Top predators - Glaucous Gull - Project topic/title:
	Effects of persistant organic pollutants (POPs) on the immune response and retinoid- and thyroid hormone status of Glaucous Gulls.
Co-ordinating institution
	Norwegian Polar Institute
Final  report
	Sagerup, Kjetil; Gabrielsen, Geir Wing; Larsen, Hans Jørgen and Skaare, Janneche Utne:  Effects of persistant organic pollutants (POPs) on the immune response and retinoid- and thyroid hormone status of Glaucous Gulls.
Summary and results
	Summary The present project includes one pilot study of wild adult glaucous gull (Larus hyperboreus) and one experimental study of glaucous gull chicks raised in captivity. The pilot study resulted in the development of a good methodology for assessment of white blood cell activities under field condition. In the experimental study a total of 39 glaucous gull chicks were hatched and raised in captivity in Svalbard, Norway. The chicks were divided into two groups. One experimental group (20 chicks) was given food that mimicked the "natural" food found in the marine environment. The control group (19 chicks) was given "clean" food. After 56 days the chicks were sacrificed in order to collect samples for analyses of organochlorines (OCs) and immune response studies. The experimental group had 2.8, 3.9, 5.0, and 6.1 time’s higher concentrations of HCB, oxy-chlordane, å DDT, and å PCB, respectively, compared to the control group at day 56. All chicks used in the experiment were immunized with various vaccines in order to test their ability to respond against foreign antigens. The demonstration of decreased ability to produce antibodies to influenza virus in growing glaucous gull chicks exposed to OC provide important information on toxic effect of OCs. In the experimental model we focused on the measurement of protective antibodies and it is therefore reasonable to assume that the OC exposure is associated with decreased resistance to infections. The effect of PCB exposure on the lymphocyte proliferation in glaucous gulls was tested following in vitro stimulation with mitogens. There was significant higher response of peripheral blood lymphocyte to PHA and LPS whereas the lymphocyte response to Con A and PWM were not influenced by the PCB exposure. Increased lymphocyte response to PHA indicates a general stimulation of the immune system following a short-term exposure to PCB that could be triggered off by increased exposure to environmental microbes. Scientific results The goals for the present project were: 1. Method development for immunological studies. 2. Effects of PCB on neonatal immune system. 3. Effects of PCB on specific immune response and resistance against infections. 4. Effects of PCB on lymphocyte proliferation. 5. Effects of in vitro exposure of lymphocytes to PCB. Most of our goals were reached in the present study. Some new methods were developed and some conclusions could be made. The chicks responded when tested against the influenza virus. In this test the mean antibody titre in the exposed group was significantly lower than the in control group, indicating depressed response ability to different diseases. In glaucous gull chicks the mitogen-induced response of circulating blood lymphocytes to PHA and LPS was increased in the exposed group when compared to the control group. In vitro exposure of lymphocytes from glaucous gull chicks to the PCB congeners 99, 126, 153, and 156 indicate that the response to the in vitro exposure vary. Significant differences for response was found between groups after stimulation with PCB 126 and ConA. The glaucous gull chicks in the experimental group received the same amount of pollutants that a naturally existing population of glaucous gull in the Svalbard area receives. The combined effects of the mixture of pollutant have a negative effect on the immune system of glaucous gull chicks. The observed suppression of the immune system is shown by a lower response to the influenza vaccine, which indicate a suppressed resistance to foreign substances such as different diseases. Relevance for monitoring (only in Norwegian) Resultatene indikerer at dagens forurensningsnivå av organiske miljøgifter kan virke negativt på polarmåkers immunforsvar. Dette betyr at vi må være observant på utviklingen av langtransporterte forurensninger. Studien er unik i den sammenheng at det ikke er brukt kommersielt fremstilte kjemikalier til forsøket. Vi har gitt fuglene deres naturlige kost med det innholdet av miljøgifter som finnes i den maten de spiser. Disse miljøgiftene har vært utsatt for alle påvirkninger som miljøet gir, fysisk, kjemisk og biologisk. Effektene må forstås som en total effekt av den miksturen av stoffer som finnes i føden til fuglene. Vi kan ikke skille ut noen av stoffene og si at et stoff er mer skadelig enn andre. Dessuten må vi inkludere at stoffene kan ha en kombinert effekt. Dette betyr at selv om to (eller flere) stoffer hver for seg ikke har noen effekt kan de to samvirke og gi effekter.
