Katja Broeg

The European technical report on aquatic effect-based monitoring tools under the water framework directive

The Water Framework Directive (WFD), 2000/60/EC, requires an integrated approach to the monitoring and assessment of the quality of surface water bodies. The chemical status assessment is based on compliance with legally binding Environmental Quality Standards (EQSs) for selected chemical pollutants (priority substances) of EU-wide concern. In the context of the mandate for the period 2010 to 2012 of the subgroup Chemical Monitoring and Emerging Pollutants (CMEP) under the Common Implementation Strategy (CIS) for the WFD, a specific task was established for the elaboration of a technical report on aquatic effect-based monitoring tools. The activity was chaired by Sweden and co-chaired by Italy and progressively involved several Member States and stakeholders in an EU-wide drafting group. The main aim of this technical report was to identify potential effect-based tools (e.g. biomarkers and bioassays) that could be used in the context of the different monitoring programmes (surveillance, operational and investigative) linking chemical and ecological status assessment. The present paper summarizes the major technical contents and findings of the report.

Effects of nanoparticles in Mytilus edulis gills and hepatopancreas : A new threat to marine life?

Every day new extraordinary properties of nanoparticles (a billionth of a meter) are discovered and worldwide millions are invested into nanotechnology and nanomaterials. Risks to marine organisms are still not fully understood and biomarkers to detect health effects are not implemented, yet. We used the filter feeding blue mussel as a model to analyse uptake and effects of nanoparticles from glass wool, a new absorbent material suggested for use in floating oil spill barriers. In both, gills and hepatopancreas we analysed uptake of nanomaterials by transmission electronmicroscopy (TEM) in unstained ultrathin sections over a period of up to 16 days. Lysosomal stability and lipofuscin content as general indicators of cellular pathology and oxidative stress were also measured. As portals of uptake, diffusion and endocytosis were identified resulting in nanoparticle accumulation in endocytotic vesicles, lysosomes, mitochondria and nuclei. Dramatic decrease of lysosomal membrane stability occurred after 12h of exposure. Lysosomal damage was followed by excessive lipofuscin accumulation indicative of severe oxidative stress. Increased phagocytosis by granulocytes, autophagy and finally apoptosis of epithelial cells of gills and primary and secondary digestive tubules epithelial cells indicated progressive cell death. These pathological responses are regarded as general indices of toxic cell injury and oxidative stress. By the combinational use of biomakers with the ultrastructural localisation of nanoparticle deposition, final evidence of cause-effect relationships is delivered.

Effects of dredging in Göteborg harbor, Sweden, assessed by biomarkers in eelpout (Zoarces viviparus).

We used a battery of biomarkers in fish to study the effects of the extensive dredging in Göteborg harbor situated at the river Göta alv estuary, Sweden. Eelpout (Zoarces viviparus) were sampled along a gradient into Göteborg harbor, both before and during the dredging. Biomarker responses in the eelpout before the dredging already indicated that fish in Göteborg harbor are chronically affected by pollutants under normal conditions compared to those in a reference area. However, the results during the dredging activities clearly show that fish were even more affected by remobilized pollutants. Elevated ethoxyresorufin-O-deethylase activities and cytochrome P4501A levels indicated exposure to polycyclic aromatic hydrocarbons. Elevated metallothionein gene expression indicated an increase in metal exposure. An increase in general cell toxicity, measured as a decrease in lysosomal membrane stability, as well as effects on the immune system also could be observed in eelpout sampled during the dredging. The results also suggest that dredging activities in the Göta alv estuary can affect larger parts of the Swedish western coast than originally anticipated. The present study demonstrates that the application of a set of biomarkers is a useful approach in monitoring the impact of anthropogenic activities on aquatic environments.

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.

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.

Detection of P-glycoprotein mediated MDR/MXR in Caranus maenas hepatopancreas by immuno-gold-silver labeling

Abstract Carcinus maenas were sampled in the German Bight for immunohistochemistry, histology and parallel chemical analysis of organochlorines. Incubations were performed with a monoclonal antibody against P-glycoprotein which mediates multixenobiotic resistance in order to investigate whether Carcinus maenas develop MXR in hepatopancreas. The data showed that MXR was highly expressed at the microvilli of epithelial cells while inside epithelial cells of the tubules MXR was restricted to a specific cell type, the transitional stage between fibrillar (F) cells and blister (B) cells. These cells were rich in lysosomes as was demonstrated by high acid phosphatase activity. In aging B cells, fusion events of the lysosomal compartment to one large vacuole were observed to be accompanied by a loss of MXR protein content. Subsequently, B cells were eliminated from the epithelium into the lumina where the cell debris was incorporated into the faeces. It is concluded that F/B cells appear to be specialized for accumulation of toxic compounds and their digestion and elimination.

Parasites of flounder ( Platichthys flesus L.) from the German Bight, North Sea, and their potential use in biological effects monitoring

