Are biochemical biomarker responses related to physiological performance of juvenile sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) caged in a polluted harbour?

ArticleinComparative Biochemistry and Physiology Part C Toxicology & Pharmacology 154(3):187-95 · May 2011with 100 Reads
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    Background: Freshwater sediments have been recognized as a long-term sink and potential source for environmen- tal pollutants released into the aquatic ecosystems. In this study, the sediment quality of Taihu Lake, which is sus- ceptible to anthropogenic contamination, was assessed by a combination of chemical analytical and biological end points. Speci cally, the snail Bellamya aeruginosa was caged in situ at two locations representing di erent pollution levels for di erent exposure times (7, 14 and 21 days). At each of these time points, biochemical parameters, i.e., phase I biotransformation enzymes ethoxyresoru n-O-deethylase (EROD), the antioxidant enzymes superoxide dismutase and catalase, reactive oxygen species, protein carbonyl content and lipid peroxidation, were evaluated in the hepato- pancreas of snails. In addition, surface sediments were collected for analysis of contaminants of concern, including inorganic pollutants, organochlorine pesticides, polychlorinated biphenyls and polybrominated diphenyl ethers. Results: Chemical analyses revealed that sediments from Taihu Lake were contaminated with trace elements and organic pollutants. Concentrations of trace elements (Cu, Ni and As) and organochlorinated pesticides (4,4′-DDE) exceeded their corresponding threshold e ect level according to the sediment quality assessment values for freshwa- ter ecosystems in Canada, indicating that adverse biological e ects may occur. All biomarkers, except EROD activity, were induced in snails during all exposure times. The integrated biomarker response index (IBR) indicated that during the initial exposure phase (7 days), B. aeruginosa were subjected to signi cant environmental stress, which diminished during later sampling time points. Conclusions: Results showed that IBR correlated well with the levels of environmental contaminants, demonstrating the applicability of this biomonitoring approach to complex environmental exposure scenarios.
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    Substances derived from anthropogenic activities induce changes in the physical and chemical characteristics of the aquatic environment. Physicochemical and biological studies are necessary to understand how changes in landscape affect the health of the aquatic environment. The main goal of this study was to evaluate how the landscape at different spatial scales affects (1) water quality and (2) the health status of Heptapterus mustelinus, based on several biomarkers. During the dry season, individuals were caught in three sites with different degrees of anthropogenic activity. The quality of the terrestrial environment was assessed using the Riparian Quality and Land Use Indices. The water quality condition was evaluated using a water quality index, and pesticides and pharmaceuticals were measured in water. The following biomarkers were analyzed in the fish: general health status (Condition Factor, Hepatosomatic index and energetic costs), enzymatic activity (GST, CAT, AchE), carbonyl content in proteins and histopathological responses in liver and gills. The most impacted sites by the presence of pesticides showed more alterations in the surrounding landscape; specially, changes in the riparian area. In this area, biomarkers denoted more damage than in sites with protected riparian zone. Conservation status of riparian ecosystems is crucial in the determination of rivers ecological quality. Our results demonstrate the importance of monitoring the environmental quality through an integrated analysis, using native fish to understand the effects of human activities on the biota. Graphical abstract Open image in new window
  • Article
    Starved Japanese flounder Paralichthys olivaceus larvae were characterized by relatively lower levels of RNA content throughout their early life stages. Significant differences in the RNA: DNA ratios were found between fed and starved fish, and appeared to increase as starvation proceeded. Ontogenetic changes in RNA: DNA ratios were clearly observed during metamorphosis, especially decreasing during the period from the late-metamorphic to postmetamorphic stages. The criteria established from these laboratory experiments, were applied to the nutritional condition of wild larvae and juveniles collected in Wakasa Bay, Sea of Japan in 1994 and 1995 by measuring RNA and DNA content. Starved fish were mainly found in stage I (settling stage) fish during the late season of settlement in 1995. This suggests that starvation could be associated with settlement in Japanese flounder.
  • Article
    A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
  • Article
    Starved Japanese flounder Paralichthys olivaceus larvae were characterized by relatively lower levels of RNA content throughout their early life stages. Significant differences in the RNA:DNA ratios were found between fed and starved fish, and appeared to increase as starvation proceeded. Ontogenetic changes in RNA:DNA ratios were clearly observed during metamorphosis, especially decreasing during the period from the late-metamorphic to post-metamorphic stages. The criteria established from these laboratory experiments, were applied to the nutritional condition of wild larvae and juveniles collected in Wakasa Bay, Sea of Japan in 1994 and 1995 by measuring RNA and DNA content. Starved fish were mainly found in stage I (settling stage) fish during the late season of settlement in 1995. This suggests that starvation could be associated with settlement in Japanese flounder.
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  • Article
    Aquatic and terrestrial animals metabolize foreign chemicals mainly by oxidation, reduction, hydrolysis and conjugation reactions catalyzed by various enzymes, predominantly localized in the liver, but also found in lower concentrations in other tissues. Xenobiotics are thus biotransformed via several different pathways to less toxic, more polar products which are then readily excreted by the liver, kidney or gill. Whereas biotransformation reactions normally result in detoxification, some chemicals also may be enzymatically converted to highly reactive, electrophilic metabolites (epoxides, free radicals, etc.) which may induce cytotoxic, teratogenic, mutagenic or carcinogenic effects through reaction with various cellular constituents. The initial biotransformation of a less toxic chemical to one or more reactive metabolites, a process commonly referred to as ‘metabolic activation’, is now recognized as an essential initial step for many chemically induced toxicities. The metabolic activation of many mutagens and carcinogens, for example, is primarily catalyzed by the cytochrome P-450-dependent mixed-function oxygenase system in the endoplasmic reticulum, with the formation of epoxides or other reactive species capable of covalently binding to nucleic acids or proteins. Whether or not a xenobiotic causes toxicity, is detoxified, or undergoes metabolic activation to more toxic products, depends not only on the chemical and physical properties of the xenobiotic and its metabolites, but also on the species, strain, age, and sex of the animal, the dose and route of exposure, and the effect of various environmental, nutritional and physiological factors.
