Field application of biochemical markers and a physiological index in the mussel,Mytilus galloprovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy)

A number of biochemical markers and a physiological index were measured in mussels, Mytilus galloprovincialis, transplanted or native to five different contaminated sites in the lagoon of Venice. Mussels from Pellestrina, a reference site in the adjacent Adriatic Sea, were transplanted for 6 weeks to areas of the lagoon where indigenous mussels were also collected. As biochemical indices, superoxide dismutase (SOD), catalase, aldehyde dehydrogenase (ADH) and NADPH cytochrome c reductase (NADPHcred) were measured in mussel digestive gland; survival in air as a physiological index was also determined. Biomarker responses varied among sites and between indigenous and transplanted animals. Significant induction of catalase and SOD was shown in animals transplanted to the urban sites of Salute and Chioggia, respectively. In indigenous mussels, induction of SOD and NADPHcred was seen in animals from the polluted site of Treporti and the heavily contaminated industrial area of Marghera. The overall biochemical data indicate significantly higher activity for ADH in transplanted animals in comparison with indigenous ones which, in contrast, present an increase in SOD. As regard survival in air, control mussels did not seem to be healthier in comparison either with transplanted or indigenous ones, suggesting that pollution has no effect on this parameter.     

Confinement during field studies may jeopardize antioxidant and physiological responses of Nile tilapia to contaminants

This work evaluates the effects of caging, a known confinement stress, in Nile tilapia (Oreochromis niloticus) during an environmental study in Cubatão river, southern Brazil. Caging animals for 7 days, regardless of being at the reference or at a contaminated site, resulted in lower levels of antioxidant-related defenses (glutathione, glutathione S-transferase, glucose-6-phosphate dehydrogenase) in liver and physiological parameters (blood glucose and lactate) as compared with free-swimming animals. Higher hepatic glutathione reductase activity and elevated Hb content could be associated to contaminant exposure. In conclusion, the confinement stress in caged Nile tilapia biochemical and physiological disturbances, acting as a confounding factor in field studies.     

Adaptation to metal toxicity: a comparison of hydrothermal vent and coastal shrimps

Rainbow vent field is one of the most metal‐contaminated hydrothermal sites on the Mid‐Atlantic Ridge near the Azores region. Two hydrothermal shrimp species dominate the fauna at the Rainbow site along with the mussel Bathymodiolus azoricus. Although the levels of essential and non‐essential metals in these shrimps have been studied, the biological consequences of a metal‐rich environment are still largely unknown. Therefore, the aim of this study was to determine the levels of metal‐binding proteins – metallothioneins (MT) and the activities of antioxidant enzymes – superoxide dismutase, catalase, total glutathione peroxidase and selenium‐dependent glutathione peroxidase in two hydrothermal vent shrimps (Mirocaris fortunata and Rimicaris exoculata) collected from the Rainbow site and to compare them with two coastal shrimps (Palaemon elegans and Palaemonetes varians) from a south Portugal lagoon (Ria Formosa) to evaluate their different adaptation strategies towards metals in their environment. Results show significant differences in MT levels and antioxidant enzymatic activities between vent and coastal shrimps and also between shrimp species collected from the same site. This suggests that biochemical responses in both vent and coastal shrimps are affected not only by the environmental characteristics but also by inter‐specific differences. Nevertheless, these responses apparently confer successful adaptation for survival in a metal‐extreme environment.     

Biochemical responses in farmed mussel Perna perna transplanted to contaminated sites on Santa Catarina Island, SC, Brazil.

