Evidence of estrogenic endocrine disruption in smallmouth and largemouth bass inhabiting Northeast U.S. national wildlife refuge waters: A reconnaissance study.

Intersex as the manifestation of testicular oocytes (TO) in male gonochoristic fishes has been used as an indicator of estrogenic exposure. Here we evaluated largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) form 19 National Wildlife Refuges (NWRs) in the Northeast U.S. inhabiting waters on or near NWR lands for evidence of estrogenic endocrine disruption. Waterbodies sampled included rivers, lakes, impoundments, ponds, and reservoirs. Here we focus on evidence of endocrine disruption in male bass evidenced by gonad histopathology including intersex or abnormal plasma vitellogenin (Vtg) concentrations. During the fall seasons of 2008-2010, we collected male smallmouth bass (n=118) from 12 sites and largemouth bass (n=173) from 27 sites. Intersex in male smallmouth bass was observed at all sites and ranged from 60% to 100%; in male largemouth bass the range was 0-100%. Estrogenicity, as measured using a bioluminescent yeast reporter, was detected above the probable no effects concentration (0.73ng/L) in ambient water samples from 79% of the NWR sites. Additionally, the presence of androgen receptor and glucocorticoid receptor ligands were noted as measured via novel nuclear receptor translocation assays. Mean plasma Vtg was elevated (>0.2mg/ml) in male smallmouth bass at four sites and in male largemouth bass at one site. This is the first reconnaissance survey of this scope conducted on US National Wildlife Refuges. The baseline data collected here provide a necessary benchmark for future monitoring and justify more comprehensive NWR-specific studies.

Retinoids in eggs and embryos of birds fed fish from the Great Lakes.

Retinoids were analyzed in 11-day chick embryos and eggs from white Leghorn hens (Gallus domesticus) fed environmentally-derived polychlorinated biphenyls (PCB) in carp (Cyprinus carpio) from Saginaw Bay. The yolks and the embryos contained all-trans-retinol, 3,4-didehydroretinol and retinyl esters. There was no significant difference in the total retinoid content in the yolks of 11-day incubated eggs among hens fed for 7 weeks diets containing 0.5-6.6 mg PCB/kg diet. However, the proportional amount of retinols in the high (6.6 mg) PCB group was significantly less than that in low (0.5 mg) PCB controls, while the amount of retinyl palmitate in the high PCB group was significantly greater than that in the controls. Retinoids in the embryos were not affected by any of the PCB levels fed to hens for 7 weeks prior to laying the eggs. The 50% reduction in the molar ratio of retinols to retinyl palmitate in the yolks of eggs as the result of the high PCB level fed to hens for 7 weeks can serve as an indicator for chronic exposure to PCB contamination at the level of 6.6 mg or higher PCB/kg diet.

Effects induced by feeding organochlorine-contaminated carp from Saginaw Bay, Lake Huron, to laying White Leghorn hens. I. Effects on health of adult hens, egg production, and fertility.

This study was conducted to determine the effects of consumption of halogenated hydrocarbon compounds, primarily polychlorinated biphenyls (PCBs), contained in Great Lakes fish by the domestic chicken (Gallus domesticus). In this article we report the results of feeding White Leghorn hens for a period of 8 wk diets that contained 31-35% ocean fish and/or carp (Cyprinus carpio) from Saginaw Bay, Lake Huron, MI, which provided 0.3 (control), 0.8 (low-dose group), or 6.6 (high-dose group) mg PCB/kg, wet weight (ww). These concentrations were analogous to 3.3, 26, or 59 pg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQs)/g diet, ww, respectively. There were no significant effects on feed consumption among the groups. An unexpectedly high incidence of fatty liver hemorrhagic syndrome (FLHS) was observed in hens from the control (78% FLHS) and low-dose (75% FLHS) groups when compared to the high-dose group (15% FLHS). Birds in the control and low-dose groups had a significant increase in liver and body weights. Significant decreases in egg production, weight, and fertility were immediate in all dose groups, with the effect being permanent in the control and low-dose groups. Although the incidence of FLHS was an unexpected complication, the fact that there were no significant effects on egg production, egg weights, or fertility in the high-dose group suggests that the no-observable-adverse-effect concentration (NOAEC) for these parameters is in excess of 26 mg total weathered PCBs/kg egg, ww. This value was the average concentration of PCBs in the high-dose group eggs during the last week of the study.

