Tumor prevalence and biomarkers of exposure in brown bullhead (Ameiurus nebulosus) from Back River, Furnace Creek, and Tuckahoe River, Maryland.

Brown bullheads (Ameiurus nebulosus) were collected from 2 locations near Baltimore, Maryland, Back River and Furnace Creek, and 1 (reference) location, Tuckahoe River, to compare the prevalence of tumors (liver and skin) and visible skin lesions (fin erosion and abnormal barbels). Cytochrome P450 activity measured as ethoxyresorufin-O-deethylase, biliary PAH-like fluorescent metabolites, and fillet contaminant concentrations were determined as indicators of exposure in a randomly selected subset of the fish. There were no significant differences in liver tumor prevalence: Back River = 8% (4/50), Furnace Creek = 0% (0/50), and Tuckahoe River = 2.6% (1/39; p = 0.20, extension of Fisher's exact test). Skin tumor prevalence was as follows: Furnace Creek = 12% (6/50), Back River = 8% (4/50), and Tuckahoe River = 0% (0/39; p = 0.063). In the Back River fish, there was a 40% (20/50) prevalence of fin erosion and a 28% (14/50) prevalence of abnormal (shortened, clubbed, or missing) barbels. Fin erosion was not observed in the other collections, and only 10% (5/50) of the Furnace Creek fish had abnormal barbels (p < 0.001 for both lesions). There were statistically significant differences in mean EROD activity, with levels in Furnace Creek and Back River fish approximately twice that found in Tuckahoe River fish. There were also significant differences in mean benzo(a)pyrene-like bile metabolite concentrations: the lowest mean was in the Tuckahoe River fish, 8 times higher in Furnace Creek fish, and 13 times higher in Back River fish. Of the 3 groups, the Back River bullheads appear to be most adversely affected by contaminant exposure because they had the highest prevalence of liver tumors, fin erosion, and abnormal barbels.

Effects of the mosquito larvicide GB-1111 on bird eggs.

Golden Bear Oil (GB-1111; legal trade name for GB-1313) is a petroleum distillate used in the United States and other countries as a mosquito larvicide. As part of an evaluation of the potential effects of GB-1111 on birds, fertile eggs of mallards (Anas platyrhynchos) and bobwhite (Colinus virginianus) were incubated in the laboratory, and treated on day 4 of incubation with external applications equivalent to either 0, 1/3, 1, 3 or 10 times the maximum rate (X) of 47 l/ha (5 gal/A) of field application of GB-1111. Hatching success was significantly reduced in mallards treated 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. Hepatic P450-associated monooxygenase activity (ethoxyresorufin-O-dealkylase; EROD) was negatively related to dose. In the 3X group there was a significant increase in the concentration of hepatic reduced glutathione (GSH) but a decrease in protein-bound thiols (PBSH). Hatching success of bobwhite was marginally reduced at the highest level of treatment (10X). Other effects at this level in bobwhite included a significant increase in incidence of abnormal embryos or hatchlings, lower body and liver weights, and a two-fold increase in hepatic microsomal EROD activity in hatchlings. The recommended maximum rate of field application of GB-1111 is unlikely to impair the survival or development of bobwhite embryos but is potentially toxic to mallard embryos under conditions of larvicide drift or spray overlap.

Effects of the mosquito larvicide GB-1111 on red-winged blackbird embryos.

Golden Bear Oil (GB-1111; legal trade name for GB-1313) is a petroleum distillate that is used in the United States and other countries as a larvicide for mosquito suppression. As part of a multi-species evaluation of the potential effects of GB-1111 on birds, red-winged blackbird eggs were collected, artificially incubated, and treated with one of five amounts of GB-1111 varying from 0 to 10 times the expected exposure from a spray application of the maximum recommended amount (X=47 l/ha, 5 gal/ac). The application of 10 X caused a significant reduction in hatching success. A dose-related reduction of hepatic microsomal monooxygenase activity (EROD) was detected. Among body weights, skeletal measurements, and age at death, only crownrump length was different among experimental groups. Overall, the potential hazard to embryos of a representative wetland passerine appears minimal until the application rate exceeds 3 X.

Tumor prevalence and biomarkers of exposure in brown bullheads (Ameiurus nebulosus) from the tidal Potomac River, USA, watershed.

