Keith A. Grasman

Reproductive and Physiological Effects of Environmental Contaminants in Fish-Eating Birds of the Great Lakes: A Review of Historical Trends

During the 1950s and 1960s, reproductive failures and population declines were observed in fish-eating birds such as gulls, terns, cormorants, herons, and eagles in the Great Lakes. DDE-induced eggshell thinning contributed to these declines, but other factors such as embryo toxicity also were implicated. With reduced releases of many pollutants, reproduction recovered in some species. However, biomonitoring during the 1980s and 1990s indicates continuing effects at highly contaminated sites. Improved analytical techniques and bioassays have allowed the assessment of the total dioxin-like toxicity of complex mixtures of organochlorines (TCDD-equivalents). Developmental defects such as embryo mortality, deformities, and edema have been associated with dioxin-like PCBs in several avian species. Improved biochemical techniques have allowed the measurement of biomarkers that detect physiological alterations associated with contaminants. Specific biomarkers studied in Great Lakes birds include cytochrome P-450 monooxygenases, highly carboxylated porphyrins, thyroxine, vitamin A, and immune function. Reproductive and physiological alterations are associated with population-level effects in Caspian terns and bald eagles that feed on highly contaminated fish. Biomonitoring using biomarkers and population-level measures in fish-eating birds will continue to be important for assessing the effects of contaminants on the Great Lakes ecosystem.

Assessing Immunological Function in Toxicological Studies of Avian Wildlife

Abstract Laboratory and field studies have demonstrated that the immune system is sensitive to environmental contaminants. Testing protocols have been developed to screen for immunotoxic effects and elucidate mechanisms of toxicity in laboratory rodents. Similar methods have been applied to wildlife species in captivity and the wild. Several epizootics in wildlife have been associated with elevated exposure to contaminants. This paper discusses immunotoxicological techniques used in studies of avian wildlife. Measurements of immunological structure include peripheral white blood cell counts and the mass and cellularity of immune organs such as the thymus, spleen, and bursa of Fabricius. While contaminants can alter these measures of immunological structure, such measures do not directly assess how the immune system functions, i.e., responds to specific challenges. The two most commonly used in vivo immune function tests in birds are the phytohemagglutinin (PHA) skin response for T cell-mediated immunity and the sheep red blood cell (SRBC) hemagglutination assay for antibody-mediated immunity. In vitro tests of immune function in avian wildlife include proliferation of lymphocytes in response to various mitogens and phagocytosis of fluorescent particles by monocytes. While optimization of in vitro techniques for wildlife species is often time-consuming, these assays usually require only a single blood sample and can elucidate mechanisms of toxicity. In immunological studies of wildlife, investigators should consider factors that may influence immune responses, including age, body condition, date, developmental stage of the immune system, and time required for the progression of immune responses.

Aquatic Pollution-Induced Immunotoxicity in Wildlife Species

The potential for chemicals to adversely affect human immunologic health has traditionally been evaluated in rodents, under laboratory conditions. These laboratory studies have generated valuable hazard identification and immunotoxicologic mechanism data; however, genetically diverse populations exposed in the wild may better reflect both human exposure conditions and may provide insight into potential immunotoxic effects in humans. In addition, comparative studies of species occupying reference and impacted sites provide important information on the effects of environmental pollution on the immunologic health of wildlife populations. In this symposium overview, Peter Hodson describes physiological changes in fish collected above or below the outflows of paper mills discharging effluent from the bleaching process (BKME). Effects attributable to BKME were identified, as were physiological changes attributable to other environmental factors. In this context, he discussed the problems of identifying true cause and effect relationships in field studies. Mohamed Faisal described changes in immune function of fish collected from areas with high levels of polyaromatic hydrocarbon contamination. His studies identified a contaminant-related decreases in the ability of anterior kidney leukocytes to bind to and kill tumor cell line targets, as well as changes in lymphocyte proliferation in response to mitogens. Altered proliferative responses of fish from the contaminated site were partially reversed by maintaining fish in water from the reference site. Peter Ross described studies in which harbor seals were fed herring obtained from relatively clean (Atlantic Ocean) and contaminated (Baltic Sea) waters. Decreased natural killer cell activity and lymphoproliferative responses to T and B cell mitogens, as well as depressed antibody and delayed hypersensitivity responses to injected antigens, were identified in seals fed contaminated herring. In laboratory studies, it was determined that rats fed freeze-dried Baltic Sea herring had higher virus titers after challenge with rat cytomegalovirus (RCMV) than rats fed Atlantic Ocean herring; perinatal exposure of rats to oil extracted from Baltic herring also reduced the response to challenge with RCMV. Keith Grassman reported an association between exposure to polyhalogenated aryl hydrocarbons and decreased T cell immunity in the offspring of fish-eating birds (herring gulls and Capsian terns) at highly contaminated sites in the Great Lakes. The greatest suppression of skin test responses to phytohemagglutinin injection (an indicator of T cell immunity) was consistently found at sites with the highest contaminant concentrations. Judith Zelikoff addressed the applicability of immunotoxicity studies developed in laboratory-reared fish for detecting altered immune function in wild populations. She presented data from studies done in her laboratory with environmentally relevant concentrations of metals as examples. Although the necessity of proceeding with caution when extrapolating across species was emphasized, she concluded that published data, and results presented by the other Symposium participants, demonstrate that assays similar to those developed for use in laboratory rodents may be useful for detecting immune system defects in wildlife species directly exposed to toxicants present in the environment.

