Increased potential for NAD(P)H-dependent reactive oxygen species production of hepatic subcellular fractions of fish species with in vivo exposure to contaminants.

The present study investigated the proposed involvement of contaminant-stimulated reactive oxygen species (ROS) production in disease processes in fish. NAD(P)H-dependent ROS production of subcellular fractions was determined by the iron/EDTA-mediated oxidation of 2-keto-4-methiolbutyric acid. Hepatic cytosolic NADPH-dependent and microsomal NAD(P)H-dependent ROS production were increased 51-160% (P < 0.05) in rainbow trout (Oncorhynchus mykiss) 15 weeks after a single i.p. injection of polychlorobiphenyl (PCB) (100 mg Clophen A50 kg-1 wet wt.). Hepatic microsomal NADH-dependent ROS production was 114% higher in perch (Perca fluviatilis) from PCB-contaminated Lake J?rnsj?n compared to clean Lake V?nern, Sweden. Hepatic mitochondrial NADH-dependent, cytosolic NADH-dependent and microsomal NADPH-dependent ROS production were variously elevated up to 160% in flounder (Platichthys flesus) at various sites along two pollution transects near to the ports of Rotterdam and Amsterdam, Netherlands. Overall the data indicate increased potential for ROS production in liver of fish exposed to field pollution, and support the hypothesis of oxidative stress as a mechanism of contaminant-mediated disease in fish.     

Assessing DNA damage in cnidarians using the Comet assay

The assessment of DNA damage by the Comet assay has been described as a useful non-specific general biomarker of stress in many marine organisms. In field situations it has successfully been employed to distinguish between reference and polluted sites and in the laboratory it has been widely used as a mechanistic tool to determine pollutant effects and mechanisms of DNA damage. To date a wide range of marine vertebrates and invertebrates have been used, however, the usefulness of this assay as a biomarker in cnidarians has not yet been assessed. The aims of this study were to optimize the Comet assay for cnidarian cells and to assess its utility for detecting genotoxic damage in these cells. Cells were isolated from the North American pacific coast temperate sea anemone Anthopleura elegantissima using a non-enzymatic dissociation procedure and viability was determined to be in excess of 90%. Cells were incubated either with (1 h acute exposures) hydrogen peroxide (H(2)O(2)), ethylmethanesulphonate (EMS) or benzo(a)pyrene (B[a]P). In comparison to other marine species, anemone cells exhibited high control or background levels of DNA strand breaks. Despite this, however, we observed dose responses for each of the study chemicals with no reduction in cell viability. This study demonstrates that anemone cells respond to known DNA damaging agents, including B[a]P which requires metabolism to exert its genotoxic effect, and that the Comet assay may prove to be a useful biomarker of stress in cnidarian species.     

Hepatic 7-ethoxyresorufin O-deethylase activity in eel (Anguilla anguilla) from the Thames Estuary and comparisons with other United Kingdom Estuaries

Hepatic microsomal 7-ethoxyresorufin O-deethylase (EROD) activities (indicative of exposure to polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs)) were measured in eel Anguilla anguilla from the Thames Estuary. Fish were collected from up to 13 sites during November 1997, May and August 1998 and October 1999. Throughout this period no clear seasonal variation could be identified at every site along the Thames. However, during the summer months, fish sampled from sites in the middle to the upper estuary (Woolwich, Greenhithe and West Thurrock) reported up to 3-fold higher EROD activities compared to sites either at the upper reaches (Richmond and Brentford) at the same time of the year, or fish sampled in winter, along the entire length of the estuary. A laboratory exposure experiment demonstrated a 3-fold elevation of EROD activity 2 days after injection with β-naphthoflavone (β-NF). However, higher levels of activity could be determined in fish sampled from the Weston canal near the Merseyside. The lowest levels of A. anguilla EROD activity were observed in fish sampled from the upper reaches of the River Tamar, Devon, and were comparable to activities determined in fish from the Wear and Humber estuaries. A. anguilla sampled along the Thames, Tyne and Tees estuaries reported between 2.5- and 7-fold higher EROD activities compared to fish collected from the Tamar. These results indicate that a low to moderate induction of A. anguilla CYP1A had occurred (indicative of low to moderate exposure to PAHs and planar PCBs) in fish collected from the Thames, Tyne, Wear, Tees, Humber and Tamar estuaries. However, the highest level of EROD activity was observed in fish from the Weston Canal (Merseyside).     

