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 of adult gulls gave us enough blood and tissue samples to develop the methods needed for immune system analysis in the laboratory experiment. 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 immunocompetence measurements. The experimental group had 2.8, 3.9, 5.0, and 6.1 time’s higher concentrations of HCB, Oxychlordane, ?DDT, and ?PCB, respectively, compared to the control group at day 56. All chicks used in the experiment were immunised with various vaccines and sera in order to test their ability to respond against foreign antigens. The experimental chicks produced low levels of virus neutralising antibodies when tested against the herpes virus and reovirus. They produced higher levels of neutralising antibodies when tested to tetanus toxoid. There was, however, no difference between the experimental groups with regard to the mean antibody titres. The chicks in both groups also responded to the influenza virus by increasing the production of specific antibodies. However, the mean antibody titre in the exposed group was significantly lower than in the control group. The mitogen-induced response of blood lymphocytes to PHA and LPS was significantly higher in the exposed group compared to the control group. The specific response of blood lymphocytes to Con A, PWM, KLH, TET, and PPD was higher in the exposed group compared to the control group. However, do to high variance in the exposed group there was no significant difference between groups with regard to the lymphocyte response to these mitogens. The results from the present study indicate a toxic effect of OCs on the glaucous gull chicks, which induced a systematic activation of the immune system. Further work on data will be performed.
Sverdrupstasjonen Norwegian Polar Institute Ny-Ålesund Svalbard
Whole animals, glaucous gull. frozen at Norwegian Polar Institute in Tromsø
Organochlorine analysis Analyses of OCs were performed at the Environmental Toxicology Laboratory at the Norwegian School of Veterinary Science in Oslo. OCs were quantified in blood samples using the methods described by Brevik (1978) with modification by Bernhoft et al. (1997). Briefly, samples were homogenised and extracted twice with cyclohexane and acetone using an ultrasonic homogeniser. Extractable fat was determined gravimetrically and cleanup was done with ultra-clean sulphuric acid. The samples were injected on a gas chromatograph with a 60-m SB-5 and SPB-1701 capillary column and an electron capture detector. Due to the strong correlations between different PCB-congeners in glaucous gull samples from the Svalbard area Henriksen et al. (1998) we considered it cost-efficient to quantify only nine of the major PCB-congeners in the blood and fish samples. High correlation between congeners would in any case prevent assigning possible effects to specific PCB-congeners. The following OCs were determined in the blood samples; hexachlorobenzene (HCB), -oxychlordane, p,p'-DDE (1,1-dichloroethylene bis[p-chlorophenyl]), p,p'-DDT (dichlorodiphenyl-trichloro-ethane), and 9 PCB-congeners (IUPAC nos.): 28, 99, 101, 118, 138, 153, 156, 170, and 180. In addition trans-chlordane, cis-chlordane, trans-nonachlordane, and PCB congeners 47, 74, 66, 149, 114, 105, 187, 128, 196, 189, 194, 206 were analysed in the blood samples from the field experiment in 1998. In the fish samples HCB, oxychlordane, p,p'-DDE, and nine PCB congeners (28, 52, 99,101, 118, 138, 153, 170, and 180) were analysed. It was decided to analyse an enlarged series for the egg samples. This to have an as good as possible overview over the exponation of OCs given. The following OCs were analysed in eggs; HCB, ?-HCH (1,2,3,4,5,6-hexachlorocyclohexanes), ?-HCH, ?-HCH, oxy-chlordane, trans-chlordane, cis-chlordane, trans-nonachlordane, p,p’-DDE, o,p’-DDD, p,p’-DDD, p,p’-DDT, mirex, and 33 PCB congeners (IUPAC numbers; 28, 31, 52, 47, 74, 66, 56, 101, 99, 87, 136, 110, 151, 149, 118, 114, 153, 105, 141, 137, 138, 187, 183, 128, 156, 157, 180, 170, 199, 196, 189, 194, and 206). Percentage recoveries were in the in the range of 80-115%. Quantification limits for individual OCs were 0.9-28 ng g-1 extractable liver lipids. The laboratory’s analytical quality has been approved in several intercalibration tests, including the four steps of the ICES/IOC/OSPARCOM intercomparison exercise on the analysis of PCBs in marine media. The laboratory is accredited (1996) by the Norwegian Accreditation as a testing laboratory for quantification of PCBs and selected chlorinated pesticides in biological matrices according to the requirements of NS-EN 45001 and ISO/IEC guide 25. Immune analysis Method developments for immunological studies on glaucous gull were started in September 1998 when 19 birds of different age were sampled. Blood and spleen lymphocytes were isolated in the field to measure their general immune response. Techniques for measuring lymphocyte proliferation were also developed. The aim of the immunological part of the project was to develop knowledge and methods to test the immune competence and the immune response in glaucous gull. By testing the immune response to specific antigens and measuring specific lymphocyte activities it would be possible to assess the ability of glaucous gulls to resist specific infections without performing challenge studies. In 1998 an operational cell culture laboratory were established in the field. The immune response of lymphocytes was measured in blood collected from glaucous gulls of different ages. Lymphocytes were isolated from peripheral blood by using different purification techniques and a method was developed for measuring lymphocyte proliferation. A whole blood technique was also tested without success in glaucous gull. The capability of the lymphocytes to respond was measured by the lymphocyte proliferation test using in vitro stimulation with different mitogens such as Phytohemagglutinin (PHA), Concanavalin A (Con A), Poke Weed mitogen (PWM), purified protein derivate of mycobacterium (PPD), lipopolysakkarid of E.coli (LPS).
Norwegian College of Veterinary Medicine, Oslo, Norway National Veterinary Institute, Oslo, Norway
Transport and effect program, phase 1 - Norwegian Polar Institute