Associations between Altered Immune Function and Organochlorine Contamination in Young Caspian Terns (Sterna caspia) from Lake Huron, 1997–1999

Previous studies of laboratory animals and wildlife species have demonstrated the immunotoxicity of organochlorines. This study confirmed that associations between organochlorines and suppressed T cell function and enhanced antibody production in young Caspian terns from the Great Lakes, first observed in the early 1990s, continued into the late 1990s. These associations were based on measurement of organochlorines in plasma of individuals and pooled egg samples. During 1997–99, immune function, hematological variables, and organochlorine contamination were measured in prefledgling Caspian terns at two Lake Huron colonies: Channel Shelter Island (Confined Disposal Facility) at the mouth of the Saginaw River in southern Saginaw Bay and Elm Island in the North Channel. Elevated organochlorine exposure, reproductive effects, and decreased recruitment have been documented previously in the Saginaw Bay colony. Concentrations of polychlorinated biphenyls (PCBs) in eggs and plasma and 1,1-dichloro-2,2-bis(p)chlorophenyl)ethylene (DDE) in plasma were consistently higher in Saginaw Bay compared to the North Channel. The mean phytohemagglutinin (PHA) skin test, a measure of T lymphocyte function, was 42% lower in Saginaw Bay. Regression analyses showed strong negative associations between the PHA response and plasma PCBs and, to a slightly lesser degree, DDE. Despite interyear differences, total antibody titers following immunization with sheep red blood cells were higher in Saginaw Bay than the North Channel. Titers were positively associated with plasma PCBs and DDE. Plasma PCBs and DDE were negatively correlated with the percentage of monocytes and positively correlated with the percentage of basophils.

Effects of methylmercury exposure on the immune function of juvenile common loons (Gavia immer)

We conducted a dose-response laboratory study to quantify the level of exposure to dietary Hg, delivered as methylmercury chloride (CH3HgCl), that is associated with suppressed immune function in captive-reared common loon (Gavia immer) chicks. We used the phytohemagglutinin (PHA) skin test to assess T-lymphocyte function and the sheep red blood cell (SRBC) hemagglutination test to measure antibody-mediated immunity. The PHA stimulation index among chicks receiving dietary Hg treatment did not differ significantly from those of chicks on the control diet (p = 0.15). Total antibody (immunoglobulin [Ig] M [primary antibody] + IgG [secondary response]) production to the SRBC antigen in chicks treated with dietary methylmercury (MeHg), however, was suppressed (p = 0.04) relative to chicks on control diets. Analysis indicated suppression of total Ig production (p = 0.025 with comparisonwise alpha level = 0.017) between control and 0.4 microg Hg/g wet food intake treatment groups. Furthermore, the control group exhibited a higher degree of variability in antibody response compared to the Hg groups, suggesting that in addition to reducing the mean response, Hg treatment reduced the normal variation attributable to other biological factors. We observed bursal lymphoid depletion in chicks receiving the 1.2 microg Hg/g treatment (p = 0.017) and a marginally significant effect (p = 0.025) in chicks receiving the 0.4 microg Hg/g diet. These findings suggest that common loon chick immune systems may be compromised at an ecologically relevant dietary exposure concentration (0.4 microg Hg/g wet wt food intake). We also found that chicks hatched from eggs collected from low-pH lakes exhibited higher levels of lymphoid depletion in bursa tissue relative to chicks hatched from eggs collected from neutral-pH lakes.

Monitoring the Elimination of Persistent Toxic Substances from the Great Lakes; Chemical and Physiological Evidence from Adult Herring Gulls

To assess progress towards virtual elimination of PCBs, DDE, dieldrin and Mirex and their associated physiological effects, we compared their concentrations in pooled livers of adult herring gulls (Larus argentatus) repeatedly sampled at 8 Great Lakes colonies and a reference colony on the Atlantic coast between 1974 and 1993. We measured the relative thyroid mass and concentrations of highly carboxylated porphyrins and retinyl palmitate in the liver of each individual. PCBs, dieldrin and mirex declined in 7 of 8 colonies while DDE decreased in six. The greatest decreases occurred pre-1985. PCBs and DDE did not decrease in gulls from Middle Island in western L. Erie. Middle Island and Saginaw Bay had the highest concentrations of PCBs of 11 Great Lakes colonies in the 1990s. Thyroids of gulls from Great Lakes colonies were slightly enlarged but the degree of enlargement has decreased over time. In 1991, gulls from Great Lakes colonies had slight to moderately elevated concentrations of highly carboxylated porphyrins. In the early 1990s, hepatic stores of retinyl palmitate were very seriously depleted in gulls from the Detroit River, western basin of Lake Erie, and Lake Ontario, reflecting decreased availability and altered storage. We conclude that PCBs and/or other persistent toxic substances in the food of herring gulls have not been virtually eliminated.

Contaminant Residues in Tissues of Adult and Prefledged Herring Gulls from the Great Lakes in Relation to Diet in the Early 1990s

In the early 1990s, herring gulls (Larus argentatus) were collected in 15 breeding colonies throughout the Great Lakes basin and in two reference colonies on Lake Winnipeg and the Bay of Fundy. Organochlorine and metal concentrations, and stable isotope ratios (15N/14N and 13C/12C) were measured in their tissues, and we qualitatively assessed their diet. Breast muscle δ15N suggested that adults fed on planctivorous or insectivorous fish at six colonies, on piscivorous fish at four, and at a lower trophic level at the remaining three. The concentrations of Co, Ni, Al, Cr, Sn, Fe, and Pb in kidneys of adults suggested anthropogenic enrichment in the Great Lakes basin. Concentrations of contaminants were highest most often in tissues of gulls from Lake Ontario and northern Lake Michigan colonies. Concentrations of Pb in adults from Hamilton Harbour and the Detroit River, and of Se in adults from the southern Lake Huron colony, were similar to published toxicity thresholds. Tissue levels of Cd have increased, while those of Pb have decreased markedly since 1983. DDE, dieldrin, mirex, and ΣPCB concentrations in livers collected from nine of these colonies revealed declines of 16 to 87% at most locations since the early 1980s. 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents were highest in adults from the offshore colonies in western Lake Erie and northern Lake Michigan, where gulls feed on piscivorous fish, and were driven by non-ortho PCBs. There was evidence of an unusually high bioavailablity of organochlorines, especially dieldrin, near the northern Lake Michigan colony during the period of chick growth, and of an ongoing loading of mercury to eastern Lake Ontario. Tissues of adult gulls from colonies on Lakes Ontario, Erie, and Michigan best reflect local conditions whereas those from Lake Superior and northern Lake Huron reflect contaminants accumulated from time spent on the lower lakes.

Effects of PCB 126 on Primary Immune Organs and Thymocyte Apoptosis in Chicken Embryos

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyl (PCB) 126 produce thymic atrophy and immunosuppression. This study explored the hypothesis that the thymic atrophy produced by developmental exposure to PCB 126 is associated with an increase in apoptotic thymocytes at the end of incubation in chicken embryos. Eggs were injected via the air cell with PCB 126 (0.05, 0.13, 0.32, 0.64, and 0.80 ng/g egg) on d 0 of incubation, and tissues were collected on d 20. Controls included noninjected and vehicle-injected (sunflower oil) eggs. Thymocytes were cultured for 6 h and analyzed by flow cytometry for decreased DNA content (propidium iodide staining) and cell size (forward scatter), which indicate apoptosis. PCB 126 induced dose-dependent mortality with an LD50 of 1.01 ng/g and lowest-observed-effect concentration (LOEC) of 0.32 ng/g. Teratogenic effects commonly associated with TCDD and planar PCBs, including cranial and foot deformities and subcutaneous edema, tended to increase with dose of PCB 126. PCB 126 reduced thymus mass by approximately 20% at 0.64 and 0.8 ng/g, the number of viable thymocytes by approximately 20–24% at and above 0.13 ng/g, and the number of bursal lymphoid cells by 57% at 0.64 ng/g. The percentage of apoptotic thymocytes increased with dose, reaching levels 2 times greater than controls at 0.8 ng/g. Electrophoresis of low-molecular-weight DNA from thymocytes of all doses demonstrated fragments in multiples of 180 bp. This DNA laddering is a hallmark of apoptosis. At all doses, thymocytes exhibited caspase-3 activation, another indicator of apoptosis. The results of this experiment supported the hypothesis that the thymic atrophy produced by developmental exposure to PCB 126 in chicken embryos is associated with an increase in apoptotic thymocytes on embryonic d 20. This project was supported by the U.S. Environmental Protection Agency (GR825216-01-0) and the Biomedical Sciences PhD Program at Wright State University. E. Lavoie, R. Garrett, A. Handy, and E. Reaves helped with laboratory work. Drs. Nancy Bigley, Thomas Brown, G. Allen Burton, and Robert Grubbs provided technical assistance and reviewed earlier drafts of this article.

