Comparison of GC-NCI MS, GC-ICP-MS, and GC-EI MS-MS for the determination of PBDEs in water samples according to the Water Framework Directive.

The Water Framework Directive (WFD) includes some polybrominated diphenyl ethers (PBDEs) in the list of priority substances that must be measured in surface waters at very low concentrations. The typical approaches applied to the determination of PBDEs in environmental samples might not meet the demanding requirements of the WFD. In this work, the instrumental capabilities of the mass-spectrometry (MS) techniques most frequently used in the determination of PBDEs, namely gas chromatography-negative-chemical-ionisation MS (GC-NCI MS) and GC-electrospray-ionisation tandem MS (EI MS-MS), are evaluated in comparison with highly sensitive GC-inductively-coupled-plasma MS (ICP-MS) for the reliable determination of PBDEs according to the WFD. Three analytical methods based on the liquid-liquid extraction of water samples and measurement of the extracts by GC-NCI MS, GC-EI MS-MS, or GC-ICP-MS are described. The priority PBDEs were quantified in different types of water sample by means of isotope-dilution mass spectrometry (IDMS) using (81)Br-labelled or (13)C-labelled PBDEs spikes, depending on the selected ionisation source. The three proposed methods met the requirements of the European legislation in terms of LOQs and expanded uncertainties. The determination method using (81)Br-labelled PBDEs and GC-ICP-MS had the highest sensitivity and the lowest instrumental limits of detection and expanded uncertainties.

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.

Isolation of a cytotoxic glycoprotein from the Scyphozoa Cyanea lamarckii by lectin-affinity chromatography and characterization of molecule interactions by surface plasmon resonance.

A biospecific lectin-affinity-based isolation process for a novel glycoprotein (ClGp1) from the venom of the pelagic jellyfish Cyanea lamarckii, is described and the isolated glycoprotein is chemically and biologically characterized according to size, molecular interaction and toxicity. The molecular mass of the isolated protein is 25.7 kDa as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). The carbohydrate content was calculated after enzymatic deglycosylation as 6.85 kDa. The glycoprotein is cytotoxic and could be isolated from cnidocysts of mesenteric and fishing tentacles. The binding behaviour of the glycoprotein to the lectins Concanavalin A (ConA) and Wheat Germ Agglutinin (WGA) was analyzed by surface plasmon resonance (SPR) and affinity constants in the range of K(D)=3.0 x 10(-7) M for ConA and 2.1 x 10(-6) M (pH 5.0) and 2.6 x 10(-6) M (pH 7.4) for WGA were obtained.

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.

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.

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.

Comparative study on the cell toxicity and enzymatic activity of two northern scyphozoan species Cyanea capillata (L.) and Cyanea lamarckii (Péron & Léslieur).

Two species of venomous pelagic cnidaria are compared according to their enzymatic, cytotoxic and haemolytic potency. The widely distributed jellyfish Cyanea capillata and Cyanea lamarckii were collected in the North Sea at the coasts of the Orkney Island and the Island of Helgoland. Purified cnidocyst extracts from fishing and mesenteric tentacles were prepared and tested for their bioactivity. The haemolysis induced by toxins of C. capillata was determined with respect to organism size and toxigenic organs. The haemolytic activity of the related species C. lamarckii was documented for the first time. Dose dependent haemolytic activities have been detected by means of protein equivalents at concentrations above 20mug(protein)/mL. Extracts of fishing tentacle cnidocysts showed a less potent haemolytic activity compared to extracts of mesenteric tentacles. In vitro studies with permanent cells of a hepatoma cell line have shown a time and concentration dependent loss of cell vitality up to 90% at 33.3mug(protein)/mL (10mug(protein)/10(5) cells). Supplementing the cell based toxicity tests an enzyme assay was performed to measure a phospholipase A(2) (PLA(2)) activity. A PLA(2)-like activity could be demonstrated in cnidocysts extracts prepared from mesenteric and fishing tentacles of both jellyfish species.

Direct comparison of capillary electrophoresis and capillary liquid chromatography hyphenated to collision-cell inductively coupled plasma mass spectrometry for the investigation of Cd-, Cu- and Zn-containing metalloproteins.

