Essential and Toxic Elements in Blood Samples of Harbor Seals (Phoca vitulina) from the Islands Helgoland (North Sea) and Anholt (Baltic Sea): A Comparison Study with Urbanized Areas.

The harbor seals (Phoca vitulina) from Helgoland (North Sea) and Anholt (Kattegat, Baltic Sea) are top predators within the marine food web and an indicator species of the environmental contamination. Furthermore, they are a main tourist attraction. Despite these important roles, little is known about the health and pollutant contamination of these seals. The objective of this study was therefore to investigate 18 essential and nonessential/toxic elements (Al, As, Be, Ca, Cr, Cu, Fe, K, Mn, Mo, Ni, P, Pb, Rb, S, Se, Sr, and Zn) in blood samples using inductively coupled plasma mass spectrometry and total X-ray-fluorescence spectrometry. Blood concentrations of mineral nutrients, such as Ca, K, P, and S, were within the reference ranges described for harbor seals. Likewise, for the trace elements, As, Be, Rb, Se, and Sr, no significant differences were observed compared with previous studies. Interestingly, blood concentrations of nine nonessential as well as essential trace metals (Al, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Zn) measured significantly lower in the offshore living seals from Helgoland and Anholt compared with results obtained from animals living close to urbanized areas, such as the Wadden Sea and Elbe estuary. This suggests that industrial emissions, sewage deposition, shipping traffic and dredging tasks might be the cause of increased metal concentrations of inshore harbor seals.

Are metal-induced hypersensitivities in harbor seals associated with liver function?

Environmental exposure to metals is believed to affect marine mammal health adversely including immunosuppression or acute as well as chronic inflammatory processes leading to hypersensitivities or autoimmune diseases. Metal-specific hypersensitivities were found in several pinnipeds of the North Sea. However, hypersensitivity is a complex phenomenon whose characteristics are still not completely understood; in particular, effects on health are not well established. In the present study, we compared basic hematological and biochemical parameters of seals with and without metal-specific hypersensitivities. We found altered hematological parameters and liver enzyme patterns in seals with a metal-induced hypersensitivity, including a reduction in macrophages, an increase in lymphocytes, and elevated levels of lactate dehydrogenase. These findings support the suggestion of a chronic influence of metal pollutants on the health of marine mammals of the North Sea.

Absolute quantification of transferrin in blood samples of harbour seals using HPLC-ICP-MS.

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 ± 0.03 g L(-1) (sum of all quantified glycoforms) has been calculated, which is in good agreement with the certified total Tf concentration of 2.35 ± 0.08 g L(-1), 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(-1)) and female German North Sea seals (1.93-2.74 g L(-1)) as well as a CDT level of 0.00-0.10 g L(-1), 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.

Metallomics approach for the identification of the iron transport protein transferrin in the blood of harbour seals (Phoca vitulina).

The health status of marine mammals such as harbour seals (Phoca vitulina) represents an indirect but powerful way for the assessment of environmental changes. The present work illustrates the first investigation and characterisation of Tf isolated from blood samples of North Sea harbour seals with a view to using changes in Tf isoform patterns as an additional parameter in extended studies of their health status. Therefore, an HPLC-ICP-MS approach has been developed which allows the highly resolved separation and fractionation of up to eight different Tf isoforms, as well as their sensitive and specific detection on the basis of their characteristic iron content. Molecule-specific detection techniques such as nanoLC-ESI-QTRAP-MS or MALDI-TOF-MS were used as complementary techniques to unambiguously identify the isolated proteins as Tf via cross species protein identification and to further characterise the molecular weight as well as the sialic acid content, which is responsible for the elution behaviour of the different isoforms during their ion exchange separation. A molecular mass above 80 kDa has been measured for the different seal Tf isoforms, which is in good agreement with the known molecular mass in other mammalian species, while the estimated pI of the different isoforms indicates some differences in comparison to other species. A number of homologies to known Tf sequences have been observed, which finally allows the cross species protein identification. The combined metallomics orientated analytical approach, which includes the complementary application of element and molecule-specific detection techniques, opens up interesting possibilities for the fast and targeted isolation and identification of a diagnostically relevant metal containing protein from an un-sequenced mammalian species prior to its utilisation in extended studies.

First health and pollution study on harbor seals (Phoca vitulina) living in the German Elbe estuary.

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.

Essential and non-essential elements in tissues of harbour porpoises (Phocoena phocoena) stranded on the coasts of the North and Baltic Seas between 2004-2006.

This study on harbour porpoises (Phocoena phocoena) stranded along the coasts of the Eastern North and Western Baltic Sea as well as in the river Elbe during 2004-2006, evaluated concentrations of 20 essential and non-essential elements (Ag, Al, As, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Ni, Pb, Se, Sn, V, Zn) in liver and muscle samples. Tissue samples of 22 porpoises were taken during post-mortem investigations at the Research and Technology Centre (FTZ) in Büsum, Germany. A multi element method utilizing microwave accelerated acid digestion for sample preparation and collision/reaction cell inductively coupled plasma mass spectrometry (CC-ICP-MS) was used for element quantification. All 20 elements investigated could be determined in liver and muscle tissues except for Al in muscle samples. Furthermore the concentrations in liver tissues were higher compared to muscle tissues. While sex specific differences were observed only for Cu concentrations in liver tissue, age-dependent relationships were obtained for nine elements. Differences between juveniles and adults were found for Ag, Al, Co, Mn, Mo, Se, Sn, Pb, and V concentrations in liver, as well as Sn concentrations in muscle tissues. Furthermore, As and Sn concentrations in liver and muscle showed differences between the stranding locations. This multi-element study on harbour porpoises gives baseline information to concentrations of essential and non-essential elements in tissue to develop reference ranges for health status determination as well as the assessment of the pollutant body burden.

