12Concentrations of 23 elements (Be, Al, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, 13Se, Rb, Sr, Mo, Pd, Cd, Sn, Pt, Pb) were evaluated in whole blood samples of live 14 harbor seals (Phoca vitulina) from two different locations in the Wadden Sea, the 15 Lorenzenplate in Germany, and the Danish island RØmØ. Elemental blood levels 16 were compared to data from literature of seals, other marine mammals and humans. 17While homeostatically controlled elements showed no differences, concentrations of 18As, Cr, Mn, Mo, Se, and V were higher than human levels. Furthermore, animals 19 from both locations showed significant geographical differences in whole blood 20 concentrations of Al, Mn, Cu, and Pt. These findings could be explained by 21 differences in feeding areas. The element pattern was not affected by gender. In 22 conclusion, these findings indicate an impact of the environment on biochemical 23 blood parameters of the harbor seals. The significant differences of elements in blood 24 samples of two groups of seals, which were associated with geographical variations 25 2 of prey support the use of element pattern in blood as tool for investigation of 1 environmental impact on seals. 2 3

Environmental pollutants may affect the immune system of marine mammals in many areas of the industrialized world. This study provides the first evidence for metal-induced hypersensitivity in harbor seals and demonstrates a relationship between this immunopathy and the level of metals in blood. The concentrations of 20 essential and nonessential elements were analyzed in the blood of 13 harbor seals from the North Sea. In addition, their T-lymphocyte response to metals in terms of hypersensitivity was investigated using a lymphocyte transformation test (LTT) according to the MELISA (memory lymphocyte immuno-stimulation assay) modification. The results showed metal hypersensitivities in 7 of 11 seals investigated in MELISA (data from two seals could not be assessed), reflecting a positive or possible positive reaction in 13 of 154 total single tests. Four animals responded to one metal and three animals to multiple metals. The sensitizing metals were molybdenum (Mo), titanium (Ti), nickel (Ni), chromium (Cr), aluminum (Al), lead (Pb), and tin (Sn). Furthermore, the seals with a Ni-, Al-,.and Cr-sensibilization showed the highest concentrations of these metals in blood. In 8 of the 13 positive cases, elevated blood metal concentrations correlated with the hypersensitivity reaction. Summarizing, we demonstrate in this first pilot study the potential immunological impact of metals in seals, a topic rarely investigated previously. Our results show the value of a combined biological and effect-monitoring tool to investigate pollution-induced immunopathies in live animals.

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The Elbe is one of the major rivers releasing pollutants into the coastal areas of the German North Sea. Its estuary represents the habitat of a small population of harbor seals (Phoca vitulina). Only little is known about the health status and contamination levels of these seals. Therefore, a first-ever seal catch was organized next to the islands of Neuwerk and Scharhörn in the region of the Hamburg Wadden Sea National Park. The investigations included a broad set of health parameters and the analysis of metals and organic pollutants in blood samples. Compared to animals of other Wadden Sea areas, the seals showed higher γ-globulin levels, suggesting higher concentrations of pathogens in this near-urban area, elevated concentrations for several metals in particular for V, Sn, Pb, and Sr, and comparable ranges for chlorinated organic contaminants, except for elevated levels of hexachlorobenzene, which indicates characteristic inputs from the Elbe.

and IL-12 were found at admission, consistent with an activated immune system, whereas the anti-inflammatory cytokine IL-4 was increased before release, suggesting recovery from infections and a mature immune system. This study indicates that cytokines detection is highly sensitive for the status of the immune system of harbor seals.

Harbour seals (Phoca vitulina) are bio-indicators for the assessment of their habitat and environmental changes. Besides population parameters and trends (survival, age structure, sex ratio), the individual health status represents a further important parameter for this assessment. The health status of seals is a complex and vague term, determined by a wide range of diagnostic parameters. Quantities of important blood proteins such as transferrin (Tf), as well as altered distribution patterns of its glycoforms, are frequently used as biomarkers in clinical diagnosis. Within this context Tf quantities and a varying pattern of its glycoforms are used as indicator for e.g. certain liver diseases, which also represents one of the most frequently observed pathological indication in harbour seals of the North Sea. Currently, most assay based quantification methods for Tf are limited since they often provide only information regarding the total Tf concentration rather than information of its different glycoforms. Due to a lack of suitable seal Tf antibodies also the application of more specific antibody based approaches is not possible. Within this background a new approach for the absolute quantification of the iron-transport protein Tf in the blood of harbour seals using its characteristic iron content and HPLC-ICP-MS detection is described. Method validation was performed using a certified human serum reference material (ERM-DA470K/IFCC). A Tf concentration of 2.33 AE 0.03 g L À1(sum of all quantified glycoforms) has been calculated, which is in good agreement with the certified total Tf concentration of 2.35 AE 0.08 g L À1 , confirming the accuracy of the proposed analytical method. Finally, different seal samples were analysed to demonstrate the suitability of the procedure for the quantification of Tf in real samples as well as to observe modified glycoform patterns. Compared to our previous studies for the first time it was possible to quantify the serum Tf baseline reference range for male (1.42-2.35 g L À1) and female German North Sea seals (1.93-2.74 g L À1 ) as well as a CDT level of 0.00-0.10 g L À1 , respectively, which provides valuable further diagnostic information regarding the health status of these specific marine mammals. Compared to assay based quantification approaches the proposed technique indicates great potential to obtain comparable and traceable absolute quantitative results, which are in particular important for long term investigations. This absolute quantification is based on an accurate, traceable element standard, while assay based approaches often show variations depending on the kit quality or changing activities of the used antibodies.

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