The effects of contaminants on the biochemical parameters of the intensively farmed mussel Perna perna, are unknown. The aim of this study was to compare biochemical responses in mussels held in clean and contaminated sites in Santa Catarina Island, Brazil. Mussels were transplanted from a farming area, Ratones Grande Island (RGI), to two contaminated sites, Itacorubi (ITAC) and Hercílio Luz Bridge (HLB). A reference group was kept at RGI. After 150 and 180 days of exposure, the digestive glands of the mussels were analyzed for catalase, glucose 6-phosphate dehydrogenase (G6PDH) and glutathione S-transferase (GST) activities. No changes were observed in the catalase activity, in both periods. Low G6PDH activity was observed in mussels transplanted for 150 days at the ITAC site. Increased GST activity was observed in mussels from ITAC and HLG sites after 180 days. These responses are probably related to the augmented discharges of domestic effluents associated with elevated rainfall index.     

Pollutant-induced cell injury in fish liver: Use of fluorescent molecular probes in live hepatocytes

This study was conducted to test whether live cells from the liver of fish could be used to detect early changes that are indicative of pollutant-induced liver damage. Low-molecular-weight fluorescent molecular probes were inserted into isolated hepatocytes from dab (Limanda limanda) from a contaminated site and a reference site in the North Sea. These included bioprobes for endoplasmic reticulum (ER), cytochrome P-450-associated 7-ethoxyresorufin-O-deethylase (EROD), oxyradicals, reduced glutathione (GSH) and microtubules (MT). Endocytosis of Texas Red-albumin was used as an integrated indicator of hepatocyte performance. Findings showed increases in ER-associated fluorescence, EROD and oxyradical generation, with a marked decrease in endocytosis in hepatocytes from fish caught at the contaminated site. These results indicate that fish from the historically contaminated site were impacted by organic xenobiotics which induced the biotransformation system, radical production and cell injury.     

DNA strand breakage in mussels (Mytilus edulis L.) deployed in intertidal and subtidal zone in Reykjavík harbour

DNA single-strand breaks were analysed in the blue mussel (Mytilus edulis L.) deployed in intertidal and subtidal zones in the PAH contaminated Reykjavík harbour and at a reference site, Hvalfj?rethur, Iceland. DNA strand breaks were analysed by Comet assay in isolated gill and haemocyte cells from six mussels from each site and depth. Increased DNA damage in both gill cells and haemocytes were observed in mussels deployed in Reykjavík harbour compared to the reference site. Intertidal mussels from Reykjavík harbour had higher DNA damage in haemocytes compared to subtidal mussels. The Comet assay seems to be useful for measuring genotoxic exposure in mussels from the field, and that DNA damage might be higher in the intertidal zone either due to higher exposure to contaminants or because of physiological and biochemical responses to variations in oxygen availability.     

Immune response of greenback flounder Rhombosolea tapirina after exposure to contaminated marine sediment and diet.

Non-specific immune response of greenback flounder, Rhombosolea tapirina, exposed to contaminated marine sediments was examined. Reference sediments from Port Sorell and contaminated sediments from Deceitful Cove, Tasmania, Australia were investigated. Hatchery-reared flounder were exposed to reference sediment, contaminated sediment or contaminated sediment and diet for 6 weeks. Phagocytic capacity and lysozyme response in flounder were examined on cessation of exposure trial. Significant differences were found in phagocytic capacity and lysozyme response between treatments. Exposure to contaminated sediment, irrespective of diet or benthic disturbance elicited inhibition of phagocytic efficiency in flounder. Disturbance of contaminated sediment stimulated lysozyme activity. The immune response in flounder indicates potential immunotoxicity of sediment from Deceitful Cove.     

Oxidative stress in two populations of killifish (Fundulus heteroclitus) with differing contaminant exposure histories

A population of killifish (Fundulus heteroclitus) inhabiting a creosote-polluted inlet of the Elizabeth River demonstrates tolerance to the acute toxic effects exerted by contaminated sediments on reference site killifish. Previous data have suggested that upregulated antioxidant defenses contribute to short-term tolerance in killifish exposed to Elizabeth River sediments. This study investigated population differences in antioxidant defenses from wild caught Elizabeth River and reference population killifish in different seasons, and after being held in the laboratory. Parameters measured in the killifish were total glutathione concentrations (GSH_T), activities of glutathione reductase (GR), glutathione peroxidase (GPx), and lipid peroxidation (LPO), all in adult hepatic tissues. The Elizabeth River population exhibited greater GSH_T, higher GPx activities, and increased LPO as compared to the reference population. Sex specific population differences were also observed in GSH_T and GPx. Both populations displayed decreased GSH_T and increased GR from early to late summer, as well as after being held in the laboratory. This study indicates that there are many factors that may contribute to differences in levels of antioxidant defenses in addition to exposure to contaminants, including reproductive status and environmental conditions.     