Effects induced by feeding organochlorine-contaminated carp from Saginaw Bay, Lake Huron, to laying White Leghorn hens. II. Embryotoxic and teratogenic effects.

Carp from Saginaw Bay, Lake Huron, MI, was fed to White Leghorn chickens for a period of 8 wk. The diets contained 0.3 (control; 0% carp), 0.8 (3.4% carp), and 6.6 (35% carp) mg polychlorinated biphenyls (PCBs)/kg diet, by wet weight (ww). These concentrations corresponded to 3.3, 26, and 59 pg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents/g diet ww, respectively. Though the diets were not acutely toxic to the adult laying hens, dose- and time-dependent responses were observed in the embryos and chicks. Toxicity was manifested as a dose-dependent increase in embryo mortality and decreased hatching rates. Furthermore, embryos and chicks displayed various deformities, including (1) head and neck edema and hemorrhage, (2) abdominal edema and hemorrhage, (3) foot and leg deformities, (4) skull and brain deformities, (5) yolk-sac deformities, and (6) miscellaneous deformities. The types of deformities observed were similar to those reported for embryos and chicks of colonial waterbirds in Saginaw Bay, as well as in controlled studies where technical mixtures or individual congeners of polychlorinated diaromatic hydrocarbons (PCDAHs) were fed to chickens. Increasing concentrations of carp also significantly affected the various organ weights in 18-d embryos and hatched chicks. At 18 d of incubation, weights of the embryos' livers were directly proportional to the concentration of PCBs in the diets. The weights of the spleens and bursae were inversely proportional to the dietary PCB concentration. After 3 additional days of incubation, significant effects in body, brain, liver, heart, and bursa weights were observed in hatched chicks. The concentrations of total PCBs, as well as 2,3,7,8-TCDD equivalents (TEQs) in the diets, were in the range of those that have been shown to cause similar adverse effects in other species. This study has shown that fish, the primary food source of colonial waterbirds in Saginaw Bay, are capable of causing adverse reproductive effects in a model avian species, the chicken. However, due to differences in the relative potency to cause effects on different endpoints in different species, the results of this study should not be used to predict the threshold for effects in other species.

Dietary exposure of mink to carp from Saginaw Bay, Michigan: 2. Hematology and liver pathology.

The effects of consumption of environmental contaminants contained in carp (Cyprinus carpio) from Saginaw Bay, Michigan on various hematological parameters and liver integrity of adult female mink (Mustela vision) were determined. Mink were fed diets that contained 0 (control), 10, 20, or 40% carp prior to and throughout the reproductive period (182 days). The diets contained 0.015, 0.72, 1.53, and 2.56 mg polychlorinated biphenyls (PCBs)/kg diet and 1.0, 19, 40, and 81 pg 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs)/g diet, respectively. Mink fed the diets containing carp showed a general dose-dependent occurrence of clinical signs commonly associated with chlorinated hydrocarbon toxicity, including listlessness, nervousness when approached, anorexia, and melena. Erythrocyte counts were less in mink exposed to Saginaw Bay carp than in controls, while the number of white blood cells was greater than in controls. Significant differences (p < 0.05) in the concentrations of neutrophils, lymphocytes, monocytes, and eosinophils were also found between the control and carp-fed groups, but are considered to be of limited clinical or biological importance. Hematocrit values for the mink fed the 20 and 40% carp diets were significantly less than those of mink in the control and 10% carp groups. There were no significant differences in hemoglobin concentrations among the groups. Necropsies revealed enlarged yellowish livers in many of the carp-fed mink, especially those fed the 40% carp diet. Liver, spleen, and lung weights of carp-fed mink were significantly greater than those of control mink.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary exposure of mink to carp from Saginaw Bay, Michigan. 1. Effects on reproduction and survival, and the potential risks to wild mink populations.