  • Article
    Results presented in an earlier paper showed that differences in temperature and salinity could explain approximately 30% of the variability in growth rates of juvenile southern flounder (Paralichthys lethostigma) in different creeks of the Pamlico River estuary. In addition, the maximal growth rate was lower than expected from published laboratory results, suggesting food limitation. In the present paper, the obtained mean and maximal growth rates are related to simultaneously collected data on the amount of food available in the creeks. Thirteen different groups of food items were observed in the stomachs of the flounder. Most of the stomachs of flounder collected outside the cages contained only mysids (65 to 75%). Inside the cages, 25 to 45% of the flounder stomachs contained only mysids, while 85 to 100% of the stomachs contained both mysids and other food categories. Individual growth rates of the founder were only significantly related to the number of mysids in the stomachs, and not to any of the other food categories. Stomachs of other fish species mostly showed food categories other than mysids. This indicates that interspecific competition for food did not occur. The four creeks of the cage experiment showed significant differences in abundance of mysids. However, in only one of the two trials the abundance of mysids was significantly related to the observed growth rates. It can be concluded that mysid abundance does not unequivocally explain the variability in growth rates of juvenile southern flounder in low salinity nursery areas of Pamlico Sound, and that the hypothesis of food limiting maximal growth is not supported by the results of this study.
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    Genetic and physiological responses of flounder Platichthys flesus populations to chemical stress were investigated in 3 highly contaminated French estuaries (the Vilaine, Loire and Gironde) and compared to those observed in the Ster estuary, considered as a reference site because of its reletively low level of contamination. In the Vilaine, Loire and Gironde populations, multi-loci heterozygosity (allozymes: PGM, GPI-2, MPI, IDHP, AAT-1, AAT-2) was lower than in the reference site. The mean growth rate was measured by back-calculation from otoliths, and the mean condition factor corresponded to the somatic weight:fish length(3) ratio. The fish from contaminated estuaries displayed lower values for both parameters than those from the Ster. Among the alleles, PGM 85, AAT-1 95, and to a lesser extent MPI 90, MPI 95 and IDHP 90, could be considered as 'resistant' to contamination. In contaminated estuaries, these alleles were carried by individuals that displayed good fitness, measured as high DNA integrity (low coefficient of variation of DNA content in blood cells) by flow cytometry. The frequencies of these alleles were also more elevated compared to fish from the reference site. Moreover, the comparative analysis of the relationships between genotypic and phenotypic responses of flounder populations to pollution in the 4 studied estuaries highlighted some specificity in relation to contamination typology.
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    We used growth rates of juvenile winter flounder Pseudopleuronectes americanus, to assess habitat quality in 3 of Rhode Island's coastal salt ponds that had differing levels of nutrients and human development. In each pond, 1 m(2) cages were placed in vegetated and unvegetated habitats and growth rates of individually marked fish were measured in three 10 to 15 d experiments from 4 June to 7 July 1997. Water temperature, salinity, dissolved oxygen, and benthic food were also measured. Stable isotopes of C and N were measured in experimental and wild fish. Growth rates were 0.06 to 0.76 mm d(-1) and decreased through the experiments. Growth rates of wild fish (0.19 mm d(-1) in Point Judith Pond and 0.21 in Ninigret Pond) were similar to the average of the 2nd and 3rd experiments (0.24 mm d(-1)). Growth rates were the same in vegetated and unvegetated sites. They were also the same in Point Judith and Ninigret ponds but lower in Green Hill Pond. An ANCOVA suggested that Green Hill's lower rates were caused by its higher temperatures, particularly during the 3rd experiment. Benthic food was similar in the different ponds, different habitat types, and in cores taken inside and outside cages. Categories of food consumed by fish were not affected by the presence of vegetation in a cage, although food consumed did differ from pond to pond. Amphipods were the preferred food in all ponds; fish consumed proportionately more amphipods and fewer polychaetes in Ninigret Pond than in the other ponds. Values of delta(15)N in the fish varied with the degree of development in the watershed but not with total nitrogen in the water column. The results of this study indicate that growth rates of fish can be used as indicators of habitat quality.