Capillary liquid chromatography (cLC) and capillary electrophoresis (CE) have been critically compared for the separation of metalloproteins when using collision-cell inductively coupled plasma mass spectrometry (ICP-CC-MS) as detection system. For cLC separation, the selected column was a C8 (0.3 mm I.D.) and the separation conditions involved a gradient up to 80% methanol in 10mM ammonium acetate buffer (pH 7.4). The low flow rate used (3 microL min(-1)) permitted the utilization of a high methanol content maintaining the sensitivity along the whole chromatographic run. For this purpose, a new low-flow interface has been developed based on a total consumption nebulizer. Similarly, CE has been studied as separation technique using a 75 microm I.D. fused silica capillary and a running buffer of 20 mM Tris-HNO3 (pH 7.4) and working at 30 kV. Metallothionein (mixture of MT-I and -II) and superoxide dismutase (SOD) have been used as protein models in order to evaluate the separation/detection capabilities using the same injection volumes in both systems (20 nL). For both hybrid systems, separation parameters such as retention factor, numbers of theoretical plates, tailing factor and resolution have been critically compared. Also, the analytical performance characteristics of both hybrid systems have been evaluated and tested by analyzing the Cu-, Zn-species present in red blood cell extracts in order to explore more adequate separation methodology for the analysis of metalloproteins in complex matrices.

Seasonal and annual variations in physiological and biochemical responses from transplanted marine bioindicator species Mytilus spp. during a long term field exposure experiment.

In a pilot field study the long term response of transplanted bioindicator organisms Mytilus spp. was analyzed on the basis of physiological indices and biochemical measurements related to the energy budget. Three different time series with deployment times of eight to twelve months were compared according to seasonality and repeatability of the responses. Test organisms were incubated at a coastal station in the anthropogenically impacted estuary of the river Elbe and at a North Sea station located in vicinity to the Island of Helgoland in the German Bight. The stations differ in their hydrological as well as chemical characteristics. They can be discriminated by statistical factor analysis based on the measured biochemical parameter. Levels of all energy budget biomarker varied between seasons; however, the degree of variation of the specific response was differently expressed. The mussels deployed at Helgoland showed a reproducible high Condition Index in each sampling series and an oscillating Gonadosomatic Index representing the reproduction cycle. The lowest available energy was recorded in mussels at the estuarine sampling station compared to the off-shore station. This may be caused by the energetically costly maintenance of osmotic balance and consequently result in a lower amount of energy available for defense again chemical stress, growth and reproduction.

Bioactivation of inert alumina ceramics by hydroxylation.

Alumina ceramics (Al(2)O(3)) are frequently used for medical implants and prostheses because of the excellent biocompatibility, and the high mechanical reliability of the material. Inauspiciously alumina is not suitable for implant components with bone contact, because the material is bioinert and thereby no bony ongrowth, and subsequently loosening of the implant occurs. Here, we present a new method to bioactivate the surface of the material. Specimens made of high purity alumina were treated in sodium hydroxide. Cell culture tests with osteoblast-like cells as well as spectroscopical and mechanical tests were performed. Aluminium hydroxide groups were detected on the surface of the treated specimens. Enhanced cell adhesion, proliferation and secretion of osteocalcin were determined after hydroxylation. The bioactivating treatment had no deteriorating effect on the short- and long-term strength behaviour. Our results indicate that the described surface technique could be used to develop a new class of osseointegrative high-strength ceramic implants.

Comparison of modified supports on the base of glycoprotein interaction studies and of adsorption investigations.

The features of matrices, suitable for affinity chromatography, have been extensively investigated and got subject for several reviews. But these investigations show, that there is still a lack in adsorbent characterization and a demand of comparative investigations of adsorbents, based on different materials with a range of different surface functionalities. In this work the performance of self-prepared silica and cellulose-based adsorbents were compared with commercially available polymeric supports. A model system was chosen comprising the lectins concanavalin A (ConA) and wheat germ agglutinin (WGA), which were covalently attached to the support matrices, combining the selectivity of the lectin-sugar interaction with the chemical and mechanical properties of the support that influence the efficacy of the prepared adsorbent. The verification of the different supports provides information about tayloring carbohydrate specific lectin adsorbents. The characterization outlines the main features of the different adsorbents and takes into account the properties of the pure supports. It encompasses immobilization kinetics and isotherms as well as the description of the binding capacity of the adsorbents by depicting adsorption isotherms. The separation performances were also investigated in terms of glycoprotein purification factors and recoveries. Further, detailed information about binding of GOD to immobilized ConA are obtained.

Distribution of elements binding to molecules with different molecular weights in aqueous extract of Antarctic krill by size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry.