Petroleum contamination and biochemical alterations in oysters (Crassostrea gigas) and plaice (Pleuronectes platessa) from bays impacted by the Amoco Cadiz crude oil spill

Following the Amoco Cadiz crude oil spill in March 1978, marine animals in coastal and estuarine environments along the north shore of Brittany, France became heavily contaminated with crude oil. The resident benthic fauna in the oil-impacted area which survived the spill were severely stressed by the pollution. The objectives of our investigation were to document long-term trends in petroleum hydrocarbon contamination and sublethal stress in oysters and plaice from the estuaries, Aber Benoit and Aber Wrac'h. Oysters in the estuaries accumulated aliphatic and aromatic petroleum hydrocarbons to high levels and remained heavily contaminated for the duration of the 27 month investigation. Plaice, on the other hand, retained only low concentrations of petroleum hydrocarbons in muscle and liver tissue. Nevertheless, the oysters exhibited very few histopathologic and biochemical responses to the oil, whereas the plaice from the estuaries exhibited a variety of sublethal histopathologic and biochemical alterations.     

Lifespan mercury accumulation pattern in Liza aurata: Evidence from two southern European estuaries

Mercury accumulation throughout the lifespan of Liza aurata (Risso, 1810) was analysed in four tissues (muscle, gills, liver and brain) in two southern European coastal ecosystems with distinct mercury contamination. Specimens from four to five age classes were captured in two sampling sites in the Ria de Aveiro (Laranjo bay and Mira), a system historically contaminated by industrial mercury, and in one site in the Mondego estuary, assumed as a mercury-free ecosystem. Mercury concentration in all tissues was found to be significantly higher in the Ria de Aveiro (Laranjo bay) compared to the Mondego, in accordance with the environmental contamination (water, sediments and suspended particulate matter). Significant differences inside the Ria de Aveiro (between the Mira and Laranjo bay) were only detected in the liver. This tissue registered the highest levels of mercury (ranging from 0.11 to 4.2 μg g⁻¹) in all sampling sites, followed by muscle, brain, and gills. In all sampling sites and tissues was denoted a mercury dilution pattern along the lifecycle (except in liver at the Mondego, the reference area where the concentrations are always very low). An exponential trend was found in the metal age variation patterns in Laranjo (the most contaminated area) and a linear trend in the Mira and the Mondego (the least contaminated areas). Organic mercury concentration in muscle generally accounted for over 95% of total mercury concentration, and followed the same accumulation pattern of total mercury. This fish species is of lesser importance in mercury transfer to adjacent coastal areas and although the consumption of fish from Laranjo may present some risk for the humans, this risk decreases with fish age/size.     

Implications of seasonal priming and reproductive activity on the interpretation of Comet assay data derived from the clam, Tapes semidecussatus Reeves 1864, exposed to contaminated sediments