  • Article
    Caging experiments were conducted to determine the growth of juvenile fishes as a measure of habitat quality under large pile-supported platforms or piers, in pile fields and in open-water habitat types in shallow areas (average depth 1.1 to 3.9 m) in the Hudson River estuary in 1994. Three 10 d caging experiments were conducted in June and early July with recently settled winter flounder Pseudopleuronectes americanus (14.3 to 40.1 mm SL) and 3 experiments of similar duration in July and August with recently settled tautog Tautoga onitis (20.6 to 48.6 mm TL). For both species, within-experiment instantaneous growth rates in weight (G(w)(-1)) were significantly higher (3-way ANOVA, Tukey's pair-wise test, p < 0.05) in pile field and open-water habitats than under the piers where fish lost weight in all of the experiments. Growth rates for individual winter flounder and tautog reached Values as high as 0.09 d(-1) and 0.11 d(-1), respectively. in open water and pile field habitats. In addition, G(w) values for both winter flounder and tautog caged under piers were not significantly different (p > 0.017, 1-sided Dunnett test with Bonferroni correction) from those of conspecifics held concurrently in the laboratory without food. These results, as wen as related studies of fish distribution and abundance in the same habitats, indicate that habitat quality under the platforms of large piers (> 20 000 m(2)) is poor for juvenile fishes when compared with nearby pile field and open-water habitat types. As a result, the impacts of these structures should be considered when estuarine shorelines are developed or renovated.
  • Article
    Estuaries are vital nursery grounds for many marine fishes. During initial estuarine residence, juvenile fishes presumably benefit greatly from rapid growth, which can diminish susceptibility to size-selective predators. To measure the degree of variability in growth rates across different estuarine sites and habitats, I conducted caging experiments with 3 species (Pseudopleuronectes americanus, Tautoga onitis, and Gobiosoma bosci) at 4 sites in New Jersey, USA, estuaries. Two sites were in an estuary with dense eelgrass Zostera marina beds (Little Egg Harbor) and 2 were in an estuary lacking eelgrass but supporting patchy accumulations of the macroalgae Ova lactuca (Great Bay). Experiments were conducted in vegetated (either Zostera or Ulva) and unvegetated habitats at each site. Relative differences in growth rates among the 4 sites and between the 2 habitats were generally maintained across experiments within species, suggesting a consistency in foraging value. For all 3 species, growth was highest at a Great Bay site and generally lowest at a Little Egg Harbor site. The presence of vegetation (either Zostera or Ulva) was beneficial to growth only for T. onitis. Observed patterns in water temperature, sediment structure, and food availability were potential factors effecting the observed differences in growth rates. Natural densities of the 3 fish species were quantified and compared to the results of growth experiments to determine if there were trade-offs between rapid growth and other attributes of habitat quality. P. americanus and T. onitis were more abundant in areas supporting faster growth, but the complete absence of tautog from unvegetated substrates suggested some degree of habitat avoidance. A clear trade-off between habitat selection and foraging quality occurred for G. bosci; gobies were most abundant inside eelgrass beds, which supported the poorest growth rates.
  • Article
    In contrast to studies relating biochemical indicators of cellular stress to exposure, little has been done to compare biochemical effects with biological effects at higher levels of biological organization. From 13 sites on a low-gradient stream in southeastern Arkansas, hepatic cytochrome P450 1A (CYP1A) and its related activity ethoxyresorufin-O-deethylase were measured in male and female carp, female largemouth bass, male bluegill, and male white crappie. Hepatic metallothionein mRNA and a 30-kDa stress protein (HSP30) protein were also measured in male and female carp, male white crappie, and female largemouth bass. A fish health index was determined for female largemouth bass and was compared with expression of hepatic bioindicators. Comparison of biochemical indicators with population-community metrics such as abundance, richness, percentage of tolerant individuals, percentage of tolerant species, and the Shannon-Weaver index failed to show significant linear correlations. However, the fish health index was significantly correlated to hepatic CYP1A, HSP30, and richness with nonsignificant relationships to abundance, percentage of tolerant individuals, and Shannon-Weaver indices. These data show that multiple bioindicators are necessary and can be used to adequately evaluate animal health, but may not accurately predict population-community effects.
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    Juvenile Solea senegalensis were exposed to different concentrations of waterborne copper (Cu) for 15 days in static conditions with continuous aerated artificial salt water at 20°C (± 0.8°C) with a normal photoperiod (10 h/14 h light/dark) and daily feeding. Several measures of exposure and effects were determined: 1) biomarkers - metallothioneins and lipid peroxidation level; 2) mass indices - growth rate and morphometric condition indices; and 3) biochemical condition indices - RNA:DNA ratio and lipid and protein content in fish tissues. Copper exposure triggered the response of the biomarkers and resulted in reduced growth and condition (RNA:DNA and lipid content), but the morphometric indices did not vary. The physiological costs of Cu contamination on condition suggested that lipid reserves were allocated as an energy source to enable exposed fish to respond to Cu toxicity as well as to maintain positive growth rates and protein synthesis throughout the experiment, although with lower growth rates than the control fish. This study showed the importance of selecting suitable biomarkers according to contaminant source, fish species and their life-history stage. In addition, the use of several biomarkers of exposure, growth and specific condition indices can improve fish health determination and should be considered in evaluations of the effects of environmental contaminants on fish.
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  • Article
    Rainbow trout (Oncorhynchus mykiss) (mean weight 180 ± 10 g) fed diets deficient in ascorbic acid, tocopherol, both, or neither were exposed to one of three cadmium concentrations (0, 2, or 4 μg Cd/L). After 181 d of exposure, liver and whole blood were sampled. Cadmium exposure was found to significantly increase the Cd content in liver, especially in fish fed a diet deficient in ascorbic acid. Hepatic stores of ascorbic acid decreased in fish exposed to Cd and in fish fed diets deficient in tocopherol and ascorbic acid. Depletion of tocopherol in liver occurred only in those fish fed a diet deficient in tocopherol and exposed to Cd. Superoxide dismutase activity in liver increased with Cd exposure and with dietary deficiencies of tocopherol and ascorbic acid, while catalase was inhibited by exposure to Cd alone. Glutathione peroxidase activity was unaffected by Cd but was lower in fish fed a diet deficient in both tocopherol and ascorbic acid. Erythrocyte fragility, measured by spontaneous hemolysis of red blood cells in physiological saline, was increased by Cd exposure and the deficiencies of dietary tocopherol and ascorbic acid.
