Assessment of the effects of discontinuous sources of contamination through biomarker analyses on caged mussels.

The present study analysed potential adverse effects of discontinuous sources of contamination, namely the discharge of a combined sewer overflow (CSO) and of runoff in an urban area, the Bay of Santander (North Iberian Peninsula). Water samples and caged mussels were used to analyse concentrations of contaminants and biological responses. Mussels (Mytilus galloprovincialis) were transplanted to a marina receiving runoff from a petrol station and to a CSO discharge site. Samples were collected in synchrony with heavy rains along 62days. Lysosomal membrane stability (LMS) and acyl-CoA oxidase (AOX) activity were measured as core biomarkers and were analysed at all sampling times. Histopathology of digestive gland and gonads, transcription levels of vitellogenin gene, volume density of black silver deposits and micronuclei formation were measured at initial and final stages of the transplant. Chemical analyses of metals, polycyclic aromatic hydrocarbons (PAHs) and endocrine disruptors were performed in water samples and mussel flesh. Mussels accumulated low concentrations of contaminants, which is in accordance with results obtained from exposure biomarkers. AOX activity decreased in all transplanted mussels after the first heavy rain, but this change seems to be related to the seasonal pattern of the enzyme activity. Mussels located close to the CSO discharge site showed a reduction in LMS after the first rain event, when compared to mussels before the transplant and to mussels from the reference location. However, this was attributable to natural environmental changes rather than to pollution. Values of the rest of analysed biomarkers were below threshold values reported for the study area.

Relationships between lines of evidence of pollution in estuarine areas: Linking contaminant levels with biomarker responses in mussels and with structure of macroinvertebrate benthic communities.

Data obtained in a pollution survey performed in estuarine areas were integrated using multivariate statistics. The sites selected for the study were areas affected by treated and untreated urban discharges, harbours or industrial activities as well as reference sites. Mussels were transplanted to each site and after different times of exposure, samples of water, sediments and mussels were collected. Biomarkers were analysed on mussels after 3 and 21 days of transplant whereas concentrations of contaminants were measured in water, sediments and mussels after 21 days of transplant. The structure of macroinvertebrate benthic communities was studied in sediment samples. Studied variables were organised into 5 datasets, each one constituting a line of evidence (LOE): contaminants in water, contaminants in sediments, contaminants accumulated by transplanted mussels, biomarkers in transplanted mussels and changes in the structure of macroinvertebrate benthic communities of each sampling site. Principal Component Analysis (PCA) identified the variables of each LOE best explaining variability among sites. In order to know how LOEs relate to each other, Pearson's correlations were performed. Contaminants in sediments were not correlated with the rest of LOEs. Contaminants in water were significantly correlated with contaminants and biomarkers in mussels and with structure of macroinvertebrate benthic communities. Similarly, significant correlations were found between contaminants and biomarkers in mussels and between biomarkers in mussels and structure of macroinvertebrate benthic communities. In conclusion, biomarker responses give relevant information on pollution in estuarine areas and provide a link between chemical and ecological statuses of water bodies in the context of the Water Framework Directive.

Measuring biological responses at different levels of organisation to assess the effects of diffuse contamination derived from harbour and industrial activities in estuarine areas.

To evaluate the effects of diffuse contamination, biological measurements were applied in a scrap cargo harbour, a marina and an industrial area. Metal accumulation and biomarkers (survival in air, digestive gland and gonad histopathology, lysosomal membrane stability, intralysosomal metal accumulation, transcription of vitellogenin and MT20, peroxisome proliferation and micronuclei formation) were measured in transplanted mussels, together with metrics of benthic invertebrates. Benthic species were classified into ecological groups and univariate indexes were calculated. The marina showed high richness (16) and percentage of opportunistic species (55.1%) and low metal accumulation. Mussels in the scrap cargo harbour showed high metal accumulation, up-regulation of MT20 transcription, reduced health status (LP<6 min) and increased micronuclei frequencies (up to 11.3‰). At the industrial area, low species richness (4) and badly organised assemblages were detected and chemical analyses indicated significant amounts of bioavailable metals. Overall, selected biological measurements showed potential for the assessment of diffuse contamination.