Development of hepatic CYP1A and blood vitellogenin in eel (Anguilla anguilla) for use as biomarkers in the Thames Estuary, UK.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goks?yr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.     

Life Histories, Salinity Zones, and Sublethal Contributions of Contaminants to Pelagic Fish Declines Illustrated with a Case Study of San Francisco Estuary, California, USA

Human effects on estuaries are often associated with major decreases in abundance of aquatic species. However, remediation priorities are difficult to identify when declines result from multiple stressors with interacting sublethal effects. The San Francisco Estuary offers a useful case study of the potential role of contaminants in declines of organisms because the waters of its delta chronically violate legal water quality standards; however, direct effects of contaminants on fish species are rarely observed. Lack of direct lethality in the field has prevented consensus that contaminants may be one of the major drivers of coincident but unexplained declines of fishes with differing life histories and habitats (anadromous, brackish, and freshwater). Our review of available evidence indicates that examining the effects of contaminants and other stressors on specific life stages in different seasons and salinity zones of the estuary is critical to identifying how several interacting stressors could contribute to a general syndrome of declines. Moreover, warming water temperatures of the magnitude projected by climate models increase metabolic rates of ectotherms, and can hasten elimination of some contaminants. However, for other pollutants, concurrent increases in respiratory rate or food intake result in higher doses per unit time without changes in the contaminant concentrations in the water. Food limitation and energetic costs of osmoregulating under altered salinities further limit the amount of energy available to fish; this energy must be redirected from growth and reproduction toward pollutant avoidance, enzymatic detoxification, or elimination. Because all of these processes require energy, bioenergetics methods are promising for evaluating effects of sublethal contaminants in the presence of other stressors, and for informing remediation. Predictive models that evaluate the direct and indirect effects of contaminants will be possible when data become available on energetic costs of exposure to contaminants given simultaneous exposure to non-contaminant stressors.     

Early Mississippian sandy siltstones preserve rare vertebrate fossils in seasonal flooding episodes

Flood‐generated sandy siltstones are under‐recognised deposits that preserve key vertebrate (actinopterygians, rhizodonts, and rarer lungfish, chondrichthyans and tetrapods), invertebrate and plant fossils. Recorded for the first time from the lower Mississippian Ballagan Formation of Scotland, more than 140 beds occur throughout a 490 m thick core succession characterised by fluvial sandstones, palaeosols, siltstones, dolostone ‘cementstones’ and gypsum from a coastal–alluvial plain setting. Sandy siltstones are described as a unique taphofacies of the Ballagan Formation (Scotland, UK); they are matrix‐supported siltstones with millimetre‐sized siltstone and very fine sandstone lithic clasts. Common bioclasts include plants and megaspores, fish, ostracods, eurypterids and bivalves. Fossils have a high degree of articulation compared with those found in other fossil‐bearing deposits, such as conglomerate lags at the base of fluvial channel sandstones. Bed thickness and distribution varies throughout the formation, with no stratigraphic trend. The matrix sediment and clasts are sourced from the reworking of floodplain sediments including desiccated surfaces and palaeosols. Secondary pedogenic modification affects 30% of the sandy siltstone beds and most (71%) overlie palaeosols or desiccation cracks. Sandy siltstones are interpreted as cohesive debris flow deposits that originated by the overbank flooding of rivers and due to localised floodplain sediment transport at times of high rainfall; their association with palaeosols and desiccation cracks indicates seasonally wet to dry cycles throughout the Tournaisian. Tetrapod and fish fossils derived from floodplain lakes and land surfaces are concentrated by local erosion and reworking, and are preserved by deposition into temporary lakes on the floodplain; their distribution indicates a local origin, with sediment transported across the floodplain in seasonal rainfall episodes. These deposits are significant new sites that can be explored for the preservation of rare non‐marine fossil material and provide unique insights into the evolution of early terrestrial ecosystems.     