  • Article
    A common assumption throughout the marine ecological and fisheries literature is that growth is a valid indicator of habitat quality and can be used as a criterion for designation of essential fish habitat (EFH). In this study, the validity of growth as an index of habitat quality was tested by examining how variability in otolith growth was related to abiotic and biotic environmental conditions and could be biased by previous growth history, density dependence, and selective mortality. The study was conducted with juvenile Atlantic croaker (Micropogonias undulatus) collected in two North Carolina, USA, estuaries during two seasons of two recruitment years. Water temperature, a component of habitat quality, explained nearly 40% of the variability in juvenile otolith growth. There was also evidence that estimates of growth could be biased by density dependence (slower growth at higher conspecific abundance) and by selective mortality (higher mortality of individuals with relatively slower larval and juvenile otolith growth). Studies using growth-based assessment of habitat quality that fail to identify factors underlying growth rate differences among habitats may reach incorrect decisions regarding quality of different habitats and assignment of EFH.
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    We used growth rates (estimated from otolith microstructure), morphometric (Fulton's K condition index) and lipid (triacylglycerols: sterols ratio, TAG:ST) condition indices measured on 0-group juveniles of common sole, to compare the habitat quality of 7 nursery grounds with differing levels of anthropogenic pressure. Along the French coast of the Southern Bight of the North Sea and the Eastern English Channel, we compared 2 sites located in intensively developed and industrialised harbour areas (Dunkerque and Calais), 1 site in a medium and less industrialised harbour (Boulogne), 1 site near a large and polluted estuary (Seine) and 3 other sites located near small estuaries less subject to human pressure (Canche, Authie and Somme). Indicators of human disturbance as well as hydrological and biotic data were used to establish between-site differences in environmental quality. Our results indicate consistent differences in the growth and condition indices of 0-group sole among the 7 sites. Sole had the
  • Article
    Long term effects of sublethal concentrations of oil on the marine environment have become of general concern. Cytochrome P4501A activity (EROD) in liver and fixed wavelength fluorescence detection of PAHs metabolites (FF) have in this study been used as biomarkers for dispersed oil exposure on a long term period of juvenile turbot (Scophthalmus maximus L.). A Continuous Flow System was used to carry out the study. The fish were continuously exposed to 0.125, 0.5 or 2.0 mg litre−1 dispersed topped crude oil for 6, 15, 24 h, 4 and 21 days followed by a 9 days recovery period in clean seawater. No induction of the cytochrome P4501A was measured. A maximum level in bile metabolites (4- to 5-fold) was recorded after 24 h of exposure revealing thereby a detoxification process, but a decline occurred from day 4 to day 21. This study demonstrated that FF detection of PAHs metabolites in bile could be a more sensitive biomarker than EROD activity in a long term exposure to sublethal concentration of oil.
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    Anchovy larvae, Engraulis mordax, were reared in the laboratory under a variety of food regimes and the triacylglycerol, cholesterol, and polar lipid contents of individual larvae were measured with the Iatroscan thin-layer chromatography/flame ionization detection (TLC/FID) system, as indicators of nutritional condition. The different food levels used in the rearings produced a clear effect on the larvae for each of the three lipid components. A comparison with field-caught larvae, which were otolith-aged and analyzed for lipid content, showed that a standard laboratory rearing produced larvae with a much too high lipid content. Larvae could be reared in the laboratory to have a sizespecific lipid content similar to the field individuals, but this required that the food concentration be very much lower. When group of larvae were starved, the triacylglycerol component decreased first, regardless of the size of the store at the onset of starvation. The polar lipid component was not as labile as triacylglycerol, while the cholesterol content remained essentially unchanged during starvation. The cholesterol and polar lipid contents exhibited a good straight-line relationship with the lipid-free weight of the larvae, as would be expected from indicators of larval tissue weight. The results revealed the lipid components to be very good indicators of the condition of the larvae.
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    Data on water temperature, RNA-DNA ratio, and growth of eight species of temperate marine fish larvae reared in the laboratory were fit to the equation: Gpi = 0.93\text \operatornameT + 4.75\text RNA - DNA - 18.18G_{pi} = 0.93{\text{ }}\operatorname{T} + 4.75{\text{ RNA - DNA}} - 18.18 where Gpi is the protein growth rate in % d-1 and T is the water temperature. Water temperature and larval RNA-DNA ratio explained 92% of the variability in growth rate of laboratory-reared larvae. The model is useful over the entire range of feeding levels (starvation to excess), temperatures (2 to 20C) and fish species studied. Estimates of recent growth of larval cod, haddock, and sand lance caught at sea based on water temperature and RNA-DNA ratio ranged from negative to 26% d-1. These data demonstrate the importance of food availability in larval fish mortality and suggest that short-term growth under favorable conditions may be considerably higher than expected from long-term indicators. RNA-DNA ratio analysis offers new possibilities for understanding larval growth and mortality, and their relation to environmental variability.