We explore the use of the clam Tapes semidecussatus Reeves 1864 as an indicator for the presence of potentially genotoxic substances in estuarine sediments. The limitations associated with the interpretation of Comet assay data (expressed as % DNA in tail) in terms of clam reproductive state, size (age) and thermal exposure history following laboratory acclimation are discussed. Hatchery-reared clams, subjected to ambient temperature fluctuations during growth, were exposed in vivo under laboratory conditions for three weeks to sediment samples collected from a polluted site and a "clean" reference site. The DNA damage observed in haemocytes, gill and digestive gland cells was significantly higher in animals exposed to contaminated sediment compared to those exposed to sediment from the reference site. The extent of DNA damage recorded was not correlated with size (age). Spawning was not observed during the experiment. Nevertheless, clams with well-developed gonads showed a statistically higher degree of DNA damage in gill and digestive gland cells- but not haemocytes, demonstrating an increased sensitivity to potential genotoxic compounds, possibly caused by impaired DNA repair capacity due to reproductive activity. Furthermore, the degree of DNA damage in clams exposed to contaminated sediments was higher in autumn and winter compared to spring and summer, suggesting an effect of seasonal priming.     

Sensitivity to stress of the estuarine bivalve Macoma balthica from areas between the Netherlands and its southern limits (Gironde)

Variation in the sensitivity to stress of Macoma balthica was measured in several French and Dutch estuaries. For adult and juvenile Macoma balthica exposed to copper under conditions of starvation, differences in mortality rate, condition, glycogen, burrowing rate and copper content were assessed. No significant differences were observed between adults and juveniles; the influence of treatment and origin was always evident. Animals from the most southern estuaries, Loire and Gironde, near to the species's southern limit of distribution, showed, in the field, the strongest deviations for the ecophysiological traits measured, and were in the experiments the most sensitive to stress.     

Tapes philippinarum seed exposure to metals in polluted areas of the Venice lagoon

The concentration changes of 12 metals (As, Hg, Cd, Cr, Co, Cu, Pb, Mn, Ni, V, Zn, Fe, and Hg) in the soft tissues of Tapes philippinarum during growth were investigated. Clams were seeded in two contaminated areas of the Venice lagoon (San Giuliano and Fusina) and in an area of the Marano lagoon (Lignano Sabbiadoro) close to the clam-farm where the seed was produced. Metal trends were very different according to the considered element, the study site and the growth period.Arsenic was always higher in clam tissues than in surface sediments and suspended particles in all the three stations. Mercury, Cd and Cu were higher in the clams from Marano and Fusina but not in those from San Giuliano. Zinc and Co in clams exceeded the concentrations in the sediment and suspended particles only at Marano. The other elements (Cr, Pb, Mn, Ni, and Fe) were always higher in SPM and sediments. In general metal concentrations in clams were more highly correlated to concentrations in the suspended particles rather than in the surface sediments and in suspended clams rather than in bottom clams, nevertheless significant differences between stations and contaminants were found. Metal concentrations in clams were always lower than the European regulatory limits.     

Protein carbonyls and antioxidant defenses in corkwing wrasse (Symphodus melops) from a heavy metal polluted and a PAH polluted site

The use of fish in environmental monitoring has become increasingly important in recent years as anthropogenic substances, many of which function as prooxidants, are accumulating in aquatic environments. We have measured a battery of antioxidant defenses as a measure of oxidative status, as well as protein carbonylation as a measure of oxidative damage, in corkwing wrasse (Symphodus melops) captured near a disused copper mine, where water and sediment are contaminated with heavy metals, and an aluminum smelter, a site contaminated with PAHs. Results were compared to two different reference sites. Fish at the heavy metal site had lower glucose-6-phosphate dehydrogenase activity and elevated protein carbonyls (1.8 times) compared to fish from the reference site. At the PAH site, EROD was increased 2-fold, while total glutathione and methemoglobin reductase concentration, were decreased. No differences were seen in protein carbonyl levels at the PAH site. Measures of both antioxidant defenses and oxidative damage should be used when assessing effects of xenobiotics on oxidative stress in fish species.     

