Associations of immunotoxicity with dioxins, PCBs, and other organochlorines
Although the immune assays in this study were not chemical specific, dioxins and PCBs clearly cause immunosuppresssion in laboratory animals 10, and environmental exposures have been associated with similar immunosuppression or increased infections in wildlife. Polychlorinated biphenyls and DDE were negatively associated with proliferation of T cells in male bottlenose dolphins (Tursiops truncatus) from the west coast of Florida 34. Young harbor seals (Phoca vitulina) fed PCB-contaminated fish from the Baltic Sea had reduced delayed-type hypersensitivity responses (T cell–mediated skin responses) and reduced T cell proliferation in vitro, supporting the role of contaminant-induced immunosuppression in an outbreak of phocine distemper virus in wild seals 35. Stranded harbor porpoises (Phocoena phocoena) dying from infections had higher tissue PCB concentrations than those dying of acute trauma 36. In glaucous gulls (Larus hyperboreus) of the Svalbard archipelago in the Barents Sea, total organochlorine exposure was positively correlated with gastrointestinal parasite load 15.
Pipping herring gull embryos from lower New York Harbor had greatly reduced numbers of developing lymphocytes both in the thymi and bursa of Fabricius, the sites of T and B cell maturation, respectively. The magnitude of lymphoid atrophy (∼40 − 50%) was similar to that observed in embryos from contaminated Great Lakes areas (Saginaw Bay and Lake Erie) (C.J. Kelly, 2003, Master's thesis, Wright State University, Dayton, OH, USA) 21. In a previous study, thymic atrophy was negatively correlated with PCB concentrations in the yolk sacs of pipping herring gull embryos (PCDDs and PCDFs were not assessed; K. Grasman, Calvin College, unpublished data). In herring gull chicks of the Great Lakes, thymic atrophy was associated with liver ethoxyresorufin-O-deethylase (EROD) activity, an indicator of exposure to PCBs and dioxins 17. In chickens, PCBs and dioxins decreased the mass and lymphoid cellularity of the thymus and bursa of Fabricius 18–20. In chicken embryos, the dioxin-like PCBs 126 and 77 and Aroclor 1254 increased apoptosis in thymocytes 1 d before hatch 20. In pipping herring gull embryos in the Great Lakes, thymocyte apoptosis was altered at sites with PCB-associated thymic atrophy (C.J. Kelly, 2003, Master's thesis, Wright State University, Dayton, OH, USA). Lymphocyte proliferation assays in New York Harbor gulls gave further evidence of disrupted immune function (Table 6 and Fig. 4). As cited earlier, altered lymphocyte proliferation has been observed in wildlife and laboratory species exposed to dioxins and PCBs.
The magnitude of suppression of the PHA skin response in New York Harbor herring gulls and black-crowned night heron chicks was among the greatest ever observed by the senior author's research team. Dioxins and PCBs exhibited strong negative correlations (r = –0.89 to –0.98) with the PHA skin response in herring gull chicks (Fig. 3). The PHA skin test integrates many important T cell functions and is recognized as one of the most sensitive assays employed by avian immunotoxicologists and ecologists 11, 17. In avian laboratory studies, elimination of T cell function by irradiation or immunosuppressive drugs reduces the PHA response by 50 to 60% (reviewed in 37). The magnitude of suppression in herring gulls and black-crowned night herons in lower New York Harbor was approximately 70 to 80% lower than at reference sites. For comparison, 30 to 50% suppression was observed in herring gull and Caspian tern chicks at contaminated Great Lakes sites 12. In Caspian tern chicks, the PHA response showed a strong negative association with total PCBs and DDE in plasma of individual birds 13.
Two related Great Lakes studies allow comparison of the PHA skin response to liver organochlorine concentrations in herring gull chicks. Livers were collected for contaminant analysis (pooled by site) from herring gull chicks at 11 colonies, including Lake Winnipeg (reference colony), Hamilton Harbour, and Saginaw Bay during 1991 and western Lake Erie during 1992 25. The same team conducted the PHA skin test on herring gull chicks at all four colonies during 1992 and additionally in 1993 and 1994 for Saginaw Bay 12. The PHA skin response was suppressed at contaminated sites (Lake Erie, Hamilton Harbour, and Saginaw Bay) with liver concentrations of 75 to 267 pg/g TEQs, 1.3 to 8.9 pg/g 2,3,7,8-TCDD, and 0.792 to 1.191 ug/g PCBs, compared to the reference site (Winnipeg) with 16 pg/g TEQs, 0.85 pg/g 2,3,7,8-TCDD, and 0.132 ug/g PCBs. (PCB concentrations are reported directly from Fox et al. 25; TEQs were recalculated to include congeners not used originally in Fox et al. 25; 2,3,7,8-TCDD is reported as unpublished data from the same data set.) Mean concentrations in New York Harbor gull chicks in the present study were 47 pg/g TEQs, 4.6 pg/g 2,3,7,8-TCDD, and 0.382 ug/g PCBs, although maximal concentrations reached 157 pg/g TEQs, 31 pg/g 2,3,7,8-TCDD, and 1.100 ug/g PCBs (Tables 2 and 3). (TEQs for the Great Lakes and New York Harbor included the same set of PCDD and PCDF congeners. TEQs for New York reported above included seven PCB congeners with relatively low contributions to TEQs that were not available for the Great Lakes dataset. If these congeners are eliminated, mean and maximal TEQs in New York were slightly lower at 44 and 148 pg/g, respectively.) Mean DDE concentrations in New York gulls (0.033 ug/g) were much lower than pooled DDE concentrations at the Great Lakes sites (0.158 − 0.505 ug/g) and marginally lower than at the reference site (0.059 ug/g) in Fox et al. 25, suggesting that the negative correlation between DDE and PHA response in New York birds may not be causal but instead the result of co-correlation between DDE and immunotoxic dioxins and PCBs. Although no tissues were collected for contaminant analysis in black-crowned night herons in the present study, significant concentrations of dioxins and PCBs have been reported previously in the eggs of this species nesting in and near the Hudson Raritan Estuary 38.