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    The purpose of this present study was to use the growth rates, RNA–DNA ratio and morphometric (Fulton’s K) condition indices measured on 0-group juveniles European flounder, as a means of comparing the quality of estuarine nursery habitats. The Seine estuary, heavily impacted by manmade modifications and one of the most contaminated in Europe, was compared to two smaller estuaries considered as clean (low domestic, agricultural and industrial effluents). Indicators of human disturbance (sediment chemical contaminants) as well as biotic data (macrobenthos abundances) were used to establish between-site differences in environmental quality. The abundance of macrobenthos (potential preys for juvenile) was high and not significantly different among the three estuaries confirming the important role played by estuaries as feeding grounds. Sediment metal concentrations (Cd, Cr, Cu, Pb, Zn) were significantly higher in the Seine than in the Canche and Authie estuaries. Otolith growth was closely related to somatic fish growth, and we used daily otolith increments from the previous 10days before capture as an indicator of 0-group flounder recent growth (RG). RG did not vary significantly among flounders from the Canche and Authie estuaries and individuals from the Seine had slower RG. RNA–DNA ratio and K values were also significantly lower in individuals from the Seine compared to those of the Authie or the Canche. Pollution (e.g. metal contaminants) is the most likely cause of growth and condition differences among estuaries. The results of this study suggest that habitat-specific fish growth and condition are useful proxies for habitat quality since they integrate the effect of multiple environmental factors.
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    Matching biological and chemical data were compiled from numerous modeling, laboratory, and field studies performed in marine and estuarine sediments. Using these data, two guideline values (an effects range-low and an effects range-median) were determined for nine trace metals, total PCBs, two pesticides, 13 polynuclear aromatic hydrocarbons (PAHs), and three classes of PAHs. The two values defined concentration ranges that were: (1) rarely, (2) occasionally, or (3) frequently associated with adverse effects. The values generally agreed within a factor of 3 or less with those developed with the same methods applied to other data and to those developed with other effects-based methods. The incidence of adverse effects was quantified within each of the three concentration ranges as the number of cases in which effects were observed divided by the total number of observations. The incidence of effects increased markedly with increasing concentrations of all of the individual PAHs, the three classes of PAHs, and most of the trace metals. Relatively poor relationships were observed between the incidence of effects and the concentrations of mercury, nickel, total PCB, total DDT and p,p′-DDE. Based upon this evaluation, the approach provided reliable guidelines for use in sediment quality assessments. This method is being used as a basis for developing National sediment quality guidelines for Canada and informal, sediment quality guidelines for Florida.
  • Article
    Bio-indicators were measured on juvenile fish to assess the quality of eight coastal and estuarine nursery grounds in the Eastern English Channel and in the Bay of Biscay during 3 years. Growth (size and otolith daily increment width), body condition (morphometric index) and abundance of juvenile common soles were analysed together with xenobiotic concentrations (heavy metals and organic contaminants).Condition indices displayed important variations and did not allow relevant estimation of environmental quality. On the contrary, growth and density indicators showed good steadiness above years but varied among sites. In spite of difficulties of interpreting these indicators on such a meso-scale approach, analyses highlighted the estuaries of Seine and Gironde. In these nursery areas, the levels of contamination were especially high, and the combination of fish growth performances and density was significantly lower than in other sites. The combination of these variables appears to provide reliable indicators of habitat quality and anthropogenic pressure on nursery grounds, especially highlighting contaminated areas. Such indicators may thus contribute to improve assessment of environmental quality of essential fish habitats with the aim of a sustainable management of fisheries resources. A study at a different scale, from this meso-scale nursery approach with more precise analyses, on local habitats, will nevertheless be necessary to optimize the relevance of these indicators for the assessment of essential fish habitat quality.
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    Specimens of sea bass (Dicentrarchus labrax) were placed in cages for 1 month in spring and autumn at different locations in the Bay of Cannes (NW Mediterranean). Biochemical markers evaluated were: ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities in fish livers and acetylcholinesterase (AChE) activity in fish muscle. EROD and GST activities were higher in front of the outlet for the wastewater plant of Cannes and in the harbour than outside the marina. High EROD and GST activities may be induced by petrol hydrocarbons and/or polychlorinated biphenyls (PCBs). AChE was low in the muscles of the fish caged in the harbour compared with samples from the other cages. Low AChE activity could suggest the presence of organophosphorus and carbamate compounds in seawater from the harbour. Mussels (Mytilus galloprovincialis) were caged off Cannes for the same periods as the fishes. Heavy metal, metallothionein (MT) concentrations and lysosomal membrane stability were evaluated in the digestive gland of the mussels. Results show low heavy metal and MT concentrations, implying low metal concentrations in the surrounding waters. High lysosomal membrane stability revealed a good physiological status of these animals after caging. The whole set of data indicates that seawater in the Bay of Cannes appeared to be unpolluted as regards pollutants which may induce the measured biomarkers, except in restricted areas.
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    Laboratory-based assessments of the potentially damaging effects of trace metals in the natural environment are difficult to validate. This is a consequence of the diverse factors which in situ affect metal bioavailability and the susceptibility of organisms to trace metal toxicity. For several years, biomarkers (biochemical responses to contaminant exposure) have been measured in the tissues, body fluids and excreta of a range of organisms to signal chemical exposure. More recently, however, the biomarker concept has been extended to include physiological and behavioural indices of both pollutant exposure and effect. In this paper, a contemporary biomarker approach is outlined and new techniques are described for measuring physiological and behavioural parameters. In particular, biomarkers which indicate changes in Darwinian fitness are discussed. Protocols are outlined concerning how simultaneous measurements of molecular, physiological and behavioural biomarkers might be utilized in biological impact assessment of trace metals (and other pollutants). The potential use of biomarkers as laboratory screening tools and in bioremediation is also addressed.