Carp (Cyprinus carpio) collected from Saginaw Bay, Michigan, containing 8.4 mg total polychlorinated biphenyls (PCBs)/kg and 194 ng of 2,3,7,8-tetrachloro-dibenzo-p-dioxin equivalents (TEQs)/kg, were substituted for marine fish at levels of 0, 10, 20, or 40% in the diets of adult ranch mink (Mustela vison). The diets, containing 0.015, 0.72, 1.53, and 2.56 mg PCBs/kg diet, or 1.03, 19.41, 40.02, and 80.76 ng TEQs/kg diet, respectively, were fed to mink prior to and throughout the reproductive period to evaluate the effects of a naturally-contaminated prey species on their survival and reproductive performance. The total quantities of PCBs ingested by the mink fed 0, 10, 20, or 40% carp over the 85-day treatment period were 0.34, 13.2, 25.3, and 32.3 mg PCBs/mink. respectively. The corresponding quantities of TEQs ingested by the mink over the same treatment period were 23, 356, 661, and 1,019 ng TEQs/mink, respectively. Consumption of feed by mink was inversely proportional to the PCB and TEQ content of the diet. The diet containing Saginaw Bay carp caused impaired reproduction and/or reduced survival of the kits. Compared to controls, body weights of kits at birth were significantly reduced in the 20 and 40% carp groups, and kit body weights and survival in the 10 and 20% carp groups were significantly reduced at three and six weeks of age. The females fed 40% carp whelped the fewest number of kits, all of which were stillborn or died within 24 hours. Lowest observable adverse effect levels (LOAEL) of 0.134 mg PCBs/kg body weight/day or 3.6 ng TEQs/kg body weight/day for adult female mink were determined. The potential effects of exposure of wild mink to contaminated Great Lakes fish were assessed by calculating "maximum allowable daily intakes" and "hazard indices" based on total concentrations of PCB residues in several species of Great Lakes fish and mink toxicity data derived from the study.

Accumulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents by double-crested cormorant (Phalacrocorax auritus, Pelicaniformes) chicks in the North American Great Lakes.

Concentrations of polychlorinated biphenyls (PCBs), and 2,3,7,8-tetrachlordibenzo-p-dioxin equivalents (TCDD-EQ) were determined in eggs and chicks of double-crested cormorants (DCC) which were collected in 1989 from eight locations in the Laurentian Great Lakes. The mean biomagnification factor (BMF) from forage fish to eggs was found to be 31.3. Absolute and relative concentrations as well as rates of accumulation of total concentrations of PCBs and TCDD-EQ were measurable in all of the samples. The concentrations of both PCBs and TCDD-EQs decreased immediately upon hatching of chicks, due to growth dilution. Initial decreases in absolute masses of TCDD-EQ in chicks were also observed, which indicates that there can be significant elimination of these compounds during early development. The initial rates of accumulation by chicks were dependent only on the mass of fish consumed. After the chicks began thermoregulating, the rates of accumulation, expressed as a concentration, normalized to body weight, became greater. Rates of accumulation of both PCBs and TCDD-EQ were correlated with their respective concentrations in forage fish consumed by the chicks. The relative potency, expressed as the ratio of the concentration of TCDD-EQ to that of total PCBs was calculated to determine if there was significant trophic-level enrichment of the TCDD-EQs, relative to total concentrations of PCBs. A significant enrichment was observed at the more and less contaminated locations, but the degree of enrichment was greater at the less contaminated locations (26 vs 72 micrograms/g).

H4IIE rat hepatoma cell bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents in colonial fish-eating waterbird eggs from the Great Lakes.

Fish-eating waterbirds from the Great Lakes of North America have shown symptoms of poisoning similar to those observed in laboratory exposures of various avian species to planar halogenated hydrocarbons (PHHs). PHHs, include among others, polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs), and have been implicated in some of the reproductive problems of Great Lakes water-birds. The objectives of this study were to assess the overall potencies of PCB-containing extracts from colonial water-bird eggs taken from the Great Lakes and to compare the potencies with the location and spatial distribution of the colonies. The potencies of the extracts were assessed by their ability to induce cytochrome P450IA1-associated ethoxyresorufin O-deethylase (EROD) activity in H4IIE rat hepatoma cells as compared to the standard, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The H4IIE bioassay-derived TCDD-equivalents (TCCD-EQs) in the waterbird eggs concur with residue analyses and biological data from other studies. The greatest concentrations of TCDD-EQs were found in waterbird eggs from historically polluted, industrialized or urbanized areas in which the reproductive impairment of colonial waterbirds was most severe. However, significant concentrations of TCDD-EQs were detected at all sites tested; with a range of 49 to 415 pg TCDD-EQ/g egg, uncorrected for extraction efficiencies. The H4IIE bioassay proved to be a useful biomonitoring tool to assess the overall potency of complex PHH mixtures in environmental samples.

