Immunological responses in the mussel Mytilus trossulus transplanted at the coastline of the northern Baltic Sea.

The applicability of immune responses in transplanted Baltic blue mussels (Mytilus trossulus) as biomarkers of immunotoxic effects was studied at differently contaminated locations in the Gulf of Bothnia (northern Baltic Sea). Here, we present a detailed report on the immune responses measured as complementary part of transplantation study by Turja et al. (2014).Various immunological endpoints such as total and differential cell count, morphological alterations,phagocytic activity, and caspase 3/7 activity of mussel haemocytes as well as haemolytic activity of the haemolymph were used. Mussels collected at a reference site at a Finnish coastal site (Hanko, H) were transplanted at the Swedish coast near industrial and urban regions of the cities Sundsvall (S1, S2) and Gävle (G1, G2), respectively. Based on the measured immunological responses, multivariate statistical analysis (PCA biplot) showed a clear separation of the most polluted site S1, indicating immunotoxic impacts of the mixture of contaminants present at this location. Based on these observations and results from Turja et al. (2014), we suggest the implementation of immunotoxic biomarkers for the evaluation of ecosystem health. However, these should be accompanied by complementary endpoints of biological effects encompassing i.e., physiological, antioxidant and bioenergetic markers.

Multi-biomarker approach using the blue mussel (Mytilus edulis L.) to assess the quality of marine environments: season and habitat-related impacts.

Using a comprehensive approach, intertidal, near- and offshore sites in the German Bight were analysed for their environmental quality by assessing the health of blue mussels (Mytilus edulis). During a ten month sampling period mussels were studied with a set of biomarkers comprising lysosomal membrane stability and accumulation of lipofuscin, supplemented by biomarkers indicating nutritional status such as neutral lipids and glycogen in the cells of the digestive gland. Data were analysed in relation to sex, gonadal status, condition index and for the presence of parasites, to determine the overall health status of mussels at the respective sites. Mussels from all sites showed clear signs of stress, indicating an inferior environmental quality throughout the southern German Bight. Further, habitat characteristics such as inundation time and growing on- or off-bottom, as well as seasonal factors, can clearly influence the response of biomarkers in mussels exposed to similar levels of chemical environmental stress.

Effect of air exposure on lysosomal tissues of Mytilus edulis L. from natural intertidal wild beds and submerged culture ropes.

Blue mussels collected from suspended culture ropes and from three natural intertidal wild beds from different areas of the German Bight were tested for their ability to cope with hypoxic conditions. During the experiment mussels were exposed to air from 0 to 72h. Mussels from all sampling sites displayed high tolerance to aerial exposure with moderate levels of mortality after 12 to 48h of exposure. Lysosomal membrane stability (LMS), a biomarker of general stress, changed notably between minimum values after 12h and maximum values after 24h of aerial exposure in intertidal mussels. In contrast, labilization times of mussels from the hanging culture increased continuously up to 48h of exposure. Intertidal mussels from the island of Heligoland exhibited significantly decreased membrane stability after 72h of air exposure, correlating to higher mortality rates. Intertidal mussels, although adapted to daily aerial exposure in their natural environment, showed a similar pattern of mortality and lower LMS values during the experiment than mussels from the suspended culture site. The increase of LMS values of mussels under hypoxic conditions at the beginning of the experiment at all sites was tested for the influence of macro-autophagic processes using immune labelling techniques. With this approach it could be demonstrated that high LMS values significantly correlate with low autophagic activity. However, hypoxic conditions do not enhance autophagic processes during the early periods of aerial exposure. Only at the end of the experiment, high values for autophagy were measured in mussels from an intertidal site accompanied with high mortalities. The results indicate that autophagic processes are not involved in the early adaptive processes that enable the mussel to cope with periods of aerial exposure.

Effects of various pollutant mixtures on immune responses of the blue mussel (Mytilus edulis) collected at a salinity gradient in Danish coastal waters.

The Baltic Sea is a semi-enclosed sea with a steady salinity gradient (3‰-30‰). Organisms have adapted to such low salinities, but are suspected to be more susceptible to stress. Within the frame of the integrated environmental monitoring BONUS + project "BEAST" the applicability of immune responses of the blue mussel was investigated in Danish coastal waters. The sampling sites were characterised by a salinity range (11-19‰) and different mixtures of contaminants (metals, PAHs and POPs), according to chemical analysis of mussel tissues. Variation partitioning (redundancy analysis) was applied to decompose salinity and contamination effects. The results indicated that cellular immune responses (total and differential haemocyte count, phagocytic activity and apoptosis) were mainly influenced by contaminants, whereas humoral factors (haemolytic activity) were mainly impacted by salinity. Hence, cellular immune functions may be suitable as biomarkers in monitoring programmes for the Baltic Sea and other geographic regions with salinity variances of the studied range.