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    In the present study the bioaccumulation of poly chlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) as well as the responses of a suite of biochemical parameters were investigated in a standardized carp (Cyprinus carpio) fish line. Carps were caged for 2 to 8 weeks at two Amsterdam freshwater sites: the relatively unpolluted Outer IJ and the heavily polluted Volgermeerpolder. The primary objective of the experiments was to develop a reliable and reproducible method to monitor the inland water pollution and to classify the environmental quality of freshwater sites. The highest tissue levels of organic trace pollutants were generally found in carp that was caged for at least four weeks at the polluted site. Hepatic phase I enzymes showed the highest pollution-induced responses, while phase II enzymes and serum transaminases were less sensitive. The antioxidant enzymes showed virtually no response to pollutants. In order to assess effects on the balance between bioactivation and detoxication the ratio between phase I (EROD) and phase II (GST or UDPGT) activities was introduced as a biotransformation index (BTI). Highest BTI values were observed in carp that was caged for 4 to 6 weeks at the polluted site. The BTI values decreased after eight weeks of exposure due to elevated phase II activities.
  • Article
    In this study, wild three-spined sticklebacks were sampled every six weeks, between April and October, in a low contaminated stream. For all fish, physiological indexes, such as condition factor, hepato-, gonado- and nephro-somatic index were calculated to determine fish condition and reproductive status. Moreover, a set of biomarkers including biotransformation enzymes, oxidative stress parameters, neurotoxicity and endocrine disruption markers was measured. The results allowed to determine biomarker variability due to fish gender or sampling season. For example, 7-ethoxyresorufin-O-deethylase activity, glutathione peroxidase as well as vitellogenin and spiggin exhibited strong gender differences. Conversely, lipoperoxidation and acethylcholinesterase activity were characterised by a lack of gender and seasonal variation, and can be considered as more robust parameters for a field application. The present work allowed to establish practical guideline for biomarker measurements in wild sticklebacks and to define a reference system which can be used to analyze variations in future monitoring studies.
  • Article
    Connectivity between estuarine fish nurseries and coastal adult habitats can be affected by variations in juvenile growth and survival. Condition indices are renowned proxies of juvenile nutritional status and growth rates and are valuable tools to assess habitat quality. Biochemical (RNA:DNA ratio) and morphometric (Fulton's condition factor K) condition indices were determined in juveniles of Solea solea, Solea senegalensis, Platichthys flesus, Diplodus vulgaris and Dicentrarchus labrax collected in putative nursery areas of nine estuaries along the Portuguese coast (Minho, Douro, Ria de Aveiro, Mondego, Tejo, Sado, Mira, Ria Formosa and Guadiana) in the Spring and Summer of two consecutive years (2005 and 2006) with distinct climatic characteristics. Individual condition showed significant variation amongst species. The combined use of both condition indices highlighted the low correlation between them and that RNA:DNA had a higher sensitivity. RNA:DNA varied between years but overall the site relative patterns in condition were maintained from one year to the other. Higher RNA:DNA values were found in Spring than in Summer in most species. Intra-estuarine variation also occurred in several cases. Species specific trends in the variability of condition amongst estuaries were highlighted. Some estuaries had higher juvenile condition for more than one species but results did not reveal an identical trend for all species and sites, hindering the hypotheses of one estuarine nursery promoting superior growth for all present species. Significant correlations were found between condition indices, juvenile densities and environmental variables (water temperature, salinity and depth) in the estuarine nurseries. These influenced juvenile nutritional condition and growth, contributing to the variability in estuarine nursery habitat quality. Management and conservation wise, interest in multi-species approaches is reinforced as assessments based on a single species may not reflect the overall nursery habitat quality.
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    Mercury is a contaminant of great concern in the marine environment, particularly in coastal environments where the formation of methylmercury (MeHg) in aquatic sediment, and its subsequent bioaccumulation in edible aquatic organisms (mainly fish), presents a major pathway for human exposure to MeHg. Consequently, it is important to determine the factors controlling MeHg production in sediment, especially in contaminated environments. This study investigates some geochemical factors affecting the speciation and distribution of Hg in estuarine sediment from two highly industrialized macrotidal salt marsh/mudflat systems in the Seine estuary, France, and in the Medway estuary, UK. Obtained data revealed that the entire sediment core from a rapidly accreting mudflat in the Seine estuary (170 cm vertical accretion over the last 10–50 a) was contaminated with Hg at concentrations which are 10–50 times higher than the Hg background for sediments of the Seine basin. In the Medway mudflat, characterized by a slow sedimentation rate (400 cm over approximately the last 800 a), near-surface sediment was significantly more contaminated (10–40 times) than sediment at greater depths, which were characterized by pre-industrial Hg concentrations. Geochemical conditions in the surface sediment of the Medway mudflat are characterized by stable anoxic redox conditions (about −200 mV), which are generally favourable for Hg methylation, whereas near-surface sediment of the Seine mudflat is characterized by more oxidizing redox conditions (about +100 mV), which are generally less conducive to Hg methylation. Consequently, MeHg concentration in the upper 10 cm of the sediment column was about four times higher in the Medway than in the Seine mudflat, in spite of similar total Hg concentrations. In surface sediment, where Hg is actively methylated, MeHg variability was associated with the activity of -reducing microorganisms (SRM) and the presence of acid volatile sulphides (AVS). A strong correlation was observed between MeHg and AVS in sediments from these mudflats, which may be a consequence of the common origin of AVS and MeHg (both produced by microorganism activity), but also can be derived from the ability of Fe monosulfides to adsorb, and thus stabilize, solid phase MeHg.