Associations between contaminant exposure and liver and skin tumor prevalence were evaluated in brown bullheads (Ameiurus nebulosus) from the tidal Potomac River, USA, watershed. Thirty bullheads (> or = age 3) were collected from Quantico embayment, near a Superfund site that released organochlorine contaminants; Neabsco Creek, a tributary with petroleum inputs from runoff and marinas; and Anacostia River (spring and fall), an urban tributary designated as a Chesapeake Bay region of concern, that was contaminated with polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and organochlorine pesticides. Fish were collected from the Tuckahoe River, as a reference. Cytochrome P450 activity, bile PAH metabolites, and muscle organochlorine pesticide and 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%; and Tuckahoe, 10%. Skin tumor prevalences were significantly different: Anacostia (spring), 37%; Anacostia (fall), 10%; Neabsco, 3%; Quantico, 3%; and Tuckahoe, 0%. Tumor prevalence in Anacostia bullheads warrants concern and was similar to those at highly contaminated sites in the Great Lakes. Evidence was found of higher PAH exposure in Anacostia fish but a cause-effect linkage could not be established. Fish tumor surveys, with histopathologic examination of internal and external organs, are recommended for monitoring the status of regions of concern.

Polycyclic aromatic hydrocarbons, aliphatic hydrocarbons, trace elements, and monooxygenase activity in birds nesting on the North Platte River, Casper, Wyoming, USA.

Tree swallow (Tachycineta bicolor) and house wren (Troglodytes aedon) eggs and chicks were collected near a refinery site on the North Platte River, Casper. Wyoming, USA and at a reference site 10 km upstream. Total polycyclic aromatic hydrocarbon (PAH) concentrations in swallow and wren chicks were higher at the refinery site than at the reference site. Polycyclic aromatic hydrocarbon concentrations in sediment and chick dietary samples were consistent with these findings. The general lack of methylated PAHs in sediment, diet, and bird carcasses suggested that the PAHs were derived from combustion and not from petroleum. The predominance of odd-numbered aliphatic hydrocarbons and the low ratios (< or =0.25) of pristane:n-C17 and phytane:n-C18 in chick and diet samples also suggested that swallow and wren chicks were not being chronically exposed to petroleum. Mean ethoxyresorufin-O-dealkylase and benzyloxyresorufin-O-dealkylase activities in tree swallow livers averaged nine times higher at the refinery site than at the reference site and were probably induced by exposure to PAHs. Trace element concentrations in eggs and livers of swallows and wrens were similar or greater at the reference site than at the refinery site. Selenium, strontium, and boron concentrations were elevated in eggs and livers of swallows and wrens at both the refinery and reference sites.

Organochlorine contaminants and biomarker response in double-crested cormorants nesting in Green Bay and Lake Michigan, Wisconsin, USA.

Double-crested cormorant (Phalacrocorax auritus) eggs at pipping and sibling 10-day-old chicks were collected from two colonies in Green Bay, WI, one colony in Lake Michigan, WI, and reference colonies in South Dakota and Minnesota. Egg contents and chicks were analyzed for organochlorine contaminants including polychlorinated biphenyl (PCB) congeners. Livers of embryos and chicks were assayed for hepatic microsomal ethoxyresorufin-O-dealkylase (EROD) activity. Eggshell thickness and the physical dimensions of embryo brains were measured. Concentrations of organochlorines, including p,p'-DDE (p,p'-dichlorodiphenyldichloroethylene), PCBs, and PCB congeners were generally an order of magnitude higher in eggs and chicks from Wisconsin than from reference locations. Total PCBs averaged 10-13 microg/g wet weight in eggs from three Wisconsin colonies compared to 0.9 microg/g PCBs from reference locations. Double-crested cormorant chicks accumulated on average 33-66 microg PCBs/day and 7-12 microg p,p'-DDE/day in the Wisconsin colonies compared to 0 microg PCBs/day and 1 microg p,p'-DDE/day in the reference colonies. At pipping, EROD activity in the livers of cormorant embryos was significantly higher in the Wisconsin colonies and significantly correlated with PCBs and the toxic equivalents (TEQs) of aryl hydrocarbon-active PCB congeners relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, in 10-day-old chicks EROD activity was not consistently different among colonies and was not correlated with PCBs or TEQs. A significant negative relationship between embryo brain asymmetry and the size of the egg suggested that physical constraint might be an important factor influencing the response of this bioindicator. Thinner eggshells in two colonies located near Door County, Wisconsin, suggested that historic p,p'-DDE residues associated with orchards are still an important source of p,p'-DDE in the local environment.

Organochlorine and metal contaminant exposure and effects in hatching black-crowned night herons (Nycticorax nycticorax) in Delaware Bay.