Behavior and brain chemistry correlates in mummichogs (Fundulus heteroclitus) from polluted and unpolluted environments

The ability of Fundulus from a polluted environment to capture a prey organism, the grass shrimp (Palaemonetes pugio), was compared to that of Fundulus from an unpolluted reference site. Fish from the unpolluted site took significantly more prey (p < 0.1) during the first 5–10 min of each trial than fish from the polluted site.Analysis of the neurotransmitters dopamine (DA) and serotonin (5-HT) and their metabolites, DOPAC, homovanillic acid (HVA), and 5-hydroxy-indolacetic acid (5-HIAA) in cerebellums and medullas was performed by HPLC. Mummichogs from the polluted site had significantly lower concentrations of 5-HT and its metabolite 5-HIAA in their medullas, but not cerebellums, than fish from the reference site (p < 0.05). Concentrations of DA, DOPAC and HVA were not different between populations in either part of the brain. Neurotransmitter concentrations were shown to be independent of size and therefore, presumably, age.Analysis of Hg in livers and brains of these mummichogs by atomic absorption spectrophotometry showed significant differences between the two populations for both organs. Earlier work showed that the behavioral deficit could be elicited by Hg; thus, this was the putative neurotoxic agent. However, there are other candidates which may contribute to neurotoxicity in the study site.     

Evaluation of the leukoproliferative response of English sole (Pleuronectes vetulus) as a biomarker for immune dysfunction

The leukoproliferative (LP) response of splenic leukocytes from the marine benthic fish, English sole (Pleuronectes vetulus), stimulated with the mitogens lipopolysaccharide (LPS), concanavalin A (Con A), and pokeweed mitogen (PWM) was examined as a biomarker of immunotoxic effects. English sole were exposed to an organic-solvent extract of a sediment contaminated with polycyclic aromatic compounds (PACs) or placed on a reference sediment modified with the PAC contaminated sediment. For both treatments, English sole had an augmented LP response to Con A, no significant change in the response to LPS, and the LP response to PWM showed no consistent relationship to exposure to PACs. In a field study, English sole from a contaminated site had a significantly augmented LP response to Con A and PWM. Fish from a non-urban site also had an augmented LP response to Con A and to LPS relative to fish from another non-urban reference site. Overall, the results demonstrated that although the LP response in splenic leukocytes of English sole to Con A was linked to contaminant exposure, the LP response to Con A did not exhibit high specificity as an indicator of chemical contaminant exposure. However, the concerted use of Con A, LPS, and PWM allowed for identification of apparent chemical-contaminant induced alterations of the LP response in English sole from an urban area of Puget Sound.     

Hepatic biomarkers of sediment-associated pollution in juvenile turbot, Scophthalmus maximus L

Hatchery-reared turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to sediment collected from polluted sites in Cork Harbour and a reference site at Ballymacoda, Co. Cork, Ireland. The potential of surficial sediment for inducing hepatic biomarkers was assessed at two levels of biological organisation: expression of cytochrome P450 [Western blotting analysis and 7-ethoxy-resorufin O-dealkylase (EROD), 7-benzoxy resorufin O-dealkylase (BROD), 7-methoxy resorufin O-dealkylase (MROD), 7-pentoxy-resorufin O-dealkylase (PROD) activities] and DNA integrity (Comet assay). Positive controls were generated, either by exposing turbot to cadmium chloride-spiked seawater (Comet assay) or to beta-naphthaflavone by intraperitoneal injection (cytochrome P450 induction). The induction of cytochrome P450 activity (EROD, MROD and PROD) in animals following a 7-day exposure to contaminated sediments was significantly higher than those exposed to reference site sediment and remained elevated thereafter; BROD was not induced. DNA single-strand breaks were also significantly higher following exposure to contaminated sediments throughout the experiment. Although no direct correlation between induction of alkoxyresorufin O-dealkylase activities and a particular chemical class was established, the induction of MROD and PROD activities in fish exposed to sediments containing complex contaminant mixtures, appeared to be more sensitive than conventional EROD activity assays. We conclude from the present laboratory study that S. maximus is a suitable sentinel species for the assessment of moderately contaminated sediments and therefore allows for the further development of this model for future, ecologically relevant, field studies.     