Assessing the effects of treated and untreated urban discharges to estuarine and coastal waters applying selected biomarkers on caged mussels.

To assess effects of urban discharges, biomarkers were measured in caged mussels in northern Iberian Peninsula. Lysosomal membrane stability and histopathology of gonad and digestive gland were analysed as general effect biomarkers. Exposure to specific pollutants was evaluated by autometallographical detection of metals, peroxisomal acyl-CoA oxidase activity, micronucleus test and transcription levels of vitellogenin and MT20 genes. Health status of mussels was impaired after 3 days of caging at the untreated outfall discharge and at the waste water treatment plant effluent discharge to the estuary. The most relevant finding was the significant up-regulation of vitellogenin gene transcription in male mussels exposed to the untreated outfall discharge. Metals and xenoestrogenic endocrine disruptors were bioavailable in some discharges and disturbed the health status of mussels. Biomarkers were effective in the assessment of effects of urban discharges and could be implemented in operative controls required to assess the risks associated to effluent discharges.

Effects of exposure to Prestige-like heavy fuel oil and to perfluorooctane sulfonate on conventional biomarkers and target gene transcription in the thicklip grey mullet Chelon labrosus.

Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can be chronically exposed to commonly released pollutants such as polycyclic aromatic hydrocarbons (PAHs) and perfluorinated compounds. These pollutants can also originate from accidental spills, such as the Prestige oil spill in 2002, which resulted in the release of a heavy fuel oil that affected coastal ecosystems in the Bay of Biscay. Peroxisome proliferation (PP), induced biotransformation metabolism, immunosuppression and endocrine disruption are some of the possible biological effects caused by such chemicals. With the aim of studying the effects of organic toxic chemicals on such biological processes at the transcriptional and at the cell/tissue level, juvenile mullets were exposed to the typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) Prestige-like heavy fuel oil for 2 and 16 days. First, fragments of genes relevant to biotransformation, immune/inflammatory and endocrine disruption processes were cloned using degenerate primers. Fuel oil elicited a significant PP response as proved by the transcriptional upregulation of palmitoyl-CoA oxidase (aox1), peroxisome proliferator activated receptor alpha (pparalpha) and retinoic X receptor, by the AOX1 activity induction and by the increased peroxisomal volume density. PFOS only elicited a significant induction of AOX1 activity at day 2 and of PPARalpha mRNA expression at day 16. All treatments significantly increased catalase mRNA expression at day 16 in liver and at day 2 in gill. Cyp1a transcription (liver and gill) and EROD activity were induced in fuel oil treated organisms. In the case of phase II metabolism only hepatic glutathione S-transferase mRNA was overexpressed in mullets exposed to WF for 16 days. Functionally, this response was reflected in a significant accumulation of bile PAH metabolites. WF treated fish accumulated mainly high molecular weight metabolites while F exposure resulted in accumulation of mainly low molecular ones. Fuel oil significantly regulated immune response related complement component C3 and hepcidin transcription followed by a significant regulation of inflammatory response related apolipoprotein-A1 and fatty acid binding protein mRNAs at day 16. These responses were accompanied by a significant hepatic inflammatory response with lymphocyte accumulations (IRLA) and accumulation of melanomacrophage centers (MMC). PFOS did not elicit any transcriptional response in the studied biotransformation and immune related genes, although histologically significant effects were recorded in IRLA and MMC. A significant reduction of lysosomal membrane stability was observed in all exposed animals. No endocrine disruption effects were observed in liver while brain aromatase mRNA was overexpressed after all treatments at day 2 and estrogen receptor alpha was downregulated under WF exposure at day 16. These results show new molecular and cellular biomarkers of exposure to organic chemicals and demonstrate that in mullets PP could be regulated through molecular mechanisms similar to those in rodents, although the typical mammalian peroxisome proliferator PFOS and heavy fuel oil follow divergent mechanisms of action.