The distribution of silver, arsenic, cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, selenium and zinc binding to species with different molecular weight in aqueous extract of krill was studied by on-line size-exclusion chromatography (SEC)/inductively coupled plasma mass spectrometry (ICP-MS). The extract was fractionated in three fractions with different molecular weight (MW) ranges (>20,000 relative molecular mass (rel. mol. mass), 2000-20,000 rel. mol. mass and <2000 rel. mol. mass), which were further analyzed by SEC with columns having different optimum fractionation ranges in order to obtain more detailed information about the MW distribution of the elements. Various distribution profiles for the target elements among different MW ranges were observed. The results obtained indicated that manganese, zinc, silver, cadmium and lead species were mostly distributed in the higher MW range (>20,000 rel. mol. mass). In the case of chromium, iron, cobalt, arsenic and selenium, most of them bind to species with lower MW (<2000 rel. mol. mass). Only copper and nickel species was predominantly present in middle MW range (2000-20,000 rel. mol. mass). Further speciation of arsenic compounds in the small MW fraction was carried out with anion exchange chromatography (AEC) coupled with ICP-MS. The results showed that the dominant arsenic species in this fraction is As(III) (63% of extractable arsenic), while As(V) (13%) and two unknown arsenic species (19% and 5%, respectively) are present in lower amounts.

Laboratory calibration and field testing of the Chemcatcher-Metal for trace levels of rare earth elements in estuarine waters.

Little knowledge is available about water concentrations of rare earth elements (REEs) in the marine environment. The direct measurement of REEs in coastal waters is a challenging task due to their ultra-low concentrations as well as the high salt content in the water samples. To quantify these elements at environmental concentrations (pg L(-1) to low ng L(-1)) in coastal waters, current analytical techniques are generally expensive and time consuming, and require complex chemical preconcentration procedures. Therefore, an integrative passive sampler was tested as a more economic alternative sampling approach for REE analysis. We used a Chemcatcher-Metal passive sampler consisting of a 3M Empore Chelating Disk as the receiving phase, as well as a cellulose acetate membrane as the diffusion-limiting layer. The effect of water turbulence and temperature on the uptake rates of REEs was analyzed during 14-day calibration experiments by a flow-through exposure tank system. The sampling rates were in the range of 0.42 mL h(-1) (13 °C; 0.25 m s(-1)) to 4.01 mL h(-1) (13 °C; 1 m s(-1)). Similar results were obtained for the different REEs under investigation. The water turbulence was the most important influence on uptake. The uptake rates were appropriate to ascertain time-weighted average concentrations of REEs during a field experiment in the Elbe Estuary near Cuxhaven Harbor (exposure time 4 weeks). REE concentrations were determined to be in the range 0.2 to 13.8 ng L(-1), where the highest concentrations were found for neodymium and samarium. In comparison, most of the spot samples measured along the Chemcatcher samples had REE concentrations below the limit of detection, in particular due to necessary dilution to minimize the analytical problems that arise with the high salt content in marine water samples. This study was among the first efforts to measure REE levels in the field using a passive sampling approach. Our results suggest that passive samplers could be an effective tool to monitor ultra-trace concentrations of REEs in coastal waters with high salt content.

Investigation on the proteome response of transplanted blue mussel (Mytilus sp.) during a long term exposure experiment at differently impacted field stations in the German Bight (North Sea).

In a pilot field study the proteome response of Mytilus sp. was analyzed in relation to the concentration of different trace metal contaminants. Over a period of eight month test organisms have been exposed at a near-shore station in the anthropogenic impacted estuary of the river Elbe and at an off-shore station in the vicinity of the Island of Helgoland in the German Bight (North Sea). The stations differ in their hydrological as well as chemical characteristics. The physiological biomarkers, such as condition index which have been continuously monitored during the experiment clearly indicate the effects of the different environmental conditions. Multiple protein abundance changes were detected utilizing the techniques of two dimensional gel electrophoresis (2dGE) and consequently proteins arising as potential candidates for ecotoxicological monitoring have been identified by MALDI-ToF and ToF/ToF mass spectrometry. Different cytoskeletal proteins, enzymes of energy metabolism, stress proteins and one protein relevant for metal detoxification have been pointed out.

Characterization of the cytosolic distribution of priority pollutant metals and metalloids in the digestive gland cytosol of marine mussels: seasonal and spatial variability.