Laboratory studies with chickens have shown that birds can be susceptible to immunotoxic effects at exposures similar to those observed in herring gulls from lower New York Harbor. In chicken embryos exposed from the beginning to the end of incubation (as in the present field study), thymic and bursal atrophy have been observed over a dose range of approximately 13 to 80 pg TEQs/g egg (converting from PCB concentrations to TEQs using World Health Organization toxic equivalency factors) 18–20. As a species, herring gulls are approximately 33 to 50 times less sensitive to 2,3,7,8-TCDD compared to chickens, and 7- to 200-fold less sensitive to other planar PCDDs, PCDFs, and PCBs 39, 40. Still, some individual gulls are as sensitive as the average chicken 39.
Studies spanning a diverse phylogeny of organisms support the biological plausibility of these low exposure effects in herring gulls of New York Harbor. Adductor muscle concentrations as low as 2.0 pg 2,3,7,8-TCDD/g altered gonadal development, egg fertilization, and embryonic development in eastern oysters (Crassostrea virginica) in both controlled dosing studies in the laboratory and field studies, where oysters from uncontaminated areas were transplanted to contaminated areas (Newark Bay and Arthur Kill) 41. Female rainbow trout (Oncorrhynkis mykiss) fed 1.8 ng 2,3,7,8-TCDD/kg food for 300 d experienced significantly reduced survival, as did their offspring 42. These effects occurred at liver concentrations less than 1 ng/kg. Following a spill of PCBs into Saglek Bay, Labrador, Canada, the PHA response in prefledgling black guillemots (Cyphus grille) was suppressed significantly, both statistically and biologically 14. The PHA response was suppressed 50% in a group with mean liver PCBs of only 111 ng/g total PCBs and 70% in a group with mean liver PCBs of 1,928 ng/g. The mean PCB concentration in reference birds was 46 ng/g. Mean liver PCBs in the New York Harbor herring gulls were 3.4 times higher than the intermediate group of guillemots that had a 50% suppressed response.
Potential associations of immunotoxicity with other contaminants
Several classes of chemicals of emerging concern, particularly polybrominated diphenyl ethers (PBDEs) and brominated dioxins and furans were not measured in the present study but likely were present in bird tissues. A survey of halogenated organic contaminants in and around New York Harbor before and after the World Trade Center (WTC) disaster showed that brominated chemicals were often found in higher concentrations than similar chlorinated chemicals 43. Concentrations of PBDEs exceeded those of PCBs in post-WTC sewage sludge, and in some pre- and post-WTC water samples and post-WTC sediment samples. World Health Organization dioxin toxic equivalents for polybrominated dibenzodioxins and furans (PBDDs and PBDFs) were higher than those of PCDDs and PCDFs in WTC runoff and many water and sediment samples. In Japan, livers and eggs of common cormorants (Phalacrocorax carbo) contained significant concentrations of PBDEs, PBDDs, PBDFs, and polybrominated biphenyls (PBBs), showing biomagnification to levels of potential concern 44. The PBDDs and PBDFs were lower than PCDDs and PCDFs. Elevated concentrations of PBDEs were found in herring gull eggs from the Great Lakes 45. The presence of 2,4,6,8-tetrachlorodibenzothiophene (TCDT) in aquatic biota in the Hudson Raritan Estuary 46–48 also suggests the need to quantify polychlorinated dibenzothiophenes (PCDTs) in birds.