Pea Patch Island in Delaware Bay is the site of the largest heronry north of Florida. From 1989-93, the population of nine species of wading birds numbered approximately 12,000 pairs, but has recently declined to about 7,000 pairs. Because Delaware Bay is a major shipping channel and receives anthropogenic releases of toxic substances from agricultural, industrial, and municipal point and nonpoint sources, contaminant exposure and effects to the heronry have been an ongoing concern. In 1997, pipping (early hatching stage) black-crowned night herons (Nycticorax nycticorax) were collected from separate nests at Pea Patch Island and from a coastal reference site, Middle Island in Rehoboth Bay, Delaware. There was no evidence of malformations or hepatic histopathological lesions in embryos, and their body and liver weights did not differ between sites. Biomarkers of petroleum hydrocarbons, polyhalogenated contaminant, and metal exposure (cytochrome P450 induction and oxidative stress responses) did not differ (p > 0.05) between sites, although activities of benzyloxy-O-dealkylase and ethoxyresorufin-O-dealkylase were somewhat elevated in 3 of the 15 embryos collected from Pea Patch Island. Concentrations of 21 organochlorine pesticides and metabolites were relatively low at both sites, with p,p'-DDE values well below the threshold associated with eggshell thinning. Although total PCB concentration was modestly elevated (p < 0.05) in Pea Patch Island heron embryos, levels of arylhydrocarbon receptor-active PCB congeners, polychlorinated dibenzodioxins and dibenzofurans, and toxic equivalents were low and did not differ between sites. Concentrations of Cd and Mn in pipping embryos from Pea Patch Island were slightly greater (p < 0.05) than values observed in Middle Island embryos, but levels of these and the other metals and metalloids (e.g., Hg and Se) were below values associated with toxicity. In conclusion, it seems unlikely that chlorinated hydrocarbon and metal contaminant exposure constitutes a direct threat to the reproductive success of black-crowned night herons at Pea Patch Island. However, low-level exposure to these contaminants may constitute one of many stressors that in combination could adversely affect the stability of the wading bird population at this large heronry.

Mixed-function oxygenases, oxidative stress, and chromosomal damage measured in lesser scaup wintering on the Indiana Harbor Canal.

During the winter of 1993-1994, male lesser scaup (Aythya affinis) were collected on the heavily polluted Indiana Harbor Canal, East Chicago, Indiana, and examined for several bioindicators of chemical exposure. Livers were analyzed for activities of three cytochrome P450-associated monooxygenases and four measures of oxidative stress. Blood and spleen were analyzed by flow cytometry for chromosomal damage. In a concurrent study, scaup tissues were analyzed for organic and inorganic contaminants. Ethoxyresorufin-O-dealkylase (EROD) activity in livers of scaup collected in January 1994 was significantly higher than in livers of scaup collected in March 1994 or in livers of reference birds. Three hepatic monooxygenase activities were each significantly correlated with polycyclic aromatic hydrocarbon (PAH) concentrations in scaup carcasses. Thiobarbituric acid (TBA) activity in scaup livers was positively correlated with iron, boron, and lead concentrations in livers and polychlorinated biphenyl concentrations in carcasses. TBA activity was negatively correlated with protein-bound thiol activity and mercury concentrations in livers. The coefficient of variation of DNA content in scaup blood cells was correlated with PAH concentrations in scaup carcasses. This is the first field study with birds to demonstrate a correlation between liver monooxygenase activity and carcass PAH concentrations and to show a direct correlation between PAH concentrations in tissues and somatic chromosomal damage in blood.

Identification of in vitro cytochrome P450 modulators to detect induction by prototype inducers in the mallard duckling (Anas platyrhynchos).

Seven modulators of mammalian monooxygenase activity were screened for their ability to selectively stimulate or inhibit in vitro monooxygenase activities of hepatic microsomes from mallard ducklings treated with phenobarbital, beta-naphthoflavone, 3,3',4,4',5-pentachlorobiphenyl or vehicle. Microsomes were assayed fluorometrically for four monooxygenases: benzyloxy-, ethoxy-, methoxy-, and pentoxyresorufin-O-dealkylase, in combination with each of the seven modulators. Four combinations: alpha-naphthoflavone and 2-methylbenzimidazole with benzyloxyresorufin, and Proadifen with methoxy- and ethoxyresorufin, respectively, were evaluated further. beta-Naphthoflavone-treated groups were clearly distinguished from the corn oil vehicle control group by all of the assays and by the effects of the modulators in three of the four assay/modulator combinations. Enzyme activities of the phenobarbital and saline groups were statistically similar (P > or = 0.05) when assayed without modulator added, but each assay/modulator combination distinguished between these groups. The PCB-treated group was distinguished from the corn oil vehicle control group only for BROD activity, with or without the presence of modulator. Graphing of per cent modulation of BROD activity versus initial BROD activity provided the clearest distinction between all of the study groups. Identification of these selective in vitro modulators may improve detection and measurement of low level cytochrome P450 induction in avian species. Also, both the monooxygenase activities induced and the impacts of the modulators indicated differences between mammalian and avian cytochromes P450.