Cytosolic profiles of several priority pollutant metals (Cu, Cd, Zn, Pb) and metalloid As were analyzed in the digestive gland of the mussel (Mytilus galloprovincialis) sampled at locations with different environmental pollution levels along the Croatian coast in the spring and summer season. Size-exclusion chromatography (SEC) connected to inductively coupled plasma mass spectrometry (ICP-MS) was used to determine selected elements bound to cytosolic biomolecules separated based on their molecular size. Copper, cadmium and zinc eluted mostly associated with high molecular weight (HMW) and medium molecular weight (MMW) biomolecules, but with a more prominent elution in the MMW peak at polluted locations which were probably associated with the 20 kDa metallothionein (MT). Elution of all three metals within this peak was also strongly correlated with cytosolic Cd as strong inducer of MT. Lead mostly eluted in HMW biomolecule range, but in elevated cytosolic Pb concentrations, significant amount eluted in low molecular weight (LMW) biomolecules. Arsenic, on the other hand eluted almost completely in LMW range, but we could not distinguish specific molecular weight biomolecules which would be predominant in detoxification mechanism. Seasonal variability in element abundance within specific peaks was present, although not in the same extent, for all elements and locations, especially for As. The results confirm the suitability of the distribution of selected metals/metalloids among different cytosolic ligands as potential indicator for metal exposure. Obtained findings can also serve as guidelines for further separation and characterization of specific cytosolic metal-binding biomolecules.

Inductively coupled plasma-mass spectrometry (ICP-MS) for quantitative analysis in environmental and life sciences: a review of challenges, solutions, and trends.

This focal point review provides an overview of recent developments and capabilities of inductively coupled plasma mass spectrometry (ICP-MS) coupled with different separation techniques for applications in the fields of quantitative environmental and bio-analysis. Over the past years numerous technical improvements, which are highlighted in this review, have helped to promote the evolution of ICP-MS to one of the most versatile tools for elemental quantification. In particular, the benefits and possibilities of using state-of-the-art hyphenated ICP-MS approaches for quantitative analysis are demonstrated with a focus on environmental and bio-analytical applications.

Isolation of a Nav channel blocking polypeptide from Cyanea capillata medusae - a neurotoxin contained in fishing tentacle isorhizas.

Jellyfish are efficient predators which prey on crabs, fish larvae, and small fish. Their venoms consist of various toxins including neurotoxins that paralyse prey organisms immediately. One possible mode of action of neurotoxins is the blockage of voltage-gated sodium (Na(v)) channels. A novel polypeptide with Na(v) channel blocking activity was isolated from the northern Scyphozoa Cyanea capillata (L., 1758). For that purpose, a bioactivity-guided multidimensional liquid chromatographic purification method has been developed. A neurotoxic activity of resulting chromatographic fractions was demonstrated by a bioassay, which based on the mouse neuroblastoma cell line Neuro2A. The purification process yielded one fraction containing a single polypeptide with proven activity. The molecular weight of 8.22 kDa was determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-ToF MS). Utilising Laser Microdissection and Pressure Catapulting (LMPC) for the separation of different nematocyst types in combination with direct MALDI-ToF MS analysis of the intact capsules, the neurotoxin was found to be present in all types of fishing tentacle isorhizas (A-isorhizas, a-isorhizas, O-isorhizas) of C. capillata medusae.

A novel proteinaceous cytotoxin from the northern Scyphozoa Cyanea capillata (L.) with structural homology to cubozoan haemolysins.

It is well known that jellyfish are producers of complex mixtures of proteinaceous toxins for prey capture and defence. Nevertheless, studies on boreal scyphozoans concerning venom composition and toxic effects are rare. Here the isolation of a novel cytotoxic protein from the fishing tentacle venom of Cyanea capillata (L. 1758) using bioactivity-guided, multidimensional liquid chromatography is described. The crude venom was purified utilising preparative size-exclusion, ion-exchange, and reversed-phase chromatography. The cytotoxicity of resulting chromatographic fractions has been proven by a dye-uptake assay with the human hepatocyte cell line HepG2. The final purification step yielded, among other fractions, a fraction containing a single protein (named CcTX-1) with a molecular weight of its main isoform of 31.17 kDa The purification process leads to an increased cytotoxic activity per protein equivalents and the finally isolated CcTX-1 caused a nearly total loss of cell viability at a protein concentration of 1.3 µg mL⁻¹ corresponding to 0.4 µg/105 cells. De novo sequencing of CcTX-1 was conducted after enzymatic digestion and subsequent matrix-assisted laser desorption ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-ToF/ToF MS/MS). The obtained sequence data provide an approximate 85% description of the amino acid sequence. This sequence information partially matched that of two known haemolytic proteins of two cubozoan species: CaTX-1 from Carybdea alata Reynaud, 1830 and CrTX-1 from Carybdea rastonii Haacke, 1886.

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.

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.