Ostracods: The ultimate survivors

Ostracods are tiny crustacean arthropods just a few millimetres long, with a bivalved carapace made of calcium carbonate that covers the whole body, and into which the animal can retreat from the world outside. Because of their diminutive size they are largely overlooked as fossils, but they have a fascinating history. Silent witnesses to life in the seas since the time of trilobites, they have a fossil record extending back to the Early Ordovician, and possibly the Cambrian. Ostracods have survived nearly 500 million years of Earth history including the ‘big five’ mass extinctions of the Phanerozoic Eon; they are true survivors. They are almost perfectly adapted for the aquatic environments in which they live, and can be found from the ocean abyssal plains to damp leaf litter. The ostracod carapace is a triumph of biological engineering that has been re‐configured into myriad different morphologies according to environment. Streamlined and agile species plough through the ocean water column, sometimes reaching a ‘giant’ size of a centimetre in length, whilst their tinier sea bottom cousins make elaborately ornamented carapaces to withstand the pressures of living at the seabed, or shape their carapaces into forms that facilitate burrowing into sediment. Ostracods are key components of aquatic ecosystems. As primary consumers they are food for larger animals both in seabed and planktonic habitats, and they recycle much of the organic detritus produced by larger animals and plants. Delve into the history of ostracods and it is possible to find pioneers who triumphed in the plankton, early colonisers of terrestrial aquatic ecosystems, and ostracods that literally conquered the land. And in more recent times, ostracods have even hitched rides on rockets into space.     

Hepatic 7-ethoxyresorufin O-deethylase Activity in eel (Anguilla Anguilla) from The Thames Estuary and Comparisons with other United Kingdom Estuaries

Hepatic microsomal 7-ethoxyresorufin O-deethylase (EROD) activities (indicative of exposure to polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs)) were measured in eel Anguilla anguilla from the Thames Estuary. Fish were collected from up to 13 sites during November 1997, May and August 1998 and October 1999. Throughout this period no clear seasonal variation could be identified at every site along the Thames. However, during the summer months, fish sampled from sites in the middle to the upper estuary (Woolwich, Greenhithe and West Thurrock) reported up to 3-fold higher EROD activities compared to sites either at the upper reaches (Richmond and Brentford) at the same time of the year, or fish sampled in winter, along the entire length of the estuary. A laboratory exposure experiment demonstrated a 3-fold elevation of EROD activity 2 days after injection with β-naphthoflavone (β-NF). However, higher levels of activity could be determined in fish sampled from the Weston canal near the Merseyside. The lowest levels of A. anguilla EROD activity were observed in fish sampled from the upper reaches of the River Tamar, Devon, and were comparable to activities determined in fish from the Wear and Humber estuaries. A. anguilla sampled along the Thames, Tyne and Tees estuaries reported between 2.5- and 7-fold higher EROD activities compared to fish collected from the Tamar. These results indicate that a low to moderate induction of A. anguilla CYP1A had occurred (indicative of low to moderate exposure to PAHs and planar PCBs) in fish collected from the Thames, Tyne, Wear, Tees, Humber and Tamar estuaries. However, the highest level of EROD activity was observed in fish from the Weston Canal (Merseyside).     

Development of symbiosis-specific genes as biomarkers for the early detection of cnidarian-algal symbiosis breakdown

Coral bleaching, i.e. the loss of dinoflagellate symbionts from cnidarian hosts, is occurring globally at increasing rates, scales, and severity. The significance of these bleaching events to the health of coral reef ecosystems is extreme, as bleached corals exhibit high mortality, reduced fecundity and productivity and increased susceptibility to disease. This decreased coral fitness leads to reef degradation and ultimately to the breakdown of the coral reef ecosystem. To date there has been little work describing the application of biomarkers to assess coral health. The most commonly applied biomarker is, in fact, the bleaching event itself. We are interested in developing early warning biomarkers that can detect coral stress before bleaching occurs. Recently, several genes that are likely to function in regulating interactions between cnidarians and their symbionts have been characterized, using the temperate sea anemone Anthopleura elegantissima as a model species. One "symbiosis gene" identified from the host genome, sym32, is expressed as a function of anemone symbiotic-state, where sym32 expression is higher in symbiotic cf. aposymbiotic (symbiont-free) anemones. Real-time quantitative RT-PCR suggested that the level of sym32 expression was correlated with the abundance of algae in the host. Furthermore, laboratory exposures of anemones to low levels of cadmium (0, 20, 100 microg(-1) CdCl2; 14 days), which caused no change in algal cell numbers, resulted in a down-regulation of sym32 compared to controls, indicating that sym32 expression may serve as a new sensitive early warning biomarker of cnidarian-algal symbiosis breakdown.