Lack of developmental and reproductive toxicity of 2,3,3',4, 4'-pentachlorobiphenyl (PCB 105) in ring-necked pheasants.

Mono-ortho PCBs are global contaminants of wildlife with the potential to produce toxicity by an aryl hydrocarbon receptor (AhR)-mediated mechanism. To determine the potency of 2,3,3',4, 4'-pentachlorobiphenyl (PCB 105) for producing reproductive and developmental toxicity, adult ring-necked pheasant hens (Phasianus colchicus) were orally dosed with 0, 0.06, 0.6, or 6 mg PCB 105/kg hen/week for 10 weeks to achieve cumulative doses of 0, 0.6, 6, or 60 mg PCB 105/kg hen after which hens were bred with untreated roosters once per week for 8 weeks. Except at week 6 of the egg-laying period when cumulative egg production in the 6 mg PCB 105/kg hen group was greater than controls, fertilized egg production was not significantly different between treatment groups. Embryo mortality and chick mortality were not significantly different between treatment groups. Total body and heart weights of all chicks 1 day posthatch (dph) were not different between groups, however, liver weights of chicks from the 60 mg/kg treatment group were greater than controls at 1 dph. The first chick to hatch from each hen was reared to 21 dph and among these birds, the total body, liver, and heart weights were not different between groups. There were no dose-related malformations of the beak or limbs, and no signs of subcutaneous edema, ascites, or pericardial edema in chicks at 1 or 21 dph. Hepatic microsomal monooxygenase activities [ethoxyresorufin-O-dealkylase (EROD), benzyloxyresorufin-O-dealkylase (BROD), and methyloxyresorufin-O-dealkylase (MROD)] were significantly elevated in chicks at 1 dph from hens given a cumulative PCB 105 dose of 6 mg/kg and in chicks at 21 dph from hens given a cumulative PCB dose of 60 mg/kg. These results indicate that a cumulative PCB 105 dose up to 60 mg/kg hen does not decrease the production of fertilized eggs or increase embryo or chick mortality in ring-necked pheasants, but does increase chick hepatic monooxygenase activity.

Contaminants in eggs of colonial waterbirds and hepatic cytochrome P450 enzyme levels in pipped tern embryos, Washington State.

Eggs of Forster's terns (Sterna forsteri) collected in 1991 from nesting colonies on Crescent Island (Columbia River) and the Potholes Reservoir in south central Washington generally contained low residues of organochlorine pesticides and metabolites, 2,3,7, 8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and polychlorinated biphenyls (PCBs). Hepatic cytochrome P450 enzyme activity in pipped embryos of Forster's terns from the two colonies seemed unaffected by contaminants. At Crescent Island, examination of 23 Forster's tern eggs with large embryos (19 viable [10 pipped] and four dead [two pipped]) revealed developmental abnormalities in two viable pipped embryos (missing maxilla and deformed pelvic girdle) and a viable prepipping embryo (shortened beak). Our limited sample sizes and number of compounds analyzed preclude us from determining whether or not the abnormalities are related to contaminants. No abnormalities were noted in 10 pipped eggs (nine viable and one dead at collection) of Forster's terns collected from the Potholes Reservoir colony. Eggs of Caspian terns (Sterna caspia) collected from Crescent Island in 1991 also contained generally low residues of contaminants, only one developmental abnormality was noted, and limited data indicated that cytochrome P450 enzyme activity apparently was unaffected by contaminants. Organochlorine contaminants were generally low in addled eggs of American white pelicans (Pelecanus erythrorhynchos) collected from Crescent Island in 1994.

Developmental toxicity of PCB 126 (3,3',4,4',5-pentachlorobiphenyl) in nestling American kestrels (Falco sparverius).

