Embryonic effects of an environmentally relevant PCB mixture in the domestic chicken.

Studies were conducted to develop methods to assess the effects of a complex mixture of polychlorinated biphenyls (PCBs) in the domestic chicken (Gallus domesticus). Treatments were administered by egg injection to compare embryonic effects of an environmentally relevant PCB congener mixture in the domestic chicken over a range of doses. Chicken eggs were injected with the PCB mixture with a profile similar to that found in avian eggs collected on the upper Hudson River, New York, USA, at doses that spanned 0 to 98 µg/g egg. Eggs were hatched in the laboratory to ascertain hatching success. In the domestic chicken, the median lethal dose was 0.3 µg/g. These data demonstrate adverse effects of an environmentally relevant PCB mixture and provide the basis for further work using in vitro and other models to characterize the potential risk to avian populations. Environ Toxicol Chem 2018;37:2513-2522. © 2018 SETAC.

Changes in white cell estimates and plasma chemistry measurements following oral or external dosing of double-crested cormorants, Phalacocorax auritus, with artificially weathered MC252 oil.

Scoping studies were designed whereby double-crested cormorants (Phalacocorax auritus) were dosed with artificially weathered Deepwater Horizon (DWH) oil either daily through oil injected feeder fish, or by application of oil directly to feathers every three days. Preening results in oil ingestion, and may be an effective means of orally dosing birds with toxicant to improve our understanding of the full range of physiological effects of oral oil ingestion on birds. Blood samples collected every 5-6 days were analyzed for a number of clinical endpoints including white blood cell (WBC) estimates and differential cell counts. Plasma biochemical evaluations were performed for changes associated with oil toxicity. Oral dosing and application of oil to feathers resulted in clinical signs and statistically significant changes in a number of biochemical endpoints consistent with petroleum exposure. In orally dosed birds there were statistically significant decreases in aspartate amino transferase (AST) and gamma glutamyl transferase (GGT) activities, calcium, chloride, cholesterol, glucose, and total protein concentrations, and increases in plasma urea, uric acid, and phosphorus concentrations. Plasma electrophoresis endpoints (pre-albumin, albumin, alpha-2 globulin, beta globulin, and gamma globulin concentrations and albumin: globulin ratios) were decreased in orally dosed birds. Birds with external oil had increases in urea, creatinine, uric acid, creatine kinase (CK), glutamate dehydrogenase (GLDH), phosphorus, calcium, chloride, potassium, albumin, alpha-1 globulin and alpha-2 globulin. Decreases were observed in AST, beta globulin and glucose. WBC also differed between treatments; however, this was in part driven by monocytosis present in the externally oiled birds prior to oil treatment.

Assessing effects of environmental chemicals on neuroendocrine systems: potential mechanisms and functional outcomes.

Environmental pollutants encompass a vast array of compounds. Most studies in birds have focused on toxicological effects, with little attention to non-lethal effects. Consequently, it has proven difficult to assess potential risk associated with exposure to endocrine disrupting chemicals (EDCs). Assessing potential adverse effects due to exposure is further complicated by the great variation that occurs across avian species. These include variations in reproductive strategies, life span, sexual differentiation, and migration. Differences in reproductive strategies, particularly in the developmental patterns and mechanisms for precocial and altricial chicks, predispose birds to wide variations in response to steroids and steroid-like EDCs. We have investigated the effects of EDCs in precocial birds including Japanese quail (Coturnix japonica) and mallard ducks (Anas platyrhynchos) as well as in wild altricial songbirds. Studies in Japanese quail characterized endogenous steroid hormone changes during development and have demonstrated that the developing embryo uses the yolk as a 'steroid hormone depot'. It appears that actual embryonic exposure is quantitatively lower than indicated by the treatment in egg injections and that the true amount of compound necessary for bioactivity may be quite low relative to the actual dosage delivered. Additionally, embryonic exposure to specific EDCs adversely affected sexual differentiation in quail, especially impacting male sexual behavior as well as neural systems, immune response, and thyroid hormones. Many of these studies considered single compounds; however, wild birds are exposed to complex mixtures and multiple compounds. We tested complex mixtures of polychlorinated biphenyls (PCBs) at concentrations that bracketed those found in eggs in contaminated regions. Results indicated that the predictive value of the toxic equivalency (TEQ), based on comparative activation of the aryl hydrocarbon receptor (AhR) relative to dioxin was not as accurate as expected. We discuss the potential of developing an endocrine disruption index (EDI) to bridge the inconsistencies observed between responses predicted by the TEQ and those observed in vivo following exposure to EDCs. Further, we will discuss how an EDI would complement the adverse outcome pathways analyses to consider the range of effects of endocrine disruptors in birds.