Immunolocalization of four antioxidant enzymes in digestive glands of mollusks and crustaceans and fish liver.

The aim of this work was to determine the immunolocalization of the antioxidant enzymes catalase, Cu,Zn-superoxide dismutase (SOD), Mn-SOD, and glutathione peroxidase (GPX) in the bivalve mollusks Mytilus galloprovincialis and Crassostrea sp., the crab Carcinus maenas, and the teleostean fish Mugil cephalus. By immunoblotting, crossreactivity between antibodies and the corresponding proteins in the digestive gland/hepatopancreas of invertebrates and the fish liver was demonstrated. Immunohistochemical studies showed that the stomach epithelium was strongly immunostained for catalase in mollusks. In crabs, ducts showed stronger immunostaining than tubules and in mullet hepatocytes the reaction appeared in discrete granules corresponding to peroxisomes. With regard to Cu,Zn-SOD, the apex of the tubule cells in mussels and crabs was distinctly immunostained, whereas in oysters the reaction was more marked in ducts and in mullet liver a uniform diffuse cytoplasmic staining was found. Mn-SOD was strongly positive in mollusk and crab ducts and in mullet periportal hepatocytes. Finally, GPX was not detected in mussels while in oysters a slight reaction was noted in all cell types. In crabs, connective tissue cells and the apex of duct cells were immunostained, but in mullet liver only erythrocytes appeared reactive. Immunoelectron microscopy revealed that catalase was localized in peroxisomes with a dense labeling in fish and less intense labeling in invertebrates. Cu,Zn-SOD was mainly a cytosolic protein although additional positive subcellular sites (peroxisomes, nuclei) were also observed, while Mn-SOD was restricted to mitochondria. GPX was localized in the cytosol, nucleus, and lysosomes, occurring also in peroxisomes of the fish liver. The results presented here provide a basis for future application of the immunodetection techniques to study the possible differential induction of antioxidant enzymes in aquatic organisms subjected to oxidative stress as a result of exposure to environmental pollutants.

Ultrastructural, immunocytochemical and morphometric characterization of liver peroxisomes in gray mullet, Mugil cephalus.

Peroxisomes of the hepatocytes of gray mullets, Mugil cephalus, were characterized cytochemically and immunocytochemically using antibodies against the peroxisomal proteins catalase and palmitoyl-coenzyme A (CoA) oxidase. In addition, morphometric parameters of peroxisomes were investigated depending on the hepatic zonation, the age of the animals and the sampling season. Mullet liver peroxisomes were reactive for diaminobenzidine, but presented a marked heterogeneity in staining intensity. Most of the peroxisomes were spherical or oval in shape, although irregular forms were also observed. Their size was heterogeneous, with profile diameters ranging from 0.2 to 3 microm. Peroxisomes tended to occur in clusters, usually near the mitochondria and lipid droplets. They also showed a very close topographical relationship to the smooth endoplasmic reticulum. Mullet liver peroxisomes did not contain cores or nucleoids as rodent liver peroxisomes, but internal substructures were observed in the matrix, consisting of small tubules about 60 nm in diameter and larger semicircles 120 nm in diameter. The volume density of peroxisomes was higher in periportal hepatocytes of mullets sampled in summer than in pericentral hepatocytes, indicating that mullet peroxisomes vary depending on physiological and environmental conditions. By immunoblotting, the mammalian antibodies cross-react with the corresponding proteins in whole homogenates of mullet liver. Paraffin sections immunostained with the antibodies against catalase and palmitoyl-CoA oxidase showed a positive reaction corresponding to peroxisomes localized in the hepatocyte cytoplasm. In agreement, the ultrastructural study revealed that catalase and palmitoyl-CoA oxidase are exclusively localized in the peroxisomal matrix in fish hepatocytes, showing a dense gold labeling. The presence of the peroxisomal beta-oxidation enzyme palmitoyl-CoA oxidase in peroxisomes indicated that these organelles play a key role in the lipid metabolism of fish liver.

