Formalin preservation of avian blood for metal and DDE analysis
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Several characteristics of plumage, including color and molt schedule, are influenced by hormonal signals, and hence may be modified by endocrine active contaminants. If so, the functions of plumage (e.g. communication for mating or territorial defense), may be compromised by exposure to such compounds. Polychlorinated biphenyls (PCBs) are ubiquitous and persistent environmental toxins that can disrupt endocrine function in laboratory animals. Captive American kestrels (Falco sparverius) were fed PCBs (Aroclor 1242) at 6 and 60 ppm, thyroxine at 10 ppm, estradiol at 1.4 ppm, and a thyroid hormone blocker (propylthiouracil: PTU) at 2000 ppm, mixed in their normal diet. Plumage characteristics measured included: the width of the black subterminal band on the tail, brightness (a composite index of hue and saturation), reflectance from 230 - 800 nm (measured on a reflectance spectrophotometer), the pattern of feather loss and regrowth on the tail and wing, and the timing of onset and the duration of molt. PCB-treated birds did not differ from controls, but birds treated with thyroxine were significantly different from those dosed with estradiol or PTU in the width of subterminal tail bands, reflectance, and duration of molt. Thus, although hormone treatments did modify some of the plumage characteristics we measured, PCB treatments at these exposure levels did not.
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A detailed study of seasonal changes in bird populationswas made at the Patuxent Research Refuge, located between Bowie and Laurel, Maryland during the years 1936-1949. The history of the Refuge is reviewed and its physical and biological characteristlcs summarized.. The methods of study used during the investigation mcluded: periodic censuses of a representative 304-acre study area over a two-year period; a census of the breeding population of the entire. Refuge during one year; detailed population studies of representative habitats during the breeding season; censuses of the wintering population of. the entire Refuge during two years; general surveys of wintering populations for seven years; and general observations of seasonal changes in bird populatlons over a fourteen-year period, including data from an extensive banding program and from many special types of censuses. The phenology of the Refuge is described in conslderable detail throughout the year, with special attention given to major fluctuatIons in bIrd populations as correlated with climatic changes and with seasonal aspectlon. of the vegetation. The component specIes of birds m the more important migration waves are listed. Figures approximating the Refuge breeding and wmtering populations are given, while indices representing the relative abundance of bird populations, based on figures from the two-year seasonal populatlon study, were obtained for the entire year. The greatest variety of species as well as the greatest number of individuals occurred on the Refuge during the migration .periods in spring and fall, the variety of species being slightly higher in spring than in fa!l, while the population of individuals was considerably higher in fall. Wmtering and breeding populations were low and relatively stable compared to the populations at other seasons.The ecological affinities of the bird populations differed greatly from one season to another. Species characteristic of edge habitats were much more numerous in winter, while forest species were predominant in summer. Insectivorous species comprised a large proportion (40 to 60 percent) of the total population during the warmer months, but were of minor importance in winter. The greatest number of species of birds on the Refuge occurred during the population peaks of insectivorous species, while the largest number of individuals was found during the population peaks of omnivorous and herbivorous species. The population peaks of insectivorous species were found to occur much later in spring and considerably earlier in fall than the. corresponding peaks of omnivorous and herbivorous species. The Fringillidae contributed the greatest number of individuals in winter, while the Parulidae was the most important family (numerically) in summer. Water birds and marsh birds were relatively unimportant throughout the year, due to the scarcity of suitable habitats. Permanent resident species were found to vary from about one-fifth to slightly less than one-half of the total population throughout the year, although many individuals of these species were either transients or part-time residents. Summer residents and winter residents were more abundant than permanent residents during their respective periods of occurrence. During the greater part of the migration period, transient species were found to comprise only 10 to 20 percent of the total population. transient individuals of all species would account for a much larger proportion of the population at this time. After comparing the results of these investigations with similar studies in other areas, it is believed that the seasonal population changes on the Patuxent Research Refuge are fairly representative of those occurring throughout the Middle Atlantic and East-central States. Yearly variations in seasonal population changes are described and the causative factors indicated, when known. Of these, food supply and weather conditions were generally the m