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    Growth impairments were studied for three juvenile fish species: the red drum (Sciaenops ocellatus), the orange-spotted grouper (Epinephelus coioides) and the marine medaka (Oryzia melastigma). Fish were subjected to long-term (i) food deprivation, (ii) exposure to benzo[a]pyrene, (iii) hypoxia, respectively. Fish growth (fork length and body weight) and scale increments (circulus number and radius length) were measured. Results of Pearson correlation analysis showed a significant positive correlation between scale increment and growth for the red drum (fast growing) and the marine medaka (small sized) (r = 0.741–0.882, p < 0.001). For the slow growing orange-spotted grouper, a significant correlation was found only between body weight and circulus number (r = 0.406, p < 0.05). Overall, scale circulus number is more consistently linked to fish growth than that of radius length. Importantly, our findings demonstrate that daily circulus deposition on scales of fast-growing red drum is feasible to indicate daily growth of the fish. Moreover, scale increment exhibited the strongest correlation with fish growth as compared to that of serum T3 level and muscle RNA:DNA ratio. Our data support the use of scale increment as a means of indicating fish growth and growth impairment, particularly for the fast growing fish.
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  • Article
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    The aim of this study was to characterise biomarker responses in three-spined sticklebacks exposed to copper. For this purpose, adult sticklebacks were exposed for 3 weeks to copper sulphate at 0, 25, 100 and 200μgL(-1) as Cu. At days 4, 8, 12 and 21, several parameters were measured including liver, gonad and spleen somatic indexes, hepatic biomarkers (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH), glutathione-S-transferase (GST) and 7-ethoxyresorufin-O-deethylase (EROD)) and hepatic copper and zinc concentrations. Copper induced a rapid and transient increase of antioxidant enzymes and a depletion of glutathione content during the first 8 days of exposure. Significant copper and zinc accumulation in fish liver were observed for the two higher exposure concentrations after 8 and 12 days, respectively. This study showed that copper induced an oxidative stress in fish liver before significant metal accumulation in the liver could be detected, suggesting the involvement of differential mechanisms in copper uptake and metabolism. Three-spined stickleback appears to be a sensitive model to study oxidative stress induced by metals.
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    In this review, a wide array of bioaccumulation markers and biomarkers, used to demonstrate exposure to and effects of environmental contaminants, has been discussed in relation to their feasibility in environmental risk assessment (ERA). Fish bioaccumulation markers may be applied in order to elucidate the aquatic behavior of environmental contaminants, as bioconcentrators to identify certain substances with low water levels and to assess exposure of aquatic organisms. Since it is virtually impossible to predict the fate of xenobiotic substances with simple partitioning models, the complexity of bioaccumulation should be considered, including toxicokinetics, metabolism, biota-sediment accumulation factors (BSAFs), organ-specific bioaccumulation and bound residues. Since it remains hard to accurately predict bioaccumulation in fish, even with highly sophisticated models, analyses of tissue levels are required. The most promising fish bioaccumulation markers are body burdens of persistent organic pollutants, like PCBs and DDTs. Since PCDD and PCDF levels in fish tissues are very low as compared with the sediment levels, their value as bioaccumulation markers remains questionable. Easily biodegradable compounds, such as PAHs and chlorinated phenols, do not tend to accumulate in fish tissues in quantities that reflect the exposure. Semipermeable membrane devices (SPMDs) have been successfully used to mimic bioaccumulation of hydrophobic organic substances in aquatic organisms. In order to assess exposure to or effects of environmental pollutants on aquatic ecosystems, the following suite of fish biomarkers may be examined: biotransformation enzymes (phase I and II), oxidative stress parameters, biotransformation products, stress proteins, metallothioneins (MTs), MXR proteins, hematological parameters, immunological parameters, reproductive and endocrine parameters, genotoxic parameters, neuromuscular parameters, physiological, histological and morphological parameters. All fish biomarkers are evaluated for their potential use in ERA programs, based upon six criteria that have been proposed in the present paper. This evaluation demonstrates that phase I enzymes (e.g. hepatic EROD and CYP1A), biotransformation products (e.g. biliary PAH metabolites), reproductive parameters (e.g. plasma VTG) and genotoxic parameters (e.g. hepatic DNA adducts) are currently the most valuable fish biomarkers for ERA. The use of biomonitoring methods in the control strategies for chemical pollution has several advantages over chemical monitoring. Many of the biological measurements form the only way of integrating effects on a large number of individual and interactive processes in aquatic organisms. Moreover, biological and biochemical effects may link the bioavailability of the compounds of interest with their concentration at target organs and intrinsic toxicity. The limitations of biomonitoring, such as confounding factors that are not related to pollution, should be carefully considered when interpreting biomarker data. Based upon this overview there is little doubt that measurements of bioaccumulation and biomarker responses in fish from contaminated sites offer great promises for providing information that can contribute to environmental monitoring programs designed for various aspects of ERA.
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    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.
