printz, growing in a chemically defined medium, was used as a model for studying adaptation of algae to a toxic metal (copper) ion. Cells exhibited lag‐phase adaptation to 0.8 μM total Cu (10 −12 M free ion concentration) after 20 generations of Cu exposure. Selenastrum adapted to the same concentration when Cu was gradually introduced over an 8‐h period using a specially designed apparatus that provided a transient increase in exposure concentration. Cu adaptation was not attributable to media conditioning by algal exudates. Duration of lag phase was a more sensitive index of copper toxicity to Selenastrum that was growth rate or stationary‐phase cell density under the experimental conditions used. Chemical speciation of the Cu dosing solution influenced the duration of lag phase even when media formulations were identical after dosing. Selenastrum initially exposed to Cu in a CuCl 2 injection solution exhibited a lag phase of 3.9 d, but this was reduced to 1.5 d when a CuEDTA solution was used to achieve the same total Cu and EDTA concentrations. Physical and chemical processes that accelerated the rate of increase in cupric ion concentration generally increased the duration of lag phase.

Environmental Toxicology and Chemistry↗

Cholinesterase inhibition in meadow voles (Microtus Pennsylvanicus) following field applications of orthene®

Brain acetylcholinesterase activity in field-caught meadow voles ( Microtus pennsylvanicus ) was depressed after a field-spray of Orthene® (acephate: acetylphosphoramidothioic acid O,S -dimethyl ester) by as much as 32% in 1982 and 38% in 1983. Short-term recovery was demonstrated and occurred in a time-dependent fashion in 1982. Plasma Cholinesterase levels were more variable but also were depressed. Residues were detected in vegetation samples and in the gastrointestinal tracts of exposed voles. Residues in vegetation were diluted or absent 7 to 8 d following the treatment.

Environmental Toxicology and Chemistry↗

Discrimination learning in adult bobwhite quail fed paraquat

Adult male bobwhite quail ( Colinus virginianus ) were fed a diet containing 0, 25 or 100 ppm paraquat dichloride. After 60 d on treated diets, discrimination learning was evaluated with acquisition and reversal tests. The three groups performed similarly on these tests. Dose-related histopathological lesions were not found in liver, kidney or lung tissues.

Environmental Toxicology and Chemistry↗

Organochlorine contaminants in passeriformes and other avian prey of the peregrine falcon in the western United States

About 1,150 individuals of 38 species of Passeriformes and other small birds were collected in 1980 at 36 locations in eight western U.S. states for organochlorine (OC) chemical analysis. The bird carcasses (less beak, tarsi, gastrointestinal tract, and feathers) were combined by species and location (4 to 15 specimens per sample) into 124 composited samples for analysis. Also analyzed were 77 single specimens of four species, to assess variation in residue concentrations in individual birds. The chemicals detected (greater than 0.05 ppm) were the following (in order of frequency): DDE, polychlorinated biphenyls (PCBs), hexachlorocyclohexane (HCH), heptachlor epoxide, oxychlordane, dieldrin and toxaphene. DDE accounted for 72% of total OC concentrations (ppm DDE/ppm total OCs) overall and PCBs accounted for 3%. DDE mean (geometric) residues were highest in tree swallows ( Tachycineta bicolor Vieillot), 12.0 ppm; killdeer ( Charadrius vociferus L.), 5.9 ppm; and Brewer's blackbirds ( Euphagus cyanocephalus Wagler), 2.7 ppm. Eight migratory species showed mean DDE residues 13 times higher than those in four resident species (1.3 vs. 0.1 ppm, p < 0.05), but PCB residues were similar (0.03 vs. 0.02 ppm). Insectivorous species showed higher DDE, PCB and total OC residues than did either omnivores or granivores ( p < 0.05). Males of some species showed higher residues of DDE, PCBs and total OCs than did females ( p < 0.05). DDE residues, but not PCBs, in killdeer, Brewer's blackbirds and violet-green swallows ( Tachycineta thalassina Swainson) were significantly related (0.01 < p < 0.08) to latitude and longitude of origin. Concentrations of DDE in the carcass fat of some individual tree swallows and killdeer would be in the lethal range if 15 to 20% of the stored DDE were rapidly mobilized to the brain. Samples of 13 species contained DDE concentrations (greater than 3 ppm) considered sufficient to inhibit the normal reproduction of avian predators feeding on them. The evidence suggests that potentially harmful OC concentrations are present in certain western migrants and pose an even greater hazard to avian predators such as the peregrine falcon ( Falco peregrinus Tunstall).

Environmental Toxicology and Chemistry↗

Differences in mortality among bobwhite fed methylmercury chloride dissolved in various carriers

Twelve-day-old bobwhite chicks were fed a diet containing 0, 5.4 or 20 ppm methylmercury chloride. The methylmercury chloride was added to the diet either in a dry, pulverized form or dissolved in acetone, propylene glycol or corn oil. Mortality was measured for 6 weeks, and samples of liver were saved for mercury analysis. Mortality was significantly lower in birds fed 20 ppm methylmercury chloride when acetone was the solvent. The reduced mortality could not be explained by effects of acetone on dietary level of mercury or on uptake of mercury into the body.

Environmental Toxicology and Chemistry↗

Reproduction and health of mallards fed endrin

Concentrations of 0, 1 and 3 ppm endrin in dry duck mash were fed to mallards ( Anas platyrhynchos ) starting in December, and health and reproduction were measured the following spring and summer. One male fed 3 ppm endrin died with 2.0 ppm endrin (wet weight), a diagnostically lethal level, in its brain. Birds fed 1 ppm endrin reproduced as well as, if not better than, controls. Birds fed 1 ppm endrin had significantly greater hatching success of fertile eggs than did those fed 0 or 3 ppm, and their clutches hatched significantly earlier than did those of birds fed 3 ppm. Mallards fed 3 ppm endrin appeared to reproduce more poorly than controls, but this finding must be regarded with caution because the results of statistical tests often were not significant. Endrin accumulated in eggs to a mean of 1.1 and 2.9 ppm (wet weight) when fed to hens at 1 and 3 ppm. The concentration of endrin in the carcasses of adults was similar to that in eggs, but the concentration in the fat of adults was about 4 to 7 times higher than in eggs.

Environmental Toxicology and Chemistry↗