Planar PCB congeners are embryotoxic and teratogenic to birds including American kestrels. The developmental toxicity of 3,3',4,4',5-pentachlorobiphenyl (PCB 126) was studied in the posthatching kestrel as a model for the eagle. Nestlings were dosed orally for 10 days with 5 microl/g body weight of corn oil (controls) or the planar PCB 126 at concentrations of 50, 250, or 1000 ng/g body weight. Dosing with 50 ng/g of PCB 126 resulted in a hepatic concentration of 156 ng/g wet weight, liver enlargement and mild coagulative necrosis, over 10-fold increases in hepatic microsomal ethoxyresorufin-O-dealkylase and benzyloxyresorufin-O-dealkylase, and approximately a 5-fold increase in methoxyresorufin-O-dealkylase. At this dose, mild to moderate lymphoid depletion of the spleen was apparent, as were decreased follicle size and content of the thyroid. At 250 ng/g, concentration of PCB 126 in the liver was 380 ng/g with increasing multifocal coagulative necrosis, decreased bone growth, decreased spleen weight with lymphocyte depletion of the spleen and bursa, and degenerative lesions of the thyroid. At 1000 ng/g, the liver concentration was 1098 ng/g, accompanied by decreased bursa weight, decreased hepatic thiol concentration, and increased plasma enzyme activities (ALT, AST, and LDH-L) in addition to the previous effects. Highly significant positive correlations were noted between liver concentrations of PCB 126 and the ratio of oxidized to reduced glutathone. These findings indicate that nestling kestrels are more susceptible to PCB 126 toxicity than adults, but less sensitive than embryos, and that planar PCBs are of potential hazard to nestling birds.

Flow cytometry for monitoring contaminant exposure in black-crowned night-herons.

The flow cytometry methods (FCM) was employed to determine cellular DNA content of black-crowned night-heron (Nycticorax nycticorax) embryos and 10-day-old chicks collected at sites differing in types of chemical contamination. The coefficient of variation of DNA content (CV) in blood collected from embryos suggested cytogenetic damage at a side in Louisiana known to be contaminated with petroleum. Blood CV from chicks suggested genetic damage at a site in Texas also known to be contaminated with petroleum. Spleen CVs in chicks were significantly lower than respective means from the reference site. The CVs of chick blood and liver and spleen negatively correlated, suggesting recovery of spleen and liver cells after exposure to a clastogenic compound. Thus, the lower CVs may also have been indicative of genetic damage. Based on the findings of this study, FCM is a potential indicator of certain environmental contaminants in black-crowned night-herons.

Biotransformation and induction: implications for toxicity, bioaccumulation and monitoring of environmental xenobiotics in fish.

Biotransformation of xenobiotics in fish occurs by many of the same reactions as in mammals. These reactions have been shown to affect the bioaccumulation, persistence, residue dynamics, and toxicity of select chemicals in fish. P-450-dependent monooxygenase activity of fish can be induced by polycyclic aromatic hydrocarbons, but phenobarbital-type agents induce poorly, if at all. Fish monooxygenase activity exhibits ideal temperature compensation and sex-related variation. Induction of monooxygenase activity by polycyclic aromatic hydrocarbons can result in qualitative as well as quantitative changes in the metabolic profile of a chemical. Induction can also alter toxicity. In addition, multiple P-450 isozymes have been described for several fish species. The biotransformation products of certain chemicals have been related to specific P-450 isozymes, and the formation of these products can be influenced by induction. Exposure of fish to low levels of certain environmental contaminants has resulted in induction of specific monooxygenase activities and monitoring of such activities has been suggested as a means of identifying areas of pollutant exposure in the wild.

Metabolism of 2-methylnaphthalene by rat and rainbow trout hepatic microsomes and purified cytochromes P-450.

The metabolism of 14C-2-methylnaphthalene by hepatic microsomes and purified cytochromes P-450 from rat and rainbow trout was studied using HPLC and radioactivity counting. The effects of pretreatment with beta-naphthoflavone and phenobarbital on the formation of three dihydrodiol metabolites and 2-hydroxymethylnaphthalene by microsomes and purified cytochromes P-450 were compared. The metabolism observed with microsomes could not adequately be accounted for by the combined metabolism observed with the purified cytochromes P-450. Differences in the microsome suspension buffers used when preparing microsomes for cytochrome purification as compared to preparing microsomes for direct use appeared to be a significant factor in these differences.

Metabolism of [14C]2-methylnaphthalene by rainbow trout (Salmo gairdneri) in vivo.

Rainbow trout were exposed to aqueous [14C]2-methylnaphthalene and their bile was examined for metabolites of [14C]2-methylnaphthalene. Trout which were pretreated with the monooxygenase inducer beta-naphthoflavone exhibited greater levels of total metabolites, glucuronide conjugates and dihydrodiol metabolites of [14C]2-methylnaphthalene in bile as compared to non-induced trout. The ratio of 2-hydroxymethyl-naphthalene to dihydrodiols found was greater in non-induced than in induced trout. These results are consistent with previously published studies on the metabolism of [14C]2-methylnaphthalene by rat and trout hepatic microsomes in vitro.