Trimethyltin chloride (TMTC) and triethyltin chloride (TETC) were fed to mallard ( Anas platyrhynchos ) ducklings for 73 to 75 d, beginning when ducklings were 3 to 4 d old. Diets were mixed to contain 0, 0.5, 5 or 50 ppm Sn as either TMTC or TETC. Mortality occurred only in the 5 ppm (2/5 ducklings) and 50 ppm (5/5 ducklings, all dying within 5 d) TMTC groups. Death was preceded by cephalic tremors, lethargy and ataxia; these clinical signs also were observed in surviving ducklings from the 5 ppm TMTC group, but not the 0.5 ppm TMTC group. Ducklings fed 5 and 50 ppm Sn as TMTC exhibited degeneration of the large neurons of the pons, medulla oblongata, gray matter of the spinal cord and pyramidal cells of the cerebral cortex. TETC-fed ducklings showed few signs of clinical toxicosis. Ducklings fed 50 ppm Sn as TETC exhibited mild to severe vacuolization of the white matter of the brain and spinal cord; these lesions were not present in ducklings fed 5 ppm Sn as TETC. Liver, spleen, kidney, thymus, bursa of Fabricius and skeletal muscle presented no abnormal histopathology for either organotin compound. Hematocrit, hemoglobin and plasma aspartate aminotransferase; alanine aminotransferase; lactate dehydrogenase; alkaline phosphatase; and cholinesterase values were determined for the 5- and 50- ppm TMTC and TETC groups, and were not different from those of controls ( p < 0.05). Lesions and clinical signs of toxicosis in ducklings were consistent with those described for mammals exposed to TMTC and TETC. The effect threshold for TMTC in ducklings appears to exceed current environmental concentrations. Environmental concentrations of TETC have not been reported.
Hepatic cytochrome P450-associated monooxygenase activities were measured in 11-day-old nestling black-crowned night-herons (Nycticorax nycticorax) collected from a reference site (next to the Chincoteague National Wildlife Refuge, Virginia) and three polluted sites (Cat Island, Green Bay, Lake Michigan, Wisconsin; Bair Island, San Francisco Bay, California; West Marin Island, San Francisco Bay, California). Activities of arylhydrocarbon hydroxylase (AHH) and benzyl-oxyresorufin-O-dealkylase (BROD) weremodestly elevated (<three-fold) in nestlings from polluted sites. Concentrations of p,p?DDE, other organochlorinepesticides and total PCBs in nestlings were greatest at contaminated sites, although much lower than found in concurrently collected eggs and pipping embryos. At these low pollutant concentrations there was little correlation between monooxygenase activity and contaminant levels in nestlings. These observations markedly contrast the pronounced monooxygenase induction (up to 85-fold) and its significant correlation with total PCBs, arylhydrocarbon receptor-active PCB congeners and toxic equivalents in concurrently collected night-heron embryos that were often siblings of the nestlings. The present findings suggest that cytochrome P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.
Nutritional and bioenergetic interactions influence exposure to environmental chemicals and may affect the risk realized when wildlife are exposed in the field. Here, food-chain analysis focuses on prairie voles (Microtus ochrogaster) and the evaluation of chemical risks associated with paraquat following 10-d dietary exposures. Reproductive effects were measured in 60-d trials that followed exposures to paraquat-tainted feed: control (untainted feed); 21 mg paraquat/kg feed; 63 mg paraquat/kg feed; and feed-restricted control (untainted feed restricted to 60% baseline consumption). Reproductive success was evaluated in control and treated breeding pairs, and a preliminary bioenergetics analysis was completed in parallel to derive exposure dose. Although reproductive performance differed among groups, feed-restriction appeared to be the dominant treatment effect observed in these 10-d feeding exposure/limited reproductive trials. Exposure dose ranged from 3.70-3.76 to 9.41-11.51 mg parquat/kg BW/day at 21 and 63 mg paraquat/kg feed stock exposures, respectively. Energetic doses as ug paraquat/kcal yielded preliminary estimates of energetic costs associated with paraquat exposure, and were similar within treatments for both sexes, ranging from 4.2-5.5 and 13.1-15.0 ug paraquat/kcal for voles exposed to 21 mg/kg feed stock and 63 mg/kg feed stock, respectively. Given the increasing likelihood that environmental chemicals will be found in wildlife habitat at 'acceptable levels', the critical role that wildlife nutrition plays in evaluating ecological risks should be fully integrated into the assessment process. Tools applied to the analysis of risk must gain higher resolution than the relatively crude methods we currently bring to the process.