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    This study investigated how Cd exposure affected oxidative biomarkers in Japanese flounder, Paralichthys olivaceus, at early life stages (ELS). Fish were exposed to waterborne Cd (0-48microg L(-1)) from embryonic to juvenile stages for 80days. Growth, Cd accumulation, activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione S-transferase (GST, EC 2.5.1.18), and levels of glutathione (GSH) and lipid peroxidation (LPO) were investigated at three developmental stages. Flounder growth decreased and Cd accumulation increased with increasing Cd concentration. In metamorphosing larvae, CAT and SOD activities were inhibited and GSH level was elevated, while LPO was enhanced by increasing Cd concentrations. CAT and GST activities of settling larvae were inhibited but GSH level was elevated at high Cd concentrations. In juveniles, SOD activity and LPO level were increased but GST activity was inhibited as Cd concentration increased. Antioxidants in flounder at ELS were able to develop ductile responses to defend against oxidative stress, but LPO fatally occurred due to Cd exposure. These biochemical parameters could be used as effective oxidative biomarkers for evaluating Cd contamination and toxicity in marine environments: CAT, SOD, GSH, and LPO for metamorphosing stage; CAT, GSH, and GST for settling stage; and SOD, GST, and LPO for juvenile stage.
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    The physical and physiological condition of larval fishes throughout their development is believed to influence their survival, and ultimately to contribute to recruitment to the adult population. The study of larval condition is therefore central to larval fish ecology. In this thesis I sought to (1) carry out an appraisal of the characteristics and the reliability of condition measures now being used, (2) assess the importance of maternal contribution to the nutritional status of larval fish, (3) evaluate the possibility that diel variability in metabolism could lead to serious biasing of the interpretation of condition measures obtained over time, and (4) assess the time course of the condition of larval fishes subjected to periods of intermittent feeding. The experiments described in chapter 2 were designed (1) to assess the impact of female nutritional status on the quality of the eggs and larvae they produced, (2) to determine which of a series of nucleic acid and protein measurements were most responsive to post-hatching starvation, and (3) to determine whether the starvation dynamics of those measures was affected by female source. No significant correlation could be found between any of the maternal traits studied and eggs and larval measures. The results did show, however, that egg size was more variable between-clutches than within-clutch, was independent of embryonic developmental rate, but was positively related to larval size. The existence and ontogeny of circadian (24 hrs.) and ultradian (<24 hrs.) oscillations in the nucleic acids and protein content of larval capelin was investigated in the laboratory experiments outlined in chapter 3. The most obvious long-terms trends occurred during the embryonic period when DNA and RNA content increased constantly, and during the post-yolk-sac period when RNA and protein decreased following sub-optimal feeding. The objectives of the study described in chapter 4 were threefold, (1) to determine which of a series of nucleic acid and protein measurements were affected by intermittent (delayed-fed and delayed-starved) feeding conditions in capelin larvae, (2) to determine the dynamics and shape of the time response, and (3) to determine whether the empirical data gathered were consistent with models developed as a consequence of the review of the literature (chapter 1). Only the dry weight, and the quantity of DNA, RNA, and protein per dry weight differed significantly between starvation and ad libitum feeding controls. Starvation dynamics were less consistent with predictions. Of the indices investigated, the dynamics of the quantity of DNA and RNA per dry weight were the most consistent dynamics with model predictions. (Abstract shortened by UMI.)
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    Full-text available
    Evaluating the ecological impact of an oil spill is a complex issue requiring coherently articulated examination of the sequence of interactions that link the cell, where contaminants exert their effects, to the ecosystem, where interactions with human activities arise. This sequence of interactions traverses the frontiers between scientific disciplines (chemistry, toxicology, physiology, and fisheries ecology). Using the common sole (Solea solea L.) as a model species for the coastal habitats polluted by the "Erika" oil spill, our research project attempted to define indices of functional integrity that characterised the consequences of fuel exposure at the different biological levels. The coupling of field observations with experimental laboratory work revealed how functional alterations which are readily observable within individuals and their organs are progressively obscured as investigation progresses towards more complex organisational levels. Some of the approaches and indices are proposed as instruments for evaluating the impact of contamination by hydrocarbons.
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    An investigative biomonitoring study was carried out in a eutrophic coastal system with a moderate contamination by metals (Obidos lagoon, Portugal), combining the evaluation of exposure concentrations with metals accumulation and oxidative stress responses in gills of the golden grey mullet (Liza aurata). Two contrasting seasons (winter and summer) were considered at three sites: Barrosa (BB) and Bom-Sucesso (BS) branches; Middle lagoon (ML). Data on the water column pointed to a higher metals and nutrients availability at BB that was reflected in the higher metal concentrations in gills, particularly in winter. Similarly, oxidative stress responses demonstrated a pro-oxidant challenge at BB (winter and summer), which was corroborated by an integrated biomarker response index (IBR). Metal concentrations in gills were higher in summer than winter, reflecting the increased environmental concentrations in combination with elevated metabolic rates. Catalase (CAT), glutathione-S-transferase (GST), total glutathione (GSH(t)) and lipid peroxidation (LPO) increases observed in winter at BB were related with metal accumulation, while summer enhancement of glutathione peroxidase (GPx), glutathione reductase (GR), GST and GSH(t) was associated with other stressors. Inter-site differences on the basis of IBR were more accentuated in winter. Gills can be considered as an important route of entry for contaminants and were demonstrated to reflect water contamination and are therefore useful in the context of environmental assessment.
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