Original project description
	Summary: High levels of POPs have been found in glaucous gulls in the Barents Sea area. Glaucous gulls on both Svalbard and Bear Island have been found dead or dying. All dying and dead birds had high levels of POPs, but no cause of death have been determined. Due to seabirds low ability to metabolise pollutants and their position in the arctic marine food web, the glaucous gull can accumulate high levels of POPs. Associations between organochlorine (OC) concentrations and suppressed immune functions have been documentet in seals and seabirds. Further, vitamin A deficiencies has been linked to immuno suppression and susceptibility to disease. Retinoid deficiency is associated with a number of reproductive parameters in birds. How the immune system and retinoid and thyroid hormone status in birds are affected by POPs are poorly understood. To find biological and ecological effects of individuals and the glaucous gull population, we want to study the immune and hormonal systems in this species after exposure to a «natural» mixture of POPs. Methods and results of this experiment will be used in a future monitoring of glaucous gull in the Barents Sea area.   Aims Characterise the effects of POPs on immune competence, immune response ability and resistance against infections in glaucous gull in order to evaluate if these biomarkers can be used in future biomonitoring. Apply retinol and retinyl-palmitate as biomarkers to study effects of persistent organic pollutants in the glaucous gull, and to use these biomarkers in biomonitoring.   Background A large number of persistent organic pollutants (POPs) accumulate in organisms of the arctic marine ecosystem (Borgå et al., 1998). Polar bear (Ursus maritimus) and glaucous gull (Larus hyperboreus), which is the top-predators in this system, are found to have the highest levels of POPs (Norstrom et al., 1988; Bernhoft et al., 1997; Gabrielsen et al., 1995; Mehlum & Daelemans, 1995). Monitoring studies of several different seabird species collected from colonies in the Barents Sea area have revealed high values of polychlorinated biphenyls (PCB) in glaucous gulls (Gabrielsen et al., 1995). Brain tissue in glaucous gulls found dead or dying in colonies in the Barents Sea area had PCB values from 1-30 ppm or 10-300 times higher than values found in normal, healthy individuals. Autopsy of the birds did not reveal any cause of death. However, it has been suggested that high levels of PCB in brain tissue have contributed to the death of glaucous gulls on Bear Island (Gabrielsen et al., 1995). In the arctic marine ecosystem the glaucous gull is the most important avian predator. They mainly eat eggs/chicks of different bird species, polar cod, amphipods and crabs (Lydersen et al., 1989; Barry and Barry, 1990). They also eat garbage near human settlements and leftovers like carcasses and blubber from seals from polar bears meals. Seabirds, included the glaucous gull have revealed relatively low ability to metabolise pollutants (Walker et al., 1984). Due to this limited ability and their position high up in the marine food web, the glaucous gull can accumulate high levels of POPs. The impacts of pollution are likely to be expressed through effects operating at the cellular level which are ultimately integrated into the physiology of the individual and population level effects. Associations between OC concentrations and suppressed immune function have been observed in studies on seals (de Swart et al. 1994; Ross et al. 1996), mice (Luebke et al. 1994), Caspian terns (Sterna caspia) and herring gulls (Larus argentatus) (Grasman et al. 1996). Harbor seals (Phoca vitulina) fed herring (Clupea harengus) from the contaminated Baltic sea had reduced natural killer cells` activity and lower in vitro T-lymphocyte response, compared to seals fed fish from the less contaminated Atlantic Ocean (Ross et al. 1996). The seals fed Baltic herring also had higher levels of circulating polymorphonuclear granulocytes, which may indicate an increase in the occurrence of bacterial infections (de Swart et al. 1994). In prefledging Caspian terns and herring gulls from the contaminated North American Great Lakes, a suppression of T-cell-mediated immunity was associated with high exposure to persistent OC contaminants (Grasman et al. 1996). In a recent study, Sagerup (1998) found positive correlations between nematode intensities and OC concentrations in glaucous gulls from Bear Island. Immunological effects of high doses of PCB have been demonstrated in experiments with captive animals. These effects may explain increased susceptibility for infections against several microbes (which is also registered). Extrapolation of results from one species to another is not necessarily correct because of differences in physiology and sensitivity. However, performing immune registrations in glaucous gulls with high and low levels of PCB will increase the understanding of what importance PCB exposure in polar regions has on the organism. Registrations of effects on immune function will also be a more sensitive parameter to measure than toxic effects on cells (histopathology). Immunisation of glaucous gull chicks with test antigens, which against they do not have maternal immunity, will give us an opportunity to measure the birds ability of immune response and relate it to a PCB level. Retinoids are important to several aspects of avian reproduction and development. Defiency of retinoids is associated with changes in secondary sexual