In the frame of an integrated biological effect monitoring programme, the parasite community of flounder (Platichthys flesus) was investigated at different locations in the German Bight from 1995 to 2000. In order to assess the impact of environmental contamination caused by anthropogenic activities on the parasite community, selected parasitological parameters that displayed significant differences between the sampling sites were subjected to correlation analyses with site-specific contamination and individual pollution loads of their fish hosts. In addition, correlation analyses were conducted with the responses of selected genetic, biochemical, histopathological, physiological and immunological parameters of fish, used as potential biomarkers. In total, 802 flounder were analysed for these parameters. Information on the chemical background at the sampling sites was derived from sediment samples and from 120 samples of blue mussel (Mytilus edulis) tissue, collected at each of the sampling sites. Based on chemical data available from the sediment and blue mussel samples, a pollution gradient could be established between the sampling sites for individual contaminants. The relative abundance of Acanthochondria cornuta, Cucullanus heterochrous and Zoogonoides viviparus, and the community measures species richness and number of heteroxenous species decreased with increasing concentrations of individual heavy metals or hydrocarbons in sediment and blue mussel samples. Most of the parasitological parameters significantly reflected the established site-specific contamination gradient, when data were pooled over all sampling campaigns. Significant correlations were also found with the contamination level of individual flounder. The parasitological parameters included the parasite species Lepeophtheirus pectoralis and Lernaeocera branchialis, which were not correlated to site-specific contamination. Several biomarkers were significantly correlated to the abundance of parasitic copepods A. cornuta, Lernaeocera branchialis and Lepeophtheirus pectoralis and to parasite community parameters. The results showed that the abundance of several metazoan parasite species, species richness and parasite diversity were reduced in contaminated habitats, and that differences between sites were not only related to natural factors, such as salinity, but also to pollution-induced stress. Thus, it can be concluded that the parasite community of fish responds to the level of pollution at a specific site as well as to residues of xenobiotics in individual fish. These findings give indications that the parasite community of fish is a valuable parameter for the assessment of ecological consequences of chemical contamination in aquatic habitats.

Multivariate statistical approach to the temporal and spatial patterns of selected bioindicators observed in the North Sea during the years 1995–1997

Abstract A comprehensive database, containing biological and chemical information, collected in the framework of the bilateral interdisciplinary MARS project (”biological indicators of natural and man-made changes in marine and coastal waters”) during the years 1995–1997 in the coastal environment of the North Sea, was subjected to a multivariate statistical evaluation. The MARS project was designated to combine a variety of approaches and to develop a set of methods for the employment of biological indicators in pollution monitoring and environmental quality assessment. In total, nine ship cruises to four coastal sampling sites were conducted; 765 fish and 384 mussel samples were analysed for biological and chemical parameters. Additional information on the chemical background at the sampling sites was derived from sediment samples, collected at each of the four sampling sites. Based on the available chemical data in sediments and black mussel (Mytilus edulis) a pollution gradient between the selected sites, was established. The chemical body burden of flounder (Platichthys flesus) from these sites, though, did not reflect this gradient equally clear. In contrast, the biological information derived from measurements in fish samples displayed significant a regional as well as a temporal pattern. A multivariate bioindicator data matrix was evaluated employing a factor analysis model to identify relations between selected biological indicators, and to improve the understanding of a regional and temporal component in the parameter response. In a second approach, applying the k-means algorithm on the data matrix, two significantly different clusters of samples, characterised by the current health status of the fish, were extracted. Using this classification a temporal, and in the second order, a less pronounced spatial effect was evident. In particular, during July 1996, a clear sign of deteriorating environmental conditions was extracted from the biological data matrix.

Environmental Mutagenesis in different zones of the Baltic Sea

Environmental mutagenesis was assessed in fish from the selected coastal areas of the Baltic SeanKvadofjarden and the Stockholm archipelago (Sweden), the KlaipodanBstingo zone (Lithua- nia), the Gulf of Gdansk (Poland) and the Wismar Bay (Germany). The frequency of micronuclei, as markers of cytogenetic damage, was evaluated in erythrocytes of 911 flounder (Platichthys flesus), 332 perch (Perca fluviatilis) and 458 eelpout (Zoarces viviparus) specimens. Fish was collected du- ring bi-annual sampling campaigns performed in 2001 and 2002. Statistically significant differences (p < 0.0001) regarding season and sampling location were recorded. The highest values of cytogenetic damage were registered in flounder from the Lithuanian coast near Palanga (autumn 2001) and from the Wismar Bay (spring 2001), in perch from the Lithuanian coast near Nemirseta (autumn 2001) and in eelpout from the Wismar Bay (autumn 2001). These values are 5n10-fold higher than the baseline level of micronucleus incidence. In general, the decrease in environmental mutagenesis was observed in 2002. Nevertheless a significant increase in micronucleus levels was determined in fish after the oil spill in the Bstingo oil terminal.

The liver of wrasse - morphology and function as a mirror of point source chemical impact.

Corkwing wrasse (Symphodus melops L.), a protogynous, non-migratory lipfish species, living close to rocky shores was chosen as an indicator species for the monitoring of biological effects of contaminants. Fish were caught by local fisherman at the Norwegian west coast in fjord sites within the framework of the EU BEEP project. The sites represented different point source impacts of (I) copper (a former copper mine), (II) polycyclic aromatic hydrocarbons (PAHs, aluminium smelter discharge), (III) formaldehyde plus PAHs (kelp-factory and influence of the aluminium smelter). Livers of wrasse were studied for histopathological alterations and compared to healthy livers of fish from a reference location. Besides liver morphology, different functional and metabolic parameters were measured to link pathological alterations to functional disorders. The integrity of the lysosomal compartment was tested by the assessment of lysosomal membrane stability (lys), and the accumulation of neutral lipids and lipofuscin. Activity and intracellular localisation of the NADPH-producing enzymes in the liver were assessed histochemically and measured by computer-assisted image analysis. Histopathological alterations were most severe at the site impacted by formaldehyde and PAHs. These findings were associated with highest tumor prevalence, lowest membrane stabilities in hepatocytes and highest accumulation rates of lipofuscin in the liver. The activities of the NADPH-producing enzymes phosphogluconate dehydrogenase (PGDH) and glucose-6-phosphate dehydrogenase (G6PDH) were significantly lower compared to unimpacted reference fish. Histopathological alterations showed clear differences dependent on the input source. Potential links between specific contaminant impact and functional and morphological disorders are discussed.