Effects of methyl-, phenyl-, ethylmercury and mercurychlorid on immune cells of harbor seals (Phoca vitulina).

Mercury (Hg) is present in the marine environment as a natural metal often enhanced through human activities. Depending on its chemical form, Hg can cause a wide range of immunotoxic effects. In this study, the influence of methyl-, ethyl- and phenylmercury as well as mercurychloride on immune functions was evaluated. Two parameters of cellular immunity, proliferation and mRNA cytokine expression of interleukin-2, -4, and transforming growth factor beta, were investigated in harbor seal lymphocytes after in vitro exposure to Hg compounds. While all Hg compounds had a suppressive effect on proliferation, differences between juvenile and adult seals were found. Lymphocytes from juveniles showed a higher susceptibility to the toxic effect compared to lymphocytes from adults. Furthermore, the degree of inhibition of proliferation varied among the four Hg compounds. The organic compounds seem to be more immunotoxic than the inorganic compound. Finally, for the cytokine expression of methylmercury-incubated lymphocytes, time-dependent changes were observed, but no dose-dependency was found. Marine mammals of the North Sea are burdened with Hg, and lymphocytes of harbor seals may be functionally impaired by this metal. The present in vitro study provides baseline information for future studies on the immunotoxic effects of Hg on cellular immunity of harbor seals.

Concentrations of selected essential and non-essential elements in blood of harbor seal (Phoca vitulina) pups of the German North Sea.

This study on harbor seal (Phoca vitulina) pups of the North Sea evaluated concentrations of 14 essential and non-essential elements (Al, As, Be, Ca, Cd, Cr, Fe, Mn, Mo, Ni, Pb, Se, Sn, and Zn) in whole blood samples. The essential elements are analyzed to give references for health status determinations of pups. The measurement of classic toxic metals, like Pb or Cd, and other elements that may be in toxic concentrations in blood, is important due to their influence on health, particularly on the immune system. Blood samples of six seal pups found on the German Wadden Sea coast of Schleswig-Holstein in 2004 and transported to the Seal Centre Friedrichskoog, Germany were collected. The blood sampling was performed three times, immediately after collection of the newborns, after 1.5 months, and after 2 months before their release back into the wild. Inductively coupled plasma mass spectrometry and total reflection X-ray fluorescence spectrometry were used to determine the element concentrations. We found higher concentrations of Al, As, Fe, Mo, and Zn in blood samples of newborns compared to samples collected later, probably due to transplacental and lactational transfer from mother to fetus. Furthermore, there is a high need for, in particular, Fe and Zn in the developing organism, which may cause reduced values after some month. In contrast, the concentrations of Be, Cd, Ca, Cr, Pb, Mn, Ni, Se, and Sn, which were low in newborns and increased during the study, may be due to the fish fed to infant pups. Compared to free-ranging adults, in pups, the concentrations of Al, Ni, and Pb were higher in contrast to lower concentrations of As, Mn, and Mo. This case study is the first report on element levels in harbor seal pups of the North Sea.

Blood metal levels and metal-influenced immune functions of harbour seals in captivity.

Immunological blood parameters and the effects of environmental pollutants on the immune system are important to assess the health status of seals. Animals living permanently in seal centres are useful for development and validation of diagnostic tools for free-ranging animals. In this study, parameters of cellular immunity as well as metal concentrations in blood and metal influence on cell proliferation of seven seals from a seal centre were investigated repeatedly using multi-element analysis and a lymphocyte proliferation assay. The metal concentrations, except for tin and chromium, were in general comparable to those of free-ranging animals of the North Sea. The unstimulated and mitogen-stimulated lymphocyte proliferation showed strong intra- and inter-individual variability, which reflected variability in activation of the immune status. Furthermore, both immunosuppressive and stimulative influences of metals on lymphocytes were found. Summarising, the methods used in this investigation provided useful information on these animals, and their application to free-ranging animals can be recommended.

Trace element concentrations in blood of harbor seals (Phoca vitulina) from the Wadden Sea.

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

Metal-induced impairment of the cellular immunity of newborn harbor seals (Phoca vitulina).

The cellular immunity of newborn harbor seals and the influence of pollutants are rarely investigated. This study evaluated the lymphocyte proliferation using a lymphocyte proliferation test (LTT) to understand the dynamics of immune response in seal pups of varying ages from the moment they arrived in a seal center after active beaching until their release into wildlife 3 months later after rehabilitation. Moreover, the effect of various metals (Ag, Al, Au, Be, Cd, Co, Cr, Cu, different Hg compounds, Mo, Ni, Pb, Pd, Pt, Sn, Ti) on lymphocyte proliferation in terms of immunosuppression and hypersensitivity was investigated. First, a strong lymphocyte proliferation in newborns as a reflection of relative immunocompetence was found. Second, different metal-induced influences on lymphocyte proliferation such as specific inhibition by Be, Cd, Hg, and Sn as well as stimulation induced by Mo and Ni were determined. For seals tested repeatedly, the suppressive effect was detected in newborns but not found in the same animals when they were older and had become immunologically competent. Summarizing, the lymphocyte proliferation used as a marker in this investigation provided useful immunological information on these developing animals, and its application for toxicological studies on pollutants can be recommended.

Immunological impact of metals in harbor seals (Phoca vitulina) of the North Sea.

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.