Microcontaminants and reproductive impairment of the Forster's tern on Green Bay, Lake Michigan--1983.

For the 1983 nesting season, Forster's tern (Sterna forsteri) reproductive success was significantly impaired on organochlorine contaminated Green Bay, Lake Michigan compared to a relatively uncontaminated inland location at Lake Poygan, Wisconsin. Compared with tern eggs from Lake Poygan, eggs from Green Bay had significantly higher median concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), other polychlorinated dibenzo-p-dioxins (PCDDs), total polychlorinated biphenyls (PCBs), total (three congeners) non-ortho, ortho' PCBs, five individual PCB congeners known to induce aryl hydrocarbon hydroxylase (AHH) and several other organochlorine contaminants. Conversions of analytical concentrations of TCDD and PCB congeners based on relative AHH induction potencies allowed for estimation of total 2,3,7,8-TCDD equivalents. Two PCB congeners, 2,3,3',4,4'- and 3,3',4,4',5-pentachlorobiphenyl (PeCB) accounted for more than 90% of the median estimated TCDD equivalents at both Green Bay and Lake Poygan. The median estimated TCDD equivalents were almost 11-fold higher in tern eggs from Green Bay than in eggs from Lake Poygan (2175 and 201 pg/g). The hatching success of Green Bay sibling eggs from nests where eggs were collected for contaminant analyses was 75% lower at Green Bay than at Lake Poygan. Hatchability of eggs taken from other nests and artificially incubated was about 50% lower for Green Bay than for Lake Poygan. Among hatchlings from laboratory incubation, those from Green Bay weighed approximately 20% less and had a mean liver weight to body weight ratio 26% greater than those from Lake Poygan. In both field and laboratory, mean minimum incubation periods were significantly longer for eggs from Green Bay compared to Lake Poygan (8.25 and 4.58 days, respectively). Mean minimum incubation time for Green Bay eggs in the field was 4.37 days longer than in the laboratory. Hatchability was greatly improved when Green Bay eggs were incubated by Lake Poygan adults in an egg-exchange experiment, but was sharply decreased in Lake Poygan eggs incubated in Green Bay nests. Nest abandonment and egg disappearance were substantial at Green Bay but nil at Lake Poygan. Thus, not only factors intrinsic to the egg, but also extrinsic factors (parental attentiveness), impaired reproductive outcome at Green Bay. The epidemiological evidence from this study strongly suggested that contaminants were a causal factor. AHH-active PCB congeners (intrinsic effects) and PCBs in general (extrinsic effects) appeared to be the only contaminants at the concentrations measured in eggs, capable of producing the effects that were observed at Green Bay.

Embryotoxicity, teratogenicity, and aryl hydrocarbon hydroxylase activity in Forster's terns on Green Bay, Lake Michigan.

Known reproductive problems, including congenital malformations and poor hatching success, exist for the state endangered Forster's tern (Sterna forsteri) in Green Bay, Wisconsin. Twenty Forster's tern eggs were collected from separate nests at a natural colony with documented reproductive problems, situated at Green Bay, Lake Michigan, and an inland colony at Lake Poygan (control) where reproduction was documented as normal. Eggs from the two locations were placed in the same laboratory incubator and candled throughout incubation. Hatching success of Green Bay eggs was 52% of that for controls. Several early embryonic deaths occurred, but most mortality occurred close to the time of hatching. Liver microsomal aryl hydrocarbon hydroxylase activity was elevated approximately threefold in Green Bay hatchlings compared to controls. Green Bay terns that hatched weighed less than controls, had an increased liver to body weight ratio, and had a shorter femur length. Two Green Bay embryos that failed to hatch had anomalies, one with a crossed beak and one with poor ossification of the foot. One Green Bay hatchling had an abnormally ossified ilium. These effects were observed in eggs where there were measureable levels of aryl hydrocarbon hydroxylase inducers including polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins.