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In response to repeated complaints from recreational and commercial coastal fishermen about declining fishing opportunities in inner Danish waters, focus was directed to inshore fish stocks. However, without data targeting inshore areas, it was not possible to investigate potential changes in fish distribution or abundances, or their causes. As a first step, a voluntary catch registration system was initiated in 2002, in collaboration with locally organized recreational fishermen. Using citizen science as a methodology, scientists and the fishermen developed a protocol for data collection, which the fishermen then implemented. The aim was to establish regular monitoring of fish catches from gill net and fyke net fisheries in coastal waters around Denmark in order to provide data that could inform management. After three years, during which time recreational fishermen could use their own gear and fish where they normally fished, the data was evaluated. As a result, the fishing method was switched in 2005 to fixed gears and fixed positions, to enable comparison between areas, years and season. The project has been very successful in recruiting highly motivated fishermen, who register their entire catch regularly. The time-series of data spans more than a decade and covers over 16,000 instances of fishing. The data from this project are now being used to create coastal fish indicators for managers to assess environmental status at a regional scale. Here we present an analysis of a subset of the data on one species, the European flounder (Platichthys flesus), to illustrate how the spatial and seasonal coverage can be utilized further for investigation of coastal ecosystems and to inform management.

Marine bony fish poses the unique ability to hydrate from imbibed seawater. They accomplish this, in part, by the precipitation of inorganic carbonate mineral in their intestine, which lowers luminal osmotic pressure and allows for water uptake. It has recently been described that in the Gulf toadfish (Opsanus beta) this Ca(Mg)CO3 precipitation occurs under the regulation of an organic matrix. To date no investigations have aimed to determine if this phenomenon applies more generally to marine fish. Here, intestinally derived precipitates were collected from gray snapper (Lutjanus griseus), white grunt (Haemulon plumieri), European flounder (Platichthys flesus), as well as Gulf toadfish, and their matrices were extracted. The ability of these matrices to regulate CaCO3 production was determined using an in vitro calcification assay, which revealed that the matrix derived from each of the tested species increased precipitation at low concentrations, while inhibiting it at higher concentrations in full agreement with the earlier studies on toadfish. Matrix extracted from European flounder precipitates was then analyzed by mass spectrometry, leading to the identification of over 50 unique proteins. When the identities of these proteins were compared to previous investigation of toadfish precipitate matrix, nearly 35% were found to overlap between the flounder and toadfish analyses, suggesting conserved mechanisms of precipitation control. The effects of using different sodium hypochlorite (NaOCl) solutions during precipitate purification on the resulting organic matrix are also discussed.

Sclerochronological studies based on hard structures of marine organisms are valuable tools—both for reconstructing past climate conditions and for predicting future impacts of environmental changes on marine resources. Existing archives, which house millions of fish otoliths (ear stones) constitute an excellent basis for such research; but, they remain underutilized. The objective of this project was to identify the factors that influence the annual growth patterns of the European flounder ( Platichthys flesus ) based on an analysis of otolith increments. We applied linear mixed models to develop a 74-year long chronology that reflects the inter-annual variations in flounder growth rates using otolith samples collected from 1957 to 2016 in the southern part of the Baltic Sea, which is considered to be highly vulnerable to global climate change. By analyzing the widths of otolith increments we revealed the existence of common environmental factors that influence fish growth. Using a mixed modeling framework, we incorporated a recent method to identify the optimal time window for climatic factors and showed that the most significant effect of the mean Baltic Sea Index occurs during August–December, while mean sea surface temperature is most significant from April–June. Change point analysis on the developed chronology identified major alterations occurred in flounder growth in 1988, 1992 and 2006. This result is in accord with published studies on regime shifts in the Baltic Sea ecosystem. This paper reports information concerning the response of the commercially important European flounder to the changing environment that may support future ecosystem-based management of fish stocks. Moreover, the results also highlight the potential for applying biochronological techniques to identify rapid regime shifts in marine ecosystems.

The spatial structure of fish species is important for stock identification and management. The European flounder ( Platichthys flesus L.) shows morphological differences across the Baltic Sea Proper. However, it is not known whether flounders cluster into several distinct areas based on morphological characters, indicating discrete sub-populations, or whether they show continuous morphological variation along space indicating a more continuous population structure. Here, we study the spatial structure of body length and length-at-age distributions of the European flounder ( Platichthys flesus L.) across the Baltic Sea Proper (International Council for the Exploration of the Sea (ICES) subdivisions 25–28) using high spatial resolution data (ICES rectangles) from fishery independent surveys 2008–2014. Our results are in agreement with genetic data suggesting a continuous gradient of decreasing body length from south-west to north-east. Further, we observed distance decay in the spatial synchrony of temporal changes in the length distributions, such that the temporal trends were correlated among adjacent ICES rectangles but independent across the whole study area. Length-at-age and maturity patterns that were calculated for each subdivision also showed a consistent spatial difference where SD 28 was significantly different from SD 25 and 26. Our results indicate that the European flounder in the Baltic Sea consists of several loosely defined sub-populations, which may warrant a reconsideration of assessment models, management targets and regulations across subdivisions.

The eastern Baltic cod (Gadus morhua) stock is at a critically low level and recruitment of the western Baltic cod stock is at the lowest observed level in over 30 years. The EU (European Union) landing obligation was introduced in 2015 for cod and in 2017 for plaice (Pleuronectes platessa). Size selective codend designs have been used in the Baltic cod trawl fishery for 25 years; however, there are still relatively high discard rates for cod, plaice and European flounder (Platichthys flesus; henceforth flounder) in the trawl fishery and landings of individuals below the minimum conservation reference size (MCRS). These problems call for additional improvements of species and size selectivity in the cod trawl fishery. In this study, two trawl codend designs were developed and tested to improve species and size selectivity in the Baltic cod trawl fishery. The codends were tested onboard a commercial trawler and the test codends were compared to the conventional Bacoma codend, which was fished simultaneously in a twin trawl rig. New codend designs were based on the Bacoma design, where the first test codend had a bottom net panel made of larger diamond meshes (nominal 130 mm mesh size) to improve selectivity in a cod fishery with high catch rates of flatfish. This codend showed a reduction of plaice and flounder, and cod below 45 cm, in the catches. The other test codend was a full square mesh codend (nominal 120 mm mesh size) developed for a cod directed fishery with low flatfish catches, and showed a marked reduction in the catch of cod below 50 cm, with no differences for flounder and plaice catches. With cod stocks at critically low levels and relatively high discard rates of cod, plaice and flounder, the simple codend designs tested in this study could benefit the sustainability of the Baltic cod trawl fishery, but with some loss of commercially targeted sizes.

Like many urban catchments, the River Thames in London is contaminated with plastics. This pollutant is recorded on the river banks, in the benthic environment and in the water column. The present study was conducted to assess the extent of microplastic ingestion in two River Thames fish species, the European flounder (Platichthys flesus) and European smelt (Osmerus eperlanus). Samples were collected from two sites in Kent, England; Erith and Isle of Grain/Sheppey, near Sheerness, with the latter being more estuarine. The results revealed that up to 75% of sampled European flounder had plastic fibres in the gut compared with only 20% of smelt. This difference may be related to their diverse feeding behaviours: European flounder are benthic feeders whilst European smelt are pelagic predators. The fibres were predominantly red or black polyamides and other fibres included acrylic, nylon, polyethylene and polyethylene terephthalate and there was no difference in occurrence between the sites sampled.

Connectivity between coastal spawning grounds and estuarine nurseries is a critical step in the life cycle of many fish species. Larval immigration and transport-associated physical–biological processes are determinants of recruitment success to nursery areas. The recruitment of the European flounder, Platichthys flesus, to estuarine nurseries located at the southern edge of the species distribution range, has been usually investigated during its juvenile stages, while estuarine recruitment during the earlier planktonic life stage remains largely unstudied. The present study investigated the patterns of flounder larval recruitment and the influence of environmental factors on the immigration of the early life stages to the Lima estuary (NW Portugal), integrating data on fish larvae and post-settlement individuals (< 50 mm length), collected over 7 years. Late-stage larvae arrived at the estuary between February and July and peak abundances were observed in April. Post-settlement individuals (< 50 mm) occurred later between April and October, whereas newly-settled ones (< 20 mm) were found only in May and June. Variables associated with the spawning, survival and growth of larvae in the ocean (sea surface temperature, chlorophyll a and inland hydrological variables) were the major drivers of flounder occurrence in the estuarine nursery. Although the adjacent coastal area is characterized by a current system with strong seasonality and mesoscale variability, we did not identify any influence of variables related with physical processes (currents and upwelling) on the occurrence of early life stages in the estuary. A wider knowledge on the influence of the coastal circulation variability and its associated effects upon ocean-estuarine connectivity is required to improve our understanding of the population dynamics of marine spawning fish that use estuarine nurseries.

Flatfish in the northern Baltic Sea are facing multiple environmental pressures due to on-going large-scale ecosystem changes linked to eutrophication and climate change. Shallow juvenile habitats of flatfishes are expected to be especially susceptible to these environmental pressures. Using previously unpublished historical and present-state data on juvenile flatfish in nursery areas along the Finnish coast we demonstrate a drastic (up to 40 ×) decline in 1-Y-O flounder densities since the 1980s and a particularly low current occurrence of both flounders and turbots in several known juvenile habitats. As a consequence of ongoing coastal eutrophication vegetation coverage and filamentous algae have generally increased in shallow areas. We examined the predicted negative effect of vegetation/algae by exploring quantitative relationships between juvenile flatfish (flounder and turbot) occurrence and vegetation/algae among other environmental factors in shallow juvenile habitats. Despite sparse occurrence of juveniles we found a significant negative relationship between flatfish abundance and vegetation cover, implicating eutrophication as a potential major driver affecting the value of juvenile habitat. Shallow littoral habitats play a particularly central role for flatfish due to the spatial concentration of fish in these areas during the critical juvenile stage. Despite their importance, these areas have been relatively poorly studied in the northern Baltic Sea, which makes it difficult to quantify overall changes in environmental conditions and to relate these changes to flatfish recruitment. The low present-state flatfish densities recorded preclude strong inferences of the role of habitat quality to be drawn. Our study does, however, provide a baseline for future assessment. Based on existing evidence, we cannot thus establish any bottlenecks but hypothesize that the current low occurrence of juvenile flatfish, and the population decline of flounder on the Finnish coast, might have resulted from a combination of altered larval supply and reduced nursery value.

The European flounder (Platichthys flesus Linnaeus, 1758) exhibits plasticity for several life traits throughout its distribution range, including ontogenetic habitat shifts during early life, as well as the timing and duration of spawning. Estuaries are preferred as nursery habitat; however, the importance of specific salinity zones for larval development is not well-understood. Therefore, we aimed to identify the significance of distinct estuarine salinity habitats (i.e., tidal freshwater, brackish) along the Minho River estuary (NW-Iberian Peninsula, Europe) for larval development by combining field observations with carbon (C) and nitrogen (N) stable isotope analysis. Sampling occurred between January 2015 and January 2016 in six sampling stations across the estuarine salinity gradient. A total of 29 larvae were collected in the Minho River estuary from March till September 2015. Spawning likely occurred near the river mouth because the highest abundance of larvae occurred in the brackish estuary. Timing for migration towards freshwater was variable with metamorphosis likely occurring in both brackish and freshwater habitats. European flounder larvae obtained their diet from the benthic food web, indicating that benthic habitat is fundamental for larval development, including prior to settlement. This study provides further evidence on the behavioral plasticity of European flounder during early life regarding both habitat use and timing of migration towards freshwater habitats. Additionally, this study demonstrates the importance of preserving estuarine connectivity for this migratory species.

article i nfo The present study aimed to investigate the feeding ecology and influence of prey-predator interactions on juve- nile flounderPlatichthys flesusinanAtlanticestuarinenurseryarea(Limaestuary,NWPortugal),focusingonprey selection and ontogenetic shifts in the diet. The relationship between prey availability and flounder distribution was also investigated. Juvenile flounder diet included 21 taxa of macroinvertebrates and fishes, sand and plant debris. According to numerical, occurrence, and gravimetric dietary indices, macroinvertebrates, namely Chiron- omidae and Corophium spp. were the main prey items. The diet diversity tended to increase as juveniles grew, although some dietary overlap occurred between the early juveniles (50-149 mm total length (TL)). In fact, the diet diversity of the newly settled juveniles (b50 mm TL) was particularly low, evidencing the importance of Chironomidae. Moreover, an ontogenetic shift was evident, since older juveniles (1 + ) presented a distinct diet, including new items absent from the diet of the 0 + juveniles, namely Teleostei, Carcinus maenas ,a nd Nemertea. The juvenile flounder presented an overall generalist behavior, feeding on the most abundant macro- invertebratesnamelyChironomidaeandCorophiumspp.,asevidencedbytheStrausslinearindex.Thespatialdis- tribution of the 0 + flounder in the Lima estuary was associated with salinity and prey (Chironomidae and Corophium spp.). These preys were characteristic of the upper estuary where most of the juveniles, especially the newly settled, were found. Hence, this study reinforces the importance of both abiotic and biotic factors as environmental driven controls of habitat use during the early phases of the demersal life of European flounder. © 2014 Elsevier B.V. All rights reserved.

Molecular responses to acute toxicant exposure can be effective biomarkers, however responses to chronic exposure are less well characterised. The aim of this study was to determine chronic molecular responses to environmental mixtures in a controlled laboratory setting, free from the additional variability encountered with environmental sampling of wild organisms. Flounder fish were exposed in mesocosms for seven months to a contaminated estuarine sediment made by mixing material from the Forth (high organics) and Tyne (high metals and tributyltin) estuaries (FT) or a reference sediment from the Ythan estuary (Y). Chemical analyses demonstrated that FT sediment contained significantly higher concentrations of key environmental pollutants (including polycyclic aromatic hydrocarbons (PAHs), chlorinated biphenyls and heavy metals) than Y sediment, but that chronically exposed flounder showed a lack of differential accumulation of contaminants, including heavy metals. Biliary 1-hydroxypyrene concentration and erythrocyte DNA damage increased in FT-exposed fish. Transcriptomic and 1H NMR metabolomic analyses of liver tissues detected small but statistically significant alterations between fish exposed to different sediments. These highlighted perturbance of immune response and apoptotic pathways, but there was a lack of response from traditional biomarker genes. Gene-chemical association annotation enrichment analyses suggested that polycyclic aromatic hydrocarbons were a major class of toxicants affecting the molecular responses of the exposed fish. This demonstrated that molecular responses of sentinel organisms can be detected after chronic mixed toxicant exposure and that these can be informative of key components of the mixture.

In this work, we analysed the latitudinal trends in the nursery habitat colonization processes of the European flounder (Platichthys flesus). This was accomplished by estimating the duration of the pelagic and metamorphic stages, aswell as the duration of the spawning period, in several nursery areas across its geographical distribution range in the European Atlantic Coast: Mondego estuary (Portugal), Vilaine estuary (France), Slack estuary (France), Wadden Sea (Netherlands) and the Sørfjord (Norway). All juvenile flounders were captured with beam trawls in June/July 2010, and otolith microstructure was used to determine the duration of each stage by means of daily growth increments. The pelagic and metamorphic stages were longer at the middle of the distribution range, and lasted in total up to two months after hatching. The spawning period occurred between mid- January and early-July over the species' distribution range, with a time lapse of nearly two months between the Mondego estuary and the Sørfjord, as a consequence of warmer water temperature earlier in the season in southern areas. In general, total length of the captured fish showed a latitudinal cline between the northernmost and southernmost sampling sites, with higher values at the middle of the distribution range. The results also suggested the existence of a countergradient growth compensation mechanism in the northernmost populations. Apart from temperature, which sets the general metabolic pace of organisms, differences between siteswere also related with local features, such as the extension of the continental platform and adaptations to transport and retention mechanisms. Filipe Martinho was funded by the Portuguese Foundation for Science and Technology (FCT), through a post-doc grant (SFRH/BPD/63527/2009).

International audience; Estuaries are important areas highly susceptible to anthropogenic degradations like pollution. Estuarine species have thus to cope with many types of constraints depending on the estuaries' characteristics. The European flounder Platichthys flesus is considered as a sentinel species for the monitoring of estuarine water quality. In this study, juvenile flounders (0+ group) were sampled from three contrasted Channel estuaries, i.e. the Seine, the Canche and the Tamar, and we characterized their liver proteomes by using a two-dimensional electrophoresis based proteomic approach. We showed that 27 protein spots differentially accumulated between the 3 populations. Six of these proteins were identified by MALDI TOF-TOF mass spectrometry. Flounders from the Seine and from the Tamar, two highly polluted estuaries, displayed common differences, i.e. an increase of the energetic- and the glutathione-metabolism. The most accumulated protein in the Seine's samples (6.7-fold) was a Vitelline Membrane Outer layer protein 1 homolog, suggesting oogenesis deregulation in these juvenile (sexually immature) flounders. Future works applying this kind of proteomic approach on flounders experimentally exposed to conditions that mimic environmental constraints will help to better understand the significance of these environmental proteomic signatures.

European flounder ( Platichthys flesus . L.) is a euryhaline flatfish species which can actively migrate towards and cope with low salinity environments. A laboratory experiment was undertaken to analyse the effect of salinity on condition and growth of metamorphosing European flounder. The working hypothesis was that flounder, which preferentially settle in low salinity habitats, would display accelerated development and/or enhanced growth and condition at lower salinities. The fish used in the experiment were in the late stages of metamorphosis. At the end of the 21 day laboratory rearing period no significant difference in ontogenetic development was found between exposures (salinity of 0, 10, 20 and 30). No significant differences in somatic growth rate, somatic condition or standard length were observed between treatments. There was no correlation between RNA:DNA ratio and somatic condition. Contrary to expectations, mean RNA:DNA ratios (measure of short-term well being) tended to increase with salinity and were significantly higher in the 30 salinity exposure compared to the 0 salinity exposure. The working hypothesis was, therefore, rejected. The results demonstrate that laboratory observations can fail to capture the complex ecological interactions at play in field environments. The preference for low salinity environments may be driven by other environmental factors such as predator/competition avoidance and food supply.

This study aims to determine the diversity of migration patterns of the European flounder (Platichthys flesus (Linnaeus, 1758)) present in the Minho estuary and in the adjacent coastal area (NW-Iberian Peninsula). Assessing the diversity of flounder migration patterns at the southern limit of its distribution allows the determination of characteristics of the species' ecology and provides useful information for fishery managers, since it is a regionally important estuarine fishery. An unexpected result of our study was that flounder appears to spawn in both estuarine and coastal areas and not just in coastal areas as was previously widely accepted. Our interpretation of otolith strontium distribution patterns from flounder specimens collected in the freshwater tidal area of the Minho estuary and in the lower estuary suggested that the flounders hatched in the estuary, while only 6.7% of those captured in the coastal area hatched in the coastal area. Ultimately, studies aimed at collecting larval stages and adult flounders must be made to confirm that flounders spawn in the estuary and to define new and better scientifically supported fishing policies, or simply to confirm the existing ones regarding temporal and spatial closures for each gear used in the Minho estuary.

In this study, new DNA markers were explored for the flounder Platichthys flesus. cDNA and genomic sequences of the genes encoding the glyceraldehyde-3-phosphate-deshydrogenase (GAPDH), the cytosolic creatine kinase (CK), the prostaglandin D synthase (PGDS) and the betaine homocysteine methyltransferase (BHMT) were characterized. The tumour suppressor p53 gene structure was already described. A PCR–SSCP (Single Strand Conformation Polymorphism) analysis was finally conducted to study the genetic polymorphism of different populations of flounders collected along the French Atlantic coast. Four highly contaminated French estuaries (Seine, Vilaine, Loire and Gironde) were sampled and compared to a reference estuary (Ster) to explore possible selective effect of the environment on specific allelic frequencies. Our results showed that two loci p53 and PGDS, could be potential markers of chemical stress: p53A allele frequency increased in contaminated systems compared to the reference system. In the Vilaine estuary, PGDS polymorphism could be related to pesticide stress.

In order to identify nursery habitats for Platichthys flesus in the Venice lagoon we developed a generalized additive model relating juvenile flounder's distribution to environmental variables. A field survey was conducted between March 2004 and June 2005 and between February and October 2008 in the central and Northern sub-basins of the lagoon. Each station was sampled by means of a beach seine net and characterized collecting the main chemico-physical variables, such as water temperature, salinity, turbidity, dissolved oxygen content and bottom grain size. Main winds fetches were also considered, and estimated in a GIS environment. A logistic model was fitted, and evaluated on an independent dataset. The response curves allowed to identify the role of the environmental parameters in explaining the distribution of the juvenile flounder: turbidity, salinity and sand content revealed to be the most important factors, showing the preference for mesohaline turbid waters together with a low sand content of the sediment. The application of this model to continuous surfaces of the environmental variables allowed the creation of potential habitat distribution maps. In this way it has been possible to recognize several areas covering a key role for the juveniles of flounder, located mainly in the inner part of the lagoon.

The brackish, bloom-forming cyanobacterium Nodularia spumigena produces a peptide called nodularin, which may induce liver damage in fish. In the summer of 2007, nodularin was detected in liver tis ...

The temporal transcriptomic responses in liver of Platichthys flesus to model environmental pollutants were studied over a 16-day time span after intraperitoneal injection with cadmium chloride (50 μg/kg in saline), 3-methylcholanthrene (25 mg/kg in olive oil), Aroclor 1254 (50 mg/kg in olive oil), tert -butyl-hydroperoxide (5 mg/kg in saline), Lindane (25 mg/kg in olive oil), perfluoro-octanoic acid (100 mg/kg in olive oil) and their vehicles, olive oil (1 ml/kg) or saline (0.9%). Statistical, gene ontology and supervised analysis clearly demonstrated the progression from acute effects, biological responses to and recovery from the treatments. Key biological processes disturbed by the individual treatments were characterised by gene ontology analyses and individual toxicant-responsive genes and pathways were identified by supervised analyses. Responses to the polyaromatic and chlorinated aromatic compounds showed a degree of commonality but were distinguishable and they were clearly segregated from the responses to the pro-oxidants cadmium and the organic hydroperoxide, as well as from the peroxisomal proliferator, perfluoro-octanoic acid. This study demonstrated the utility of the microarray technique in the identification of toxicant-responsive genes and in discrimination between modes of toxicant action.

The objective of the present study was to study the release and effect of TNT from dumped ammunition. Cleaved artillery shells were placed in static brackish water systems for 5 months, and another 12 months with 5 cm sediment burial. The toxicity was determined in bioassays with crustaceans (Nitocra spinipes and Hyalella azteca) and/or European flounder (Platichtys flesus). The water phase was analysed for TNT using colorimetric method and GC-MS. This study showed a rapid release of TNT to acutely toxic concentrations when the cleaved ammuniton was not covered with sediment under static conditions, but that the release was effectively inhibited by sediment burial of the artillery shells. Hence, at least in a short-term perspective, acute adverse effects of sediment-buried ammunition on aquatic organisms should be greatly reduced.

The caudal neurosecretory system (CNSS) of the euryhaline flounder Platichthys flesus has suggested roles in osmoregulatory, reproductive and nutritional adaptation, as fish migrate between seawater (winter) and brackish/freshwater (summer) environments. This study examined seasonal changes in mRNA expression profile of functionally important genes in the CNSS. cDNAs encoding neuropeptides, receptors and ion channels were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) and screening of a flounder CNSS cDNA library. The expression profile of cloned genes was determined by real-time RT-PCR at 2-month intervals throughout the year in CNSS from seawater-adapted fish. Plasma cortisol (measured by radioimmunoassay) showed a peak in April, the time of spawning. Expression levels of mRNA for peptides urotensins I and II (UI, UII) and corticotropin releasing factor (CRF) all showed a seasonal cycle, with lowest expression in April and highest in August-October. The expression of CRF2(UI), UT(UII) and CRF1 receptors was not correlated with corresponding peptide expression. Receptors for potential neuromodulators of CNSS activity also displayed a seasonal mRNA expression profile. Glucocorticoid, 5-hydroxytryptamine, kappa-opioid and glutamate receptor expression peaked around April, suggesting that modulation of electrical activity of the neurosecretory Dahlgren cells is of particular importance at this time. Expression of mRNA for L-type Ca(2+) and Ca-activated K(+) channels was lower during the summer months. These channels underlie electrical bursting activity in Dahlgren cells. Ion channel mRNA expression was also lower in CNSS from flounder fully adapted to freshwater as opposed to seawater, consistent with previously reported observations of reduced bursting activity in Dahlgren cells from freshwater-adapted CNSS. These findings support the hypothesis that the CNSS is functionally reprogrammed to cope with changes in physiological challenge as fish migrate between sea and estuaries in winter and spring.

The induction of hepatic cytochrome P450 1A (CYP 1A) is an important step in the response to contaminants such as polycyclic aromatic hydrocarbons (PAHs), and has been used as a biomarker of exposure in fish. Several consensus response elements have been identified, including eight potential xenobiotic response elements (XREs) in the promoter region of the European flounder cytochrome P450 1A gene. However not all of these sequences are necessarily active. To help elucidate the molecular regulation of this important gene, site directed mutagenesis and dual-luciferase reporter gene assays were employed to characterize the consensus transcription factor binding sites of the CYP 1A 5′ flanking region. Mutation of response elements situated −1103, −859, −709 and −172 bases upstream of the transcription start site reduced the induction to 2.75, 1.51, 3.25 and 3.05 fold, respectively, compared with the full-length promoter (4.0-fold induction) on exposure to the PAH 3-methylcholanthrene (3MC) (1.0 μM). These results indicate that four out of eight different XREs are functional in the control of CYP 1A in the flounder. The activity of these response elements adds to the evidence for considerable diversity in vertebrate CYP 1A regulation.

In respect to the differences found for vitellogenin (VTG) expression in male and immature flounder, general biological aspects and a set of chemical residues in the liver were compared between flounder from the Mersey and Dee estuaries (UK). Except for alpha-HCH, all pollutant chemicals analysed in flounder liver differed highly significantly between Mersey and Dee fish. Overall, the higher liver contaminant concentrations were found in VTG-induced Mersey flounder. The biomarkers studied were not found to indicate significant differences between Mersey and Dee fish. However, when all of the minor differences are taken into consideration, it appears that the slightly lower growth rates after age 2 in Mersey flounder, lower gonadosomatic index in both mature male and female animals, less precise seasonal patterns of condition factor (CF), hepatosomatic index and gonadosomatic index, and lower CF in immature Mersey fish may well be signs of a contaminant-affected Mersey population.

Whole animal studies using seawater European flounder (Platichthys flesus) revealed that increasing intestinal [Ca(2+)] to 20 mM stimulated net HCO(3)(-) base secretion by 57%, but this was effectively balanced by an increase in net acid secretion, likely from the gills, to maintain whole animal acid-base status. Higher Ca(2+) concentrations (40 and 70 mM) in ambient seawater resulted in reduced plasma total CO(2). This indicates (1) imperfect acid-base compensation, and (2) that endogenous metabolic CO(2) is insufficient to fuel intestinal HCO(3)(-) secretion, under hyper-stimulated conditions. Bicarbonate secretion plays an important role in preventing calcium absorption by precipitating a large fraction of the imbibed calcium as CaCO(3). Indeed, under high Ca(2+) conditions (20 mM), up to 75% of the intestinal Ca(2+) is precipitated as CaCO(3) and then excreted. This is undoubtedly important in protecting the marine teleost kidney from the need for excessive calcium excretion and risk of renal stone formation. Using an in vitro pH-stat technique with the isolated intestinal epithelium, the replacement of serosal CO(2) with a HEPES buffered saline had no effect on HCO(3)(-) secretion, indicating that the endogenous supply of HCO(3)(-) from CO(2) hydration within epithelial cells is adequate for driving baseline secretion rates. Further, in vitro data demonstrated a stimulatory effect of low pH on intestinal HCO(3)(-) secretion. Thus, both luminal Ca(2+) and H(+) can regulate HCO(3)(-) secretion but the precise mechanisms and their potential interaction are currently unresolved.

The role of glucose-6-phosphate dehydrogenase (G6PDH) in oxidative stress responses was investigated in isolated intact living hepatocytes of immature female and male European flounder (Platichthys flesus L.) because it is the major provider of NADPH needed as reducing power for various detoxification pathways. Hepatocytes were exposed to sublethal concentrations of effective prooxidants such as 100 muM hydrogen peroxide (H2O2), 100, 200 and 400 muM benzo[a]pyrene (B[a]p) and 50 muM nitrofurantoin (NF) during culture. Since there is evidence that 17-beta-estradiol inhibits certain pathways of xenobiotic biotransformation, we tested also the effects of different concentrations of 17-beta-estradiol (0.2, 1 and 2 muM) alone and 1 muM in combination with 100 muM B[a]p on G6PDH activity. After short-term (I day) and long-term (9 days) exposure, G6PDH activity was quantified in intact living hepatocytes by a tetrazolium salt method using tetranitroblue tetrazolium salt (TNBT). Hepatocytes obtained from male fish generally showed higher G6PDH activity than those of females. We observed significant inhibition of G6PDH activity by all oxidative stressors and 17-beta-estradiol in both sexes of fish independently of culture conditions, but inhibition was stronger in cells of females than in cells of males. A cumulative effect of the steroid and B[a]p was not found. Our results indicate a sex-dependent inhibitory effect of all stressors and 17-beta-estradiol on G6PDH activity in flounder hepatocytes independent of prooxidant activity of the specific compound. Consequently, NADPH supply for xenobiotic detoxification and other cellular antioxidative defence mechanisms may be different in livers of female and male flounder. The strongly decreased supply of NADPH in hepatocytes of females may explain the reduced and/or delayed NADPH-dependent activity of xenobiotic biotransformation systems such as cytochrome P450 (CYP450) and a lower capacity of non-enzymatic defence systems such as reduced glutathione that is particularly observed in female flounder. Moreover, the strong inhibition of G6PDII in livers of female flounder may explain higher susceptibility for xenobiotic toxicity and, therefore, potentially a higher risk to develop liver cancer. (C) 2002 Elsevier Science B.V. All rights reserved

Chronic (5 weeks) exposure of freshwater-adapted European flounder, Platichthys flesus (L.), to environmental concentrations of sediment-associated tri-n-butyltin chloride (TBTCl) and triphenyltin chloride (TPhTCl) caused significant changes to hydromineral fluxes and membrane permeability, mechanisms that maintain osmotic homeostasis. The half-time of exchange of tritiated water (THO) in TBTCl- and TPhTCl-exposed fish was significantly increased during the first 2 weeks of the experiment and then decreased steadily, eventually reaching the level that the control group had constantly maintained throughout the experiment. This change in apparent water permeability was accompanied by a significant decrease in diffusional water flux across the membranes. Passive Na(+)-efflux across the gills was increased significantly but effluxes in the control group were near constant over the same time span. Drinking rates in the organotin groups increased significantly while the rate of urine production did not change. This lead to an increased net water balance in the organotin groups and consequently to a significant reduction of the blood osmolality of both organotin groups when compared to a control. There would appear to be a metabolic cost attached to the changes produced by exposure to environmental levels of organotin compounds which are manifested as a minimal increase in body length compared to the controls.