Development of an expert system for the integration of biomarker responses in mussels into an animal health index.

Biomarkers on sentinel organisms are utilised worldwide in biomonitoring programs. However, the lack of effective interpretational capacity has hampered their uptake for use for assessment of risk in environmental management. The aim of the present study was to develop and test an objective decision-support or expert system capable of integrating biomarker results into a five-level health-status index. The expert system is based on a set of rules derived from available data on responses to natural and contaminant-induced stress of marine mussels. Integration of parameters includes: level of biological organization; biological significance; mutual interrelationship; and qualitative trends in a stress gradient. The system was tested on a set of biomarker data obtained from the field and subsequently validated with data from previous studies. The results demonstrate that the expert system can effectively quantify the biological effects of different levels of pollution. The system represents a simple tool for risk assessment of the harmful impact of contaminants by providing a clear indication of the degree of stress syndrome induced by pollutants in mussels.

The "bioeffect assessment index" (BAI). A concept for the quantification of effects of marine pollution by an integrated biomarker approach.

The "bioeffect assessment index" (BAI) is based on the integration of several pathological endpoints measured in the liver of European flounder (Platichthys flesus (L.)) during a long term study of biological effects of pollution in the German Bight. The BAI represents a modification of the "health assessment index" since it includes solely validated biomarkers reflecting toxically induced alterations at different levels of biological organisation in order to quantify the effects of environmental pollution. The concept of the BAI is based on the observation of progressive deleterious effects from early responses to late effects. Specific "key events" were detected, representing progressive stages of functional deterioration. The biomarkers selected from a whole battery of cellular markers for the BAI calculation reflect deleterious effects of various classes of contaminants such as heavy metals, organochlorines, pesticides, PAHs, and therefore reflect general toxicity in an integrative manner. Selected biomarkers were: lysosomal perturbations (reduced membrane stability), storage disorders (lipid accumulation) as early markers for toxic effects of liver cells, and the size of macrophage aggregates and their acid phosphatase activity. The latter two markers are indicative for the modulation of non-specific immune response which represents longer time scale responses after chronic exposure.

The Elbe flood 2002--toxic effects of transported contaminants in flatfish and mussels of the Wadden Sea.

Cellular changes in livers from flounder (Platichthys flesus L.) and digestive glands of blue mussels (Mytilus edulis) were studied to analyze whether the Elbe flood catastrophe in August 2002 had any deleterious effects on animal health. Flatfish and mussels were sampled at different locations in the Elbe estuary and the Wadden Sea after the flood wave had reached the German Bight. Internationally used core biomarkers (lysosomal membrane stability, biotransformation enzymes) were applied to assess the toxic effects of putatively increased pollution levels. In comparison to earlier data from long-term studies at the same stations, we found a significant impairment in the function of cell organelles (lysosomes) involved in the detoxification and elimination of pollutants in fish liver. Concentrations of relevant contaminants (organochlorines, polychlorinated biphenyls) were analyzed in parallel with cellular biomarkers, and conspicuously raised concentrations of insecticides metabolites were detected. Cell recovery and a clear reduction in contaminant concentrations were observed in fish livers five months after the flood at all sampling sites except the Helgolander Tiefe Rinne.

Disorder and recovery of environmental health monitored by means of lysosomal stability in liver of European flounder (Platichthys flesus L.).

From 1995 to 2000 biological effects were studied in liver of flounder (Platichtysflesus L.) from the German Bight. During the study period deleterious consequences of acute discharges of DDT and PCBs in early spring 1996 and after 1998 due to remobilization of contaminants from riverbed deepening of the River Elbe became evident. As core biomarker which reflects toxically induced liver pathologies and integrates effects of various classes of pollutants we measured the integrity of lysosomal membranes in individual flounder liver. During the study period, twice statistically significant disturbances of lysosomal function was detected in fish from the River Elbe: in summer 1996 and in spring 1999. Yet, the detrimental contaminant effects were not only restricted to individuals from the Elbe but expanded to those flounder inhabiting formerly less polluted reference areas. In contrast to flounder of the Elbe, their ability to recover from the lysosomal disorders were limited. While in autumn 2000 Elbe individuals showed clear signs of recovery, those fish caught in areas more distant to the source of toxicant input still maintained significantly decreased lysosomal membrane integrity. It can be speculated that fish populations which are not continuously exposed to chronic anthropogenic stress may have a lower potential or need a longer period to recover from the effects of pollution.