characteristics, testes weight, spermatogenesis, egg laying, egg size, embryo survival, incubation time and deformities (Moore, 1957; Thompson, 1970; Thompson, 1976). Vitamin A defiency has also been linked to immuno suppression and susceptibility to disease (Boily et al., 1994). Persistent organic pollutants have been demonstrated to affect the retinoid-status in birds (Boily et al., 1994; Murvoll, 1996; Spear et al., 1990). Even though, the mechanisms involved have been linked to the Ah-receptor, to PCB-metabolites, to induction and/or inhibition of liver enzymes, and to disruption of plasma-transport proteins (Liu et al., 1995; Chen et al., 1992; Brouwer and van den Berg, 1986), the mechanisms involved are yet poorly understood, especially in birds. It is therefore important to establish knowledge on the effects of persistent pollutants on arctic top-predators. In a study on heron eggs in relation to St Lawrence river contamination, Boily et al. (1994) demonstrated that there were significant differences in retinoid status in the yolk between breeding colonies. It was also demonstrated that effects on retinol status was observed at very low concentrations in the eggs. It was concluded that retinyl palmitate was a useful sensitive and non-invasive biomarker for monitoring organochlorine contaminant effects. The hypothesis for this proposal is that understanding the immune system in glaucous gull with high levels of organochlorines will provide insight into the effects of pollution. Studies of retinol and retinyl-palmitate levels in glaucous gull exposed to POPs have demonstrated that these may be used as biomarkers. The methods used will hopefully provide a monitoring method for assessing population status over time. In the future these methods are meant to be tested on glaucous gulls at Bear Island in order to find good monitoring methods for effects of OCs.   Experiment Animals A total of 40 individuals is needed for this experiment, further divided in two groups with 20 birds each. One group will be a control group, the other an POP exposed group. Experimental design Up to 60 glaucous gull eggs will be collected in Isfjorden and Kongsfjorden and placed in a hatching machine at the Norwegian Polar Institute Research Station in Ny-Ålesund. It will be an advantage to collect more eggs than needed, in case not all the eggs hatch. The first five days the chicks will be held indoors under heat lamps at 37 ° C. Later they will be placed outdoors, but protected against wind and rain, in two cages with a size of 3 m x 4m x 2m each. The chicks will be divided into two groups with minimum 20 individuals in each. One group will be «clean» (control) and one is supposed to be polluted through the food. All the chicks will have a basic diet consisting of polar cod, water and vitamins (»fish-eater tablets», Mazuri Zoo Foods in England). The «polluted» group will also be fed with eggs of gulls. Seabirds and seabird eggs are suggested to be one of the most polluted ingredients in the diet of glaucous gull. To ensure a diet as equal as possible, the «clean» group will have hen eggs when the other group has gull eggs. The chicks will be fed ad lib 5 times a day. By giving an appropriate amount gull egg to the chicks in the polluted group, we can give the chicks about the same amount of organochlorines as an adult bird has in its body. To attain this, each chick should have about 4 kg gull egg during the experiment which will last for 8 weeks. A similar experiment was performed the summer 1997 by Espen O. Henriksen, a graduating fellow at the Norwegian Polar Institute. He attained 100 % survival of the chicks after completed hatching. Except one chick which did not eat properly, all the chicks also grew normally for 30 days in captivity. Cand. scient Nina Skjegstad was field assistant and participated in all tasks during the experiment as feeding, cleaning, blood and tissue sampling, and ending of the experiment.   Methodology/objectives The main project is divided in two separate parts; one concerning immunological studies, including some method development; and one concerning endocrinology in relation to immunology and reproduction.   I ) Immunology The project consists of five independent experimental elements which here are defined as objectives: Method development for immunity studies. The effects of PCB on neonatal immunity. The effects of PCB on specific immune response and resistance against infections. The effects of PCB on lymphocyte proliferation. The effects of in vitro exposure of lymphocytes to PCB.   Objective 1: Method development for immunity studies. Method development for immunity studies on glaucous gull. Will be performed during the autumn 1998. Purification of IgG from glaucous gull and production of polyclonal antibody serum Establishment of method for quantification of serum IgG in glaucous gull.   Objective 2. The effects of PCB on neonatal immunity. Effects of PCB on the immunoglobulin (IgG) levels in chicks. Measurements of serum IgG constitute the chicks total amount of antibodies of this type of immunoglobulin in blood. Because of uptake of maternal IgG via the egg, the chicks will have high levels of IgG in serum 2-7 days after hatching after which serum IgG will be reduced (week 4) until the self production is significant (week 6-8). The level of serum IgG will be built up over time after exposure to micro organisms in the environment. SerumIgG is supposed to protect the chick against reinfections but has also a certain effect against primary infections. To a certain degree the IgG level also will reflect parts of local immunity. Measures of serum IgG will give an indication of if the PCB burden affects the production of immunoglobulin which is important for the neonatal immunity. Effect of PCB on the specific antibody titer against environmental microbes. Measuring of specific antibodies against environmental microbes at different times will give indications of neonatal immune competence.   