Structure of peroxisomes and activity of the marker enzyme catalase in digestive epithelial cells in relation to PAH content of mussels from two Basque estuaries (Bay of Biscay): seasonal and site-specific variations.

The aim of the present work was to study the seasonal as well as the site-specific variations in the structure of peroxisomes and in the activity of the peroxisomal marker enzyme catalase in digestive epithelial cells of mussels to validate the potential use of these parameters as early biomarkers of environmental organic pollution in estuarine ecosystems. For this purpose, mussels were sampled monthly for 14 months in two Basque estuaries (Bay of Biscay) with different degrees of pollution. Stereological procedures were applied to detect changes in peroxisome structure, and microspectrophotometry was used to quantify changes in catalase activity. The animals from the two studied sampling sites presented differences in polycyclic aromatic hydrocarbon (PAH) burdens, mussels from Plentzia generally showing lower total PAH contents than mussels from Galea. The peroxisome structure of the animals from the two estuaries suffered seasonal variations that were of different kind and intensity in both sites. In this way, a strong peroxisome proliferatory response was found in mussels sampled in Plentzia during the summer months, while mussels from Galea presented few variations along the year. Catalase activity behaved similarly in the animals sampled in the two estuaries, with higher values in spring. It appeared that mussels exposed chronically to PAHs and other pollutants, such as those from Galea, lost their ability to respond to this exposure in terms of peroxisome proliferation. In contrast, mussels collected in Plentzia effectively responded to an increased bioavailability of organic pollutants during the summer by increasing peroxisome volume and surface and numerical densities in digestive epithelial cells. However, these increases were transient because elevated PAH body burdens detected in mussels sampled in Plentzia in autumn were not accompanied by a peroxisome proliferatory response. Further studies are needed before changes in peroxisomal structure and in the activity of catalase could be used as early biomarkers to assess environmental quality in pollution monitoring programs like the Mussel Watch.

Induction of peroxisomal oxidases in mussels: comparison of effects of lubricant oil and benzo(a)pyrene with two typical peroxisome proliferators on peroxisome structure and function in Mytilus galloprovincialis.

Marine mussels are used as bioindicators of water pollution in marine and estuarine environments in the so-called "Mussel Watch" programs because of their capacity to accumulate numerous organic xenobiotics including aromatic hydrocarbons. In this study, we have analyzed the effects of two xenobiotics [benzo(a)pyrene and the water accommodated fraction of a lubricant oil] and two typical (rodent) peroxisome proliferators (clofibrate and dioctyl phthalate) on structure and function of peroxisomes in digestive glands of mussels Mytilus galloprovincialis, either following water exposure (for 1, 7, and 21 days) or after direct injection through the adductor muscle (for 1 and 7 days). The activities of catalase (CAT), acyl-CoA oxidase (AOX), and D-amino acid oxidase were determined in whole homogenates of digestive glands. In addition, stereological methods were applied on sections stained histochemically for demonstration of catalase activity in order to quantify the morphological changes of peroxisomes. The peroxisomal acyl-CoA oxidase and D-amino acid oxidase were increased in mussels injected for 7 days with benzo(a)pyrene, phthalate, and clofibrate and a similar trend was noted for benzo(a)pyrene and lubricant oil in water exposure experiments (21 days). The catalase activity was reduced or unchanged depending on the mode of exposure of animals. By stereology, significant increases of numerical and volume densities of peroxisomes were found in animals injected for 7 days with lubricant oil or clofibrate. These observations indicate that peroxisomal oxidases in mussels are induced at moderate rates in response to different xenobiotics and that their determination could provide a (sensitive) marker for detection of effects of some toxic pollutants, particularly the lubricant oils which in addition induce significant structural alterations of mussel peroxisomes.