Lead is a known environmental toxicant, and poisoning resulting from the ingestion of lead shot has been well-documented in many species of waterfowl. However, much less is known regarding exposure and effects of free environmental lead in species of birds other than waterfowl. In an attempt to evaluate toxicity of lead to herons and to determine the usefulness of feathers as a non-invasive exposure-monitoring tool, black-crowned night-heron nestlings were dosed with lead to determine its distribution among tissues, and its effects on biochemical biomarkers, growth, and survival. Five-day-old heron nestlings (one per nest) at Chincoteague Bay, Virginia were given a single intra-peritoneal injection of dosing vehicle (control; N=7) or one of three lead solutions (as lead nitrate) (10, 50, or 250 mg/kg body weight of nestling; N=7 per dose) chosen to represent levels below, at, and above those found in moderately-polluted environments. All nestlings treated with lead exhibited dose-dependent inhibition of delta-aminolevulinic acid dehydratase (ALAD) activity compared to controls, and nestlings treated with the highest concentration showed a reduced carcass weight compared to controls. Of several measures of oxidative stress that were analyzed, significant differences were found between low- and high-dosed nestlings in hepatic total thiol and protein-bound sulfhydryl concentrations. No differences in survival were detected between dosed nestlings, controls, or uninjected siblings. Lead concentrations in several matrices, including feathers, are being determined to assess distribution among tissues and will also be examined for relationships with measures of effect.
Six month old Lesser Scaup and nestling Tree Swallows were injected intraperitoneally with beta-naphthoflavone (BNF) or vehicle. Nestling Tree Swallows were also collected from five sites with differing levels of contaminants. Liver samples were taken and stored at -80C until microsome preparation and monooxygenase (MO) assay. Skin and heart samples were placed in buffered formalin until immunohistochemical (IMHC) analysis for cytochrome P4501A (CYP1A). Scaup treated with BNF at 20 or 100 mg/kg body weight showed approximately 20- to 65-fold increases in four MOs. Responses of two of the four MOs were as high at 20 mg/kg as at 100mg/kg. There was no IMHC response in the vehicle-injected ducks, while in skin the IMHC response was the same for both dose levels of BNF and in heart there was response in two of four samples at 20 mg/kg and in all five samples at 100mg/kg. Tree Swallows injected with BNF at 100, but not at 20 mg/kg showed significant increases (ca.5-fold) in two MO activities. There was no IMHC response in control swallows. In skin and heart there were IMHC responses in one of five swallows at 20 mg/kg and four of five swallows at 100mg/kg. There was poor correlation between individual skin IMHC responses and MO activities and PCB concentrations in 47 field-collected Tree Swallow samples, but 14 of the 16 skin samples with positive IMHC responses were from the location with the highest MO activities and PCB concentrations. Although present data do not allow construction of significant dose response curves, the responses in skin make it well worth continuing study on this potential nonlethal technique for biomonitoring contaminant exposure of birds.
Polychlorinated biphenyls are known to cause adverse health effects to biological systems; however, limited data is available on their effects on the immune system of wild species. Previous work by our lab found that 4 and 6-week old white-footed mice (Perornyscus leucopus) born from dams injected with a single dose (300 mg/kg) of Aroclor 1254, had altered immunological, hematological, and biochemical responses. The present study examines various immunological parameters of 22-week old white footed mice born from dams chronically exposed to Aroclor 1254 at concentrations equivalent to those at contaminated sites. Females were fed diets containing either Aroclor 1254 in corn oil or corn off only, for 3 months, then bred; pups were maintained on the same diets as their mothers. At 22 weeks of age, 31 of the young Peromyscus were analyzed. Body and organ weights were taken and immune function was evaluated by assessing blood profiles, cellularity of thymus and spleen, antibody response to the antigen DNP-KLH, and the in vitro proliferative response to the T-cell mitogen Conconavalin A (Con A). Liver weights and liver to body weight ratios in the treated mice were significantly higher compared to controls, while the combined weights of the adrenal glands were significantly lower. In addition, the number of thymocytes in the treated mice was significantly lower than that of the controls; however, thymocytes of treated mice had a higher degree of proliferation to Con A. Taken together, these results and those obtained from our previous study, indicate that monitoring of vulnerable immunological parameters in white-footed mice may be a useful indicator of exposure.
High concentrations of mercury from past mining activities have accumulated in the food chain of fish-eating birds nesting along the mid to lower Carson River. Activities of nine plasma and tissue enzymes, and concentrations of other plasma and tissue constituents were measured for black-crowned night-heron, Nycticorax nycticorax, (BCNH) and snowy egret, Egretta thula, (SE) nestlings from two high mercury sites and one low mercury site. Geometric mean blood Hg concentrations for BCNHs at the high mercury sites were 2.6 and 2.8 ppm (ww) and 0.6 ppm at the low mercury site. Blood concentrations for SEs were 3.6 and 1.9 ppm at the high mercury sites and 0.7 ppm at the low mercury site. In BCNHs plasma glutathione peroxidase (GSH peroxidase) activity was lower in both high mercury sites relative to the low mercury site. Butyryl cholinesterase (BuChe), ALT, glutathione reductase (GSSG-reductase) and LDH-L activities were lower in one high mercury site. In SEs significant differences were lower BuChe and LDH-L, but elevated GGT activities. Evidence of renal stress in both species at high mercury sites included increased plasma uric acid, blood urea nitrogen, and creatinine concentrations as well as oxidative stress in the kidney tissue itself where oxidized glutathione increased. A number of the mercury site-related effects, including decreased plasma GSH-peroxidase and hepatic G-6-PDH activities, higher GSSG-reductase activity, and lower hepatic concentrations of reduced thiols have been reported in methylmercury feeding studies with great egrets and mallards. These findings suggest the utility of herons and egrets for monitoring mercury sites.
Golden Bear Oil or GB-1111 is a petroleum distillate that is used throughout the United States as a larvicide for mosquito pupae. The oil forms a barrier at the air-water interface, which suffocates air-breathing insects. There are few published studies on non-target effects of GB-1111 but the product label warns that ?GB-1111 is toxic to fish and other aquatic organisms.? Fertile eggs of mallards (Anas platyrhynchos) and bobwhite (Colinus virginianus) were incubated in the laboratory, and treated on days 4 or 11 of incubation with external applications equivalent to either 0, 1/3, 1, 3, or 10 times the maximum rate (5 gal/A) of field application of GB-1111. Hatching success was significantly reduced in mallards treated on day 4 or day 11 at 3 and 10 times the maximum field application, with a calculated approximate LD50 of 1.9 times the maximum field application. Most mortality occurred within a week of treatment. Hatching success of bobwhite was only reduced at the highest level of treatment. Other effects at this level in bobwhite included a significant increase in incidence of abnormal embryos/ hatchlings, lower body and liver weights of hatchlings and a two-fold increase in hepatic microsomal P450-associated monooxygenase activity (EROD) in hatchlings. Recommended rates of field application of GB-1111 are potentially toxic to mallard embryos, especially under conditions of larvicide drift or spray overlap, but unlikely to impair the survival or development of bobwhite embryos.
Several characteristics of plumage, including color and molt schedule, are influenced by hormonal signals, and hence may be modified by endocrine active contaminants. If so, the functions of plumage (e.g. communication for mating or territorial defense), may be compromised by exposure to such compounds. Polychlorinated biphenyls (PCBs) are ubiquitous and persistent environmental toxins that can disrupt endocrine function in laboratory animals. Captive American kestrels (Falco sparverius) were fed PCBs (Aroclor 1242) at 6 and 60 ppm, thyroxine at 10 ppm, estradiol at 1.4 ppm, and a thyroid hormone blocker (propylthiouracil: PTU) at 2000 ppm, mixed in their normal diet. Plumage characteristics measured included: the width of the black subterminal band on the tail, brightness (a composite index of hue and saturation), reflectance from 230 - 800 nm (measured on a reflectance spectrophotometer), the pattern of feather loss and regrowth on the tail and wing, and the timing of onset and the duration of molt. PCB-treated birds did not differ from controls, but birds treated with thyroxine were significantly different from those dosed with estradiol or PTU in the width of subterminal tail bands, reflectance, and duration of molt. Thus, although hormone treatments did modify some of the plumage characteristics we measured, PCB treatments at these exposure levels did not.
The robust redhorse is a ?Species-at-Risk? in the lower Oconee River, GA. The population is composed of aging adults with little natural recruitment. Factors contributing to the loss of early-life stages are unknown, but contaminants associated with fine sediments may play a role. The objectives of this study were to determine toxicities of sediments and pore waters from the Oconee River to early-life stages of robust redhorse and to establish toxic thresholds of metals (Cd, Cu, Mn, Zn) and ammonia, elements potentially threatening this species. Depositional sediments were collected from the only known spawning site and three sites downstream of major tributaries. Sediment pore waters were extracted in the laboratory from all sites and in situ at two sites. Toxicity tests with sediments, pore waters and metal solutions were initiated with eggs, yolk-sac fry and swim-up fry to determine effects on the life stage initially exposed as well as effects manifested in later developmental stages. Survival and growth were test endpoints, and toxicity was observed in both sediments and pore waters. Although the yolk- sac stage was the most sensitive across all tests, sediment toxicity was elicited only in tests initiated with eggs that developed through the yolk-sac stage. Toxicity appeared to be due to Mn in sediment and pore water exposures, but was more prevalent in pore waters. Sediment handling and the associated effects on redox potential contributed to the elevated concentrations of Mn in pore waters. Pore waters extracted in situ had significantly less Mn and were less toxic than laboratory-extracted pore waters. These data suggest that sediment-associated Mn may impact early-life stages of robust redhorse in the Oconee River.
Rapid Bioassessment Protocols (RBP) and the Sediment Quality Triad (SQT) were used to evaluate the biological effects of a municipal waste-water treatment facility (WWTF) on a small southern stream. During major storm events, raw sewage from the WWTF is released directly into the stream. The headwaters of the stream also receive non-point surface runoff from urban areas. RBP analyses, which included benthos, fish and habitat evaluations, and SQT, including the benthos (from the RBP), contaminant analyses (metals, organochlorine pesticides, PCBs and PAHs) andl toxicity tests of depositional sediment (exposures of Hyalella azteca to solid-phase sediment and pore water) were conducted at five sites on the stream (two upstream of the WWTF and three downstream). The stream has been channelized throughout its entire length, resulting in high, unstable banks, degraded stream channel, and unstable substratum. RBP analyses indicated that the two stations upstream of the WWTF were degraded due to poor physical habitat quality (unstable benthic substratum and lack of fish habitat). The SQT also showed reduced habitat quality at the two stations above the WWTF, but the cause was attributed to high concentrations of PAHs and metals in the sediments. The increased discharge and stabilized base flow provided by the WWTF improved habitat quality downnstream, although conditions were still impaired due to the habitat alteration. Though the causes of degradation were attributed to different factors (physical habitat vs. contamination), there was close concordance between the RBP and SQT in identifying the degraded sites in this stream. The combination of these two procedures provides a robust examination of environmental quality utilizing the weight of evidence approach.
Associations between contaminant exposure and liver and skin tumor prevalence were evaluated in brown bullheads (Ameiurus nebulosus) from the tidal Potomac River watershed. Thirty bullheads (>260 mm) were collected from (1) Quantico embayment, near a Superfund site which released organochlorine contaminants; (2) Neabsco Creek, a tributary with petroleum inputs from runoff and marinas; and (3) Anacostia River (spring and fall), an urban tributary designated as a Chesapeake Bay Region of Concern, contaminated with PCBs, PAHs, and organochlorine pesticides. Fish were collected from the Tuckahoe River, as a reference. Cytochrome P450 activity, bile PAH metabolites, and muscle organochlorine pesticide/PCB concentrations were measured in randomly-selected individuals and sediment contaminants were analyzed. We found statistically significant differences in liver tumor prevalences: Anacostia (spring)-50%, Anacostia (fall)-60%, Neabsco-17%, Quantico-7%, Tuckahoe-10%. Skin tumor prevalences were significantly different: Anacostia (spring)-37%, Anacostia (fall)-10%, Neabsco-3%, Quantico-3%, Tuckahoe-0%. Tumor prevalence in Anacostia bullheads warrants concern and was similar to those at highly contaminated sites in the Great Lakes. There was evidence of higher PAH exposure in Anacostia fish but a cause-effect linkage cannot be established. Fish tumor surveys, with histopathological examination of internal and external organs, are recommended for monitoring the status of Regions of Concern.
Birds have extremely varied reproductive strategies. As such, the impact of endocrine disrupting chemicals (EDCs) can greatly differ across avian species. Precocial species, such as Japanese quail appear to be most sensitive to EDC effects during embryonic development, particularly sexual differentiation. A great deal is known about the ontogeny of Japanese quail (Coturnix japonica) relative to endocrine, neuro-endocrine, and behavioral components of reproduction. Therefore, this species provides an excellent model for understanding effects of EDCs on reproductive biology with exposure at specific stages of the life cycle. The purpose of these experiments was to conduct a 1- or 2- generation experiment with positive or negative control chemicals and to determine changes in selected end points. Japanese quail embryos were exposed to estradiol benzoate (EB; positive control) in a 2-generation design or to fadrozole (FAD; negative control) in a 1-generation design. Embryonic EB treatment resulted in significant reductions (p< 0.5) in hen day production (90.2 vs 54.1; control vs EB, resp.) and fertility (85.3 vs 33.4%, control vs EB, resp.). Males showed sharply reduced courtship and mating behaviors as well as increased lag time (26 vs 148 sec; control vs EB) in behavioral tests. Fadrozole exposure resulted in reduced hatchability of fertile eggs, particularly at higher doses. There were no significant effects on courtship and mating behavior of males although males showed an increased lag time in their responses, nally, a behavioral test for studying motor and fear responses in young chicks was used; chicks exposed to an estrogenic pesticide (methoxychlor) showed some deficits. In summary, the use of appropriate and reliable end points that are responsive to endocrine disruption are critical for assessment of EDCs. Supported in part by EPA grant R826134.
Although lead can attain high concentrations in feathers, interpretation of the biological significance of this phenomenon is difficult. As part of an effort to develop and validate non-invasive methods to monitor contaminant exposure in free-ranging birds, lead uptake by feathers of nestling black-crowned night-herons (Nycticorax nycticorax) was evaluated in a controlled exposure study. Four to six day-old heron nestlings (one/nest) at Chincoteague Bay, Virginia, received a single intraperitoneal injection of dosing vehicle (control; n=7) or a dose of lead nitrate in water (0.01, 0.05, or 0.25 mg Pb/g body weight of nestling; n=6 or 7/dose) chosen to yield feather lead concentrations found at low to moderately polluted sites. Nestlings were euthanized at 15 days of age. Lead accumulation in feathers was associated with concentrations in bone, kidney, and liver (r = 0.32 - 0.74, p < 0.02), but exhibited only modest dose-dependence. Blood delta-aminolevulinic acid dehydratase activity was inhibited by lead, although effects on other biochemical endpoints were marginal. Tarsus growth rate was inversely related to feather lead concentration. Culmen growth rate was depressed in nestlings treated with the highest dose of lead, but not correlated with feather lead concentration. These findings provide evidence that feathers of nestling herons are a sensitive indicator of lead exposure and have potential application for the extrapolation of lead concentrations in other tissues and the estimation of environmental lead exposure in birds.