Objective 3. The effects of PCB on specific immune response and resistance against infections. Measures of antibody production is based on immunisation. One chooses protective antibody response against selected antigens which makes us able to draw conclusions about effects of PCB on the chicks infection resistance. Immunisation may start at an age of 2-3 weeks. What antigen combination and concentration one chooses depends on this time. Blood samples will be taken to analyse content of antibodies after 3-4 weeks in order to measure primary response. At this time the chicks cam be immunised once more in order to measure secondary response two weeks later. Primary and secondary immune responses represent complex but different immunological events which provide different information about effects of PCB. Measuring specific antibody titer against environmental microbes as Pasteurella multocida will prevail how the individuals have responded in the time between the sampling times. Seen together with IgG measures one will have an impression of how PCB affect the infection resistance of the chicks.   Objective 4. The effects of PCB on lymphocyte proliferation. The effect of PCB on lymphocyte proliferation has to be assayed within 6 hours after sampling. Measures of the general ability of lymphocytes to proliferate after in vitro stimulation with mitogens may document if PCB affect normal maturing of the immune system in chicks. Measures of the lymphocytes specific ability of proliferation after immunisation with antigens will demonstrate effect of PCB on specific cellular immune response. Measures of the lymphocytes proliferation after in vitro stimulation with antigens following immunization will demonstrate effect of PCB on specific cellular immune response. Hemathology will be a part of the measuring methods used to evaluate effect on cellular immune system.   Objective 5. The effects of in vitro exposure of lymphocytes to PCB. The effect of in vitro exposure with PCB on the lymphocyte proliferation (see above), and will be performed on lymphocytes from gulls belonging to both groups.   I I ) Endocrinology The study concerning retinol and retinyl-palmitate is divided into two parts, one on adult birds and one on eggs. Adults - Retinol and retinyl-palmitate will be analysed in plasma samples from adult gulls. These are gulls that are sacrified for other reasons in other parts of the project (se above). Information on retinoids are important in order to interpret the possible effects of POPs observed on other variables such as immuno competence. Eggs - Retinol and retinyl-palmitate will be analysed in the yolk of glaucous gull eggs from different locations (10 eggs from each location), and the concentrations will be related to PCB-concentrations in the yolk. It will be important to standardise sampling procedures in order to collect eggs at the same age. This part of the project will give information on the possible effects on the developing embryo caused by POPs, as well as beeing an important part of a monitoring programme (especially when linked to pollutant levels). It will also be possible to link this monitoring project to data on individual birds in the different colonies, on their population variables such as breeding success, mortality etc. Sampling of the eggs will be performed by other teams in the field who will pack and store the samples as instructed. The methods for analysing retinol in plasma and yolk of birds have been established through several projects at Allforsk and the Department of Zoology, NTNU (Henriksen et al., 1998; Murvoll, 1996; Nilssen, 1997). Some additional work will be needed to establish the method for analysing retinol palmitate.   Time frame for project (1998/1999)   Sep 98 Mar 99 A M J J A S O Nov 99 Sampling for method development x                   Planning   x x x x           Collecting eggs for food       x             Collecting eggs for hatching         x           Experiment         x x x       Analysis             x x x x Reporting                 x x   Total budget NOK 500 000,-. In this project NP will contribute with an equity finance of 400,000,- NKR. Norwegian College of Veterinary Medicine will contribute with about 150,000,- NKR, and NTNU will contribute with 25,000,- NKR.   References Barry, S.J. and Barry, T.W., 1990. Food habits of glaucous gulls in the Beaufort Sea. Arctic, 23 (1): 43-49. Bernhoft, A., Wiig, Ø. and Skaare, J.U., 1997. Organochlorines in polar bears (Ursus maritimus) at Svalbard. Environ. Pollut., 95 (2): 159-175. Boily, M. H., Champoux, L., Bourbonnais, D. H., DesGranges, J. L., Rodrigue, J. & Spear, P. A., 1994. ß-carotene and retinoids in eggs of great blue herons (Ardea herodias) in relation to St Lawrence River contamination. Ecotoxicology, 3, 271-286. Borgå, K., Gabrielsen, G.W., Hop, H. and Skaare, J.U., 1998. Organochlorines and trophic positions in a marine pelagic food chain leading to seabirds in the Norwegian Arctic. Organohalogen Compounds, 39: 431-434. Brouwer, A. & van den Berg, K. J., 1986. 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