Effects of mining wastes on a seagrass ecosystem: metal accumulation and bioavailability, seagrass dynamics and associated community structure

Two different Cymodocea nodosa (Ucria) Ascherson beds growing in mining-contaminated sediments were compared with two reference beds in the Mar Menor coastal lagoon. The accumulation of Zn, Pb and Cd in different fractions of the plant, the sediment parameters that regulate the availability of metals, the seabed structure and dynamics of each seagrass bed and its associated macroinvertebrate community were studied. C. nodosa accumulates metals from the sediments and reflects their bioavailability for this seagrass. At each station, the metal content of the rhizomes was lower than that of leaves and roots. The concentration of acid-volatile sulfides does not seem to influence the availability of metals to the seagrass, possibly due to oxygen transport to underground tissues. The highest metal concentration in all the contaminated stations was found in the leaf-biofilm, due to the formation of complexes between metals and the extracellular polymeric substances that form the biofilm. All the seagrass beds were seen to be undergoing expansion, those growing in contaminated sediments accumulating great quantities of metals and showing highest photosynthetic leaf surface area and highest leaf biomass. However, these structural parameters were not seen to be responsible for the differences in the faunal composition observed between contaminated and reference beds. Moreover, the multivariate analysis identified the metal content of leaves, biofilm and sediments as important variables that may be responsible for these differences in faunal composition. In this study we have demonstrated that both the seagrass C. nodosa and the biofilm on the plant leaves may be used as environmental tools in the Mar Menor lagoon. The former is an useful indicator of sediment contamination, whereas the latter seems to be a good sentinel of water quality.     

Preliminary results of a field study on some stress-related parameters in Tapes philippinarum naturally infected by the protozoan Perkinsus sp.

The aim of the present work was to investigate the possible use of the clam Tapes philippinarum—a filter-feeding bivalve living on soft bottoms—in the monitoring of sediment pollution in the Lagoon of Venice (North-East Italy). To this end, two clam populations, exposed to different environmental conditions (a relative clean site and an industrial polluted area), have been monitored for the prevalence of the protozoan parasite Perkinsus sp. as well as for several biological parameters commonly used as determinants of contaminants exposure. First results showed a low prevalence, higher in the clean site, but a very diffuse distribution of the parasite throughout the two populations. Nevertheless, greater digestive cells height and longer lysosomal latency time were observed in clams from the clean area. As for the physiological stress-related parameters, even if clearance rates were always higher in these clams, scope for growth values indicated a more complicated biological response.     

Spatial variation in elemental composition of otoliths of three species of fish (family Sparidae)

Determining the nursery habitat of fishes that have moved from estuarine nursery habitats is difficult. The elemental fingerprints of otoliths of three species of sparids were determined to investigate their utility as a natural tag of the nursery habitat. Juvenile Pagrus auratus (snapper), Rhabdosargus sarba (tarwhine) and Acanthopagrus australis (bream) were collected from two sites in each of 15, six and three estuaries, respectively, and their otoliths analysed by solution-based inductively coupled plasma-mass spectrometry. Significant differences in otolith chemistry were found for all three species of juveniles collected from different estuaries. The same patterns among estuaries were not seen for all species, although it was not possible to sample the same sites within an estuary for all species. For bream, significant differences in otolith chemistry were found among all three estuaries, whereas for tarwhine the six estuaries were separated into three groups. For snapper, a number of estuaries could be separated, but there was some overlap for other estuaries. All three species were collected from the same site within one estuary and their otoliths analysed. Significant differences were found among species, but the implication of this finding remains unclear as the three species show differences in microhabitat use and may also differ in age. Because the elemental fingerprints of juveniles vary among estuaries or groups of estuaries, the nursery or recruitment estuary of adult fish could now be determined by analysing the juvenile region of adult otoliths. Thus, connectivity between estuaries and open coastal populations could be determined. Such information will have major implications for fisheries management because it will provide information on the distance that fish have moved from their recruitment estuary and the number of estuaries that contribute to each adult population.