The PHA skin response was negatively correlated with liver Se concentrations, but correlations with most dioxins and PCBs were stronger (Table 2). Liver concentrations of Se in herring gull chicks in New York Harbor (mean of 3.4 ug/g dry wt, range 2.6 − 4.7; Table 5) appeared to be below concentrations associated with immunotoxicity, reproductive impairment, and growth retardation. Normal concentrations of Se are generally 12 to 16 ug/g dry weight in bird livers 49 and 7.86 ug/g for herring gulls in the United Kingdom (50 as described by Ohlendorf et al. 51). New York Harbor concentrations were similar to control Se concentrations in laboratory studies. In mallard drakes (Anas platyrhynchos) exposed to sodium selenite in drinking water for 12 weeks, no immunotoxicity was found at exposures that produced mean liver concentrations of 5 to 6 ug Se/g dry weight 52. Controls had liver concentrations of 4 ug/g. Selenomethionine in drinking water increased liver Se to 15 ug/g and significantly reduced T cell function (delayed-type hypersensitivity response to tuberculin antigen) 52. In young mallards fed 10 ug Se/g as sodium selenite or selenomethionine, minimal or no effects on growth were observed 53. Livers of these birds accumulated 12 ug Se/g dry weight (3.5 ug/g wet wt) in one week of exposure and 20 ug/g dry weight (6 ug/g wet wt) after six weeks. Liver Se concentrations in controls were 0.7 to 1.3 ug/g dry weight (0.2 − 0.4 ug/g wet wt).
Contaminants and disease susceptibility. The relationship among pollutant exposure, immune status, and susceptibility to pathogenic diseases or parasitic infestation is a significant concern. In laboratory animals contaminant-induced immunosuppression defined by immune function assays is usually associated with increased morbidity and mortality caused by challenge infections 22. Similarly, field studies have demonstrated associations between contaminants and increased infections in free-living wildlife. More specifically, the PHA skin test results from the present study indicate a strong inverse relationship of T cell function with dioxin-like and total PCB as well as p,p′-DDE residues in prefledgling herring gull chicks. Mice exposed to 2,3,7,8-TCDD exhibited a dose-responsive increase in mortality following an otherwise nonlethal influenza virus infection 54. Low pathogenic avian influenza has been positively reported in many bird species, with low global prevalence in herring gulls (1.4%) 55. No herring gulls tested in New Jersey have been reported positive for low pathogenic avian influenza (55; supporting online material at: www.sciencemag.org/cgi/content/full/312/5772/384/DC1). To date, several reports have found highly pathogenic avian influenza, specifically the H5N1 subtype, in herring gulls from Denmark (U.S. Geological Survey, http://www.nwhc.usgs.gov/disease_information/avian_influenza/affected_species_chart.jsp). Virus-host challenge protocols are available for use in gulls, including H5N1 56, 57, providing a potential avenue for future assessments. Other potential studies might investigate interactions between contaminant-induced immunosuppression and health consequences of dermestid beetles, ectoparasites, whose presence has been identified in black-crowned night heron nests in New York Harbor and other colonies on the northeastern Atlantic coast 58.
Summary of major findings and ecological significance
The present study has demonstrated poor prefledgling survival in herring gulls and suppressed immune function in herring gull and black-crowned night heron chicks on islands in lower New York Harbor. Altered immunological endpoints included severely suppressed T lymphocyte function (PHA skin response) in both species, significantly reduced numbers of developing lymphocytes in the thymus and bursa of Fabricius of herring gull embryos, and altered in vitro lymphoproliferation responses in herring gull chicks. The observed immunosuppression was consistent with the immunological effects of dioxins and PCBs, although exposure was generally lower than in Great Lakes gulls that were also immunosuppressed 12. In herring gull chicks, however, measures of dioxin and PCB exposure exhibited strong negative correlations (r = –0.89 to –0.98) with the PHA skin response (Fig. 3), suggesting that these chemicals contributed to the immunosuppression in New York Harbor birds. Immunological impairments and low prefledgling survival were consistent with the previously reported reduced breeding population numbers in the lower Newark Bay − Arthur Kill area 1. The impaired T and B cell development and function observed in fish-eating birds of lower New York Harbor is consistent with exposure to organochlorines, especially PCDDs and PCBs, although definitive causal associations cannot be made without further investigation.
The biological and ecological relevance of suppressed immune function responses is a significant consideration. Immunotoxicity screening studies in laboratory rodents have shown that immune function endpoints are excellent indicators of immunotoxicity and decreased host resistance in challenge infection experiments 21–23, 59. The present field study of colonial waterbirds from New York Harbor demonstrated alterations in lymphoproliferation responses as well as suppression of the PHA skin response, which is one of the most sensitive integrative tests of T cell function in wild birds 11, 17. In a review of 12 immune function studies, nine of which employed the PHA test, immune responses were the most significant predictors of subsequent survival of young wild birds 60. In an assessment of 280 introduction attempts in 38 avian species, immunocompetence as assessed by the PHA skin response was an important positive predictor of the ability of birds to colonize new areas (i.e., found new local populations) 61. Hence, the immunological endpoints measured in the present field study are some of the best available for detecting immunotoxicity, and the observed changes in these endpoints are likely to have significant effects on disease resistance, survival, and other measures of ecological fitness.