Absorption and biotransformation of polybrominated diphenyl ethers DE-71 and DE-79 in chicken (Gallus gallus), mallard (Anas platyrhynchos), American kestrel (Falco sparverius) and black-crowned night-heron (Nycticorax nycticorax) eggs.

We recently reported that air cell administration of penta-brominated diphenyl ether (penta-BDE; DE-71) evokes biochemical and immunologic effects in chicken (Gallus gallus) embryos at very low doses, and impairs pipping (i.e., stage immediately prior to hatching) and hatching success at 1.8mugg(-1) egg (actual dose absorbed) in American kestrels (Falco sparverius). In the present study, absorption of polybrominated diphenyl ether (PBDE) congeners was measured following air cell administration of a penta-BDE mixture (11.1mug DE-71g(-1) egg) or an octa-brominated diphenyl ether mixture (octa-BDE; DE-79; 15.4mug DE-79g(-1) egg). Uptake of PBDE congeners was measured at 24h post-injection, midway through incubation, and at pipping in chicken, mallard (Anas platyrhynchos), and American kestrel egg contents, and at the end of incubation in black-crowned night-heron (Nycticorax nycticorax) egg contents. Absorption of penta-BDE and octa-BDE from the air cell into egg contents occurred throughout incubation; at pipping, up to 29.6% of penta-BDE was absorbed, but only 1.40-6.48% of octa-BDE was absorbed. Higher brominated congeners appeared to be absorbed more slowly than lower brominated congeners, and uptake rate was inversely proportional to the log K(ow) of predominant BDE congeners. Six congeners or co-eluting pairs of congeners were detected in penta-BDE-treated eggs that were not found in the dosing solution suggesting debromination in the developing embryo, extraembryonic membranes, and possibly even in the air cell membrane. This study demonstrates the importance of determining the fraction of xenobiotic absorbed into the egg following air cell administration for estimation of the lowest-observed-effect level.

Effects of estradiol on the development of the bursa of Fabricius in Japanese quail.

Effects of androgens on the development of the bursa of Fabricius are better understood than those of estradiol, despite the known sensitivity of the bursa to estradiol early in embryogenesis. The goal of this study was to determine the effects of one-time yolk injections of estradiol at day 4 of incubation on the development of the bursa and spleen as indices of treatment effects on the immune system. Follicle size and numbers in hatchling bursas were significantly reduced at 50 and 500 microg/egg, respectively. Additionally, distorted plicae and thicker epithelial layers surrounding the plicae were observed in day-old chicks at the same treatment levels. Adult bursas from birds embryonically exposed to estrogen were significantly larger than controls, suggesting an inhibition of natural bursal regression. Although estradiol altered the development of the bursa, the spleen appeared to be unaffected. The observed effects of estradiol on the development of the bursa indicate that this lymphoid organ may be a target for developmental disruption by estrogenic endocrine disrupting chemicals, though long-term consequences of embryonic exposure on immune function remain unknown.

Toxicity of polybrominated diphenyl ethers (DE-71) in chicken (Gallus gallus), mallard (Anas platyrhynchos), and American kestrel (Falco sparverius) embryos and hatchlings.

Embryonic survival, pipping and hatching success, and sublethal biochemical, endocrine, and histological endpoints were examined in hatchling chickens (Gallus gallus), mallards (Anas platyrhynchos), and American kestrels (Falco sparverius) following air cell administration of a pentabrominated diphenyl ether (penta-BDE; DE-71) mixture (0.01-20 microg/g egg) or polychlorinated biphenyl (PCB) congener 126 (3,3',4,4',5-pentachlorobiphenyl; 0.002 microg/g egg). The penta-BDE decreased pipping and hatching success at concentrations of 10 and 20 microg/g egg in kestrels but had no effect on survival endpoints in chickens or mallards. Sublethal effects in hatchling chickens included ethoxyresorufin-O-dealkylase (EROD) induction and histological changes in the bursa, but these responses were not observed in other species. Polychlorinated biphenyl congener 126 (positive control) reduced survival endpoints in chicken and kestrel embryos and caused sublethal effects (EROD induction, reduced bursal mass and follicle size) in chickens. Mallards were clearly less sensitive than the other species to administered penta-BDE and PCB 126. In a second experiment, the absorption of penta-BDE (11.1 microg/g egg, air cell administered during early development) into the contents of chicken and kestrel eggs was determined at various intervals (24 h postinjection, midincubation, and pipping). By pipping, 29% of the penta-BDE administered dose was present in the egg contents in chickens, and 18% of the administered dose was present in kestrel egg contents. Based on uptake in kestrels, the lowest-observed-effect level on pipping and hatching success may be as low as 1.8 microg total penta-BDE/g egg, which approaches concentrations detected in eggs of free-ranging birds. Because some penta-BDE congeners are still increasing in the environment, the toxic effects observed in the present study are cause for concern in wildlife.

Egg incubation position affects toxicity of air cell administered polychlorinated biphenyl 126 (3,3',4,4',5-pentachlorobiphenyl) in chicken (Gallus gallus) embryos.

The avian egg is used extensively for chemical screening and determining the relative sensitivity of species to environmental contaminants (e.g., metals, pesticides, polyhalogenated compounds). The effect of egg incubation position on embryonic survival, pipping, and hatching success was examined following air cell administration of polychlorinated biphenyl (PCB) congener 126 (3,3',4,4',5-pentachlorobiphenyl [PCB 126]; 500-2,000 pg/g egg) on day 4 of development in fertile chicken (Gallus gallus) eggs. Depending on dose, toxicity was found to be up to nine times greater in vertically versus horizontally incubated eggs. This may be due to enhanced embryonic exposure to the injection bolus in vertically incubated eggs compared to more gradual uptake in horizontally incubated eggs. Following air cell administration of PCB 126, horizontal incubation of eggs may more closely approximate uptake and toxicity that has been observed with naturally incorporated contaminants. These data have implications for chemical screening and use of laboratory data for ecological risk assessments.

Immunotoxicity of trenbolone acetate in Japanese quail.

Trenbolone acetate is a synthetic androgen that is currently used as a growth promoter in many meat-exporting countries. Despite industry laboratories classifying trenbolone as nonteratogenic, data showed that embryonic exposure to this androgenic chemical altered development of the immune system in Japanese quail. Trenbolone is lipophilic, persistent, and released into the environment in manure used as soil fertilizer. This is the first study to date to assess this chemical's immunotoxic effects in an avian species. A one-time injection of trenbolone into yolks was administered to mimic maternal deposition, and subsequent effects on the development and function of the immune system were determined in chicks and adults. Development of the bursa of Fabricius, an organ responsible for development of the humoral arm of the immune system, was disrupted, as indicated by lower masse, and smaller and fewer follicles at day 1 of hatch. Morphological differences in the bursas persisted in adults, although no differences in either two measures of immune function were observed. Total numbers of circulating leukocytes were reduced and heterophil-lymphocyte ratios were elevated in chicks but not adults. This study shows that trenbolone acetate is teratogenic and immunotoxic in Japanese quail, and provides evidence that the quail immune system may be fairly resilient to embryonic endocrine-disrupting chemical-induced alterations following no further exposure posthatch.

Toxicity and hazard of vanadium to mallard ducks (Anas platyrhynchos) and Canada geese (Branta canadensis).

A recent Canada goose (Branta canadensis) die-off at a petroleum refinery fly ash pond in Delaware was attributed to vanadium (V) toxicity. Because of the paucity of V toxicity data for wild birds, a series of studies was undertaken using the forms of V believed to have resulted in this incident. In 7-d single oral dose trials with mallard drakes (Anas platyrhynchos), the estimated median lethal dose (LD50) for vanadium pentoxide was 113 mg/kg body weight, while the LD50 for sodium metavanadate was 75.5 mg/kg. Sodium metavanadate was found to be even more potent (LD50 = 37.2 mg/kg) in male Canada geese. The most distinctive histopathological lesion of both forms of V was lympho-granulocytic enteritis with hemorrhage into the intestinal lumen. Vanadium accumulation in liver and kidney was proportional to the administered dose, and predictive analyses based on these data suggest that V concentrations of 10 microg/g dry weight (dw) in liver and 25 microg/g dw in kidney are associated with mortality (>90% confidence that exposure is >LD50) in mallards acutely exposed to sodium metavanadate. Chronic exposure to increasing dietary concentrations of sodium metavanadate (38.5 to 2651 ppm) over 67 d resulted in V accumulation in liver and kidney (25.2 and 13.6 microg/g dw, respectively), mild intestinal hemorrhage, blood chemistry changes, and evidence of hepatic oxidative stress in mallards, although some of these responses may have been confounded by food avoidance and weight loss. Dietary exposure of mallards to 250 ppm sodium metavanadate for 4 wk resulted in modest accumulation of V in liver and kidney (<5 microg/g dw) and mild intestinal hemorrhage. Based on these data and other observations, it is unlikely that chronic low-level dietary exposure to V poses a direct lethal hazard to wildlife. However, point sources, such as the V-laden fly ash pond encountered by geese at the petroleum refinery in Delaware, may pose a significant hazard to water birds.