Lung function and airway inflammation in rats following exposure to combustion products of carbon-graphite/epoxy composite material: comparison to a rodent model of acute lung injury.

Pulmonary function and inflammation in the lungs of rodents exposed by inhalation to carbon/graphite/epoxy advanced composite material (ACM) combustion products were compared to that of a rodent model of acute lung injury (ALI) produced by pneumotoxic paraquat dichloride. This investigation was undertaken to determine if short-term exposure to ACM smoke induces ALI; and to determine if smoke-related responses were similar to the pathogenic mechanisms of a model of lung vascular injury. We examined the time-course for mechanical lung function, infiltration of inflammatory cells into the lung, and the expression of three inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2) and interferon-gamma (IFN-gamma). Male Fischer-344 rats were either exposed to 26.8-29.8 g/m(3) nominal concentrations of smoke or were given i.p. injections of paraquat dichloride. Measurements were determined at 1, 2, 3, and 7 days post exposure. In the smoke-challenged rats, there were no changes in lung function indicative of ALI throughout the 7-day observation period, despite the acute lethality of the smoke atmosphere. However, the animals showed signs of pulmonary inflammation. The expression of TNF-alpha was significantly increased in the lavage fluid 1 day following exposure, which preceded the maximum leukocyte infiltration. MIP-2 levels were significantly increased in lavage fluid at days 2, 3, and 7. This followed the leukocyte infiltration. IFN-gamma was significantly increased in the lung tissue at day 7, which occurred during the resolution of the inflammatory response. The paraquat, which was also lethal to a small percentage of the animals, caused several physiologic changes characteristic of ALI, including significant decreases in lung compliance, lung volumes/capacities, distribution of ventilation, and gas exchange capacity. The expression of TNF-alpha and MIP-2 increased significantly in the lung tissue as well as in the lavage fluid. Increased MIP-2 levels also preceded the maximum neutrophil infiltration. The differences in the time-course and primary site of TNF-alpha, MIP-2, and IFN-gamma expression; and the differences in the temporal relationship between their expression and infiltration of inflammatory cells may have accounted for the differences in lung function between paraquat treated and ACM smoke exposed animals.

Study of the Reproductive Effects in Rats Surgically Implanted With Depleted Uranium for Up to 90 Days

In 2001, the Naval Health Research Center Toxicology Detachment was funded by the U.S. Army Medical Research Acquisition Activity (USAMRAA) to conduct a study of the effects of surgically implanted depleted uranium (DU) pellets on adult rat reproductive success and development across two successive generations. This article presents some of the findings for the group of offspring from adult rats mated at 30 d post surgical implantation of DU pellets. Adult male and female Sprague-Dawley rats (P1 generation) were surgically implanted with 0, 4, 8, or 12 DU pellets (1 × 2 mm). The P1 generation was then cross-mated at 30 d post surgical implantation. Urine collected from P1 animals at 27 d post surgical implantation showed that DU was excreted in the urine of DU-implanted animals in a dose-dependent manner. DU surgical implantation did not have a negative impact on P1 reproductive success, survival, or body weight gain through post surgical implantation d 90. There were no statistically significant differences in F1 birth weight, survival, and litter size at postnatal day (PND) 0, 5, and 20. No gross physical abnormalities identified in the offspring were attributable to neonatal DU exposure. A series of neurodevelopment and immune function assessments were also conducted on F1 offspring. No group differences were observed that were related to parental DU exposure. Studies are ongoing on the impact of leaving DU embedded in soft tissue for 120 d on rat reproduction and subsequent offspring survival and development.