The phagocytic activity of macrophage aggregates (MAs) in the liver of fish is characterized by high acid phosphatase activity and generation of reactive oxygen species (ROS). Question of the present study was whether these activities were associated with damage of adjacent and surrounding liver cells and the macrophages itself. Thus, the lysosomal membrane stabilities (LMS) of two different populations of lysosomes (LMS1, short destabilization periods, and LMS2, longer destabilization periods) of liver cells close to MAs of different phagocytic activity were measured as indicator for cellular integrity by computer assisted image analysis. They were then compared to the LMS of hepatocytes remote from any MA. In addition, LMS was also assessed inside the MAs. Studies were performed on adult European flounder (Platichthys flesus) and corkwing wrasse (Symphodus melops) caught at clean and polyaromatic hydrocarbon (PAH)-contaminated field locations of the German Bight and the Karmoy peninsula at the West coast of Norway. In the hepatocytes adjacent to MAs which showed medium to high phagocytic activity, LMS was significantly decreased with a reduction of both, first (LMS1), and second (LMS2) destabilization periods in both species. LMS1 inside of these MAs was extremely low, whereas LMS2 was sometimes even higher compared to the liver cells. We suggest that LMS1 represents phagosomes which had contact to ROS and LMS2 represents less affected primary lysosomes. In single flounder and wrasse with liver tumors, MAs in the vicinity of these tumors showed low phagocytic activity and were not associated with adverse effects on adjacent tumor cells. Inside these MAs, LMS1 was higher compared to MAs with high phagocytic activity in wrasse from the reference site, indicating that cytotoxicity was not involved in their phagocytic deficiency.

Immunohistochemical and histopathological studies were conducted in two marine teleosts, Atlantic cod ( Gadus morhua ) and European flounder ( Platichthys flesus ), caged for three months on contaminated sediments in a Norwegian fjord. Cellular localization of CYP1A was analyzed immunohistochemically in liver, intestine, heart, gill and kidney, as part of an extensive study that included a number of chemical and biological measurements. Both species exhibited marked CYP1A induction when caged at contaminated sites. CYP1A induction was observed in liver as well as in several extrahepatic organs, and with increased expression at the more contaminated sites. Staining was particularly strong in vascular endothelium. Induction responses were also observed in epithelial cells, including hepatocytes, biliary epithelial cells, mucosal epithelial cells in the intestine, and renal tubular epithelial cells. Histopathological examination of the liver did not reveal major cellular abnormalities. The immunohistochemical data indicate a strong relationship between CYP1A induction and exposure to sediment-associated industrial contaminants (polycyclic aromatic hydrocarbons). Interesting species differences in localization of CYP1A expression in various cell types in cod and flounder were demonstrated immunohistochemically.

The inductive response of metallothionein (MT) synthesis to heavy-metal exposure in many Phyla is well documented; however, before this response can be used and interpreted as an environmental biomarker of exposure, the kinetics and dose responses must be known. We have carried out a series of experiments where we have studied these parameters of MT mRNA and MT protein induction in different tissues (liver, kidney, gills) of European flounder after intraperitoneal (ip) injection of Cd (1–320 μg/kg), Cu (25–200 μg/kg), Hg (235–100 μg/kg) and Zn (1–20 mg/kg). Basal hepatic levels of 320 μg MT/g wet tissue for the wild caught flounder used were five times higher than in fish from a pristine area and 10–20 times higher than in plaice or turbot obtained from pristine areas. Transient increases in renal MT mRNA levels were only observed 4–10 days post injection (p.i.) of Cu or Hg and there were no significant increases in MT protein levels in any tissue. Doses of >5 mg/kg Zn induced MT mRNA levels three- to four-fold in all tissues 2–4 days p.i. but only produced a transient elevation in MT levels at 4 days p.i. There was no measurable response to low doses of Cd, doses of >80 μg/kg Cd had no significant effect on hepatic MT levels but caused a significant elevation in gill and kidney levels 10 and 21 days p.i. MT mRNA levels in gill and kidney were induced four- to five-fold 24 h p.i. of Cd (and remained elevated for >10 days) whilst a response was only observed in liver (two-fold) 4 and 10 days p.i. These laboratory results, obtained with a wild population exhibiting moderately elevated basal MT levels indicate that the response to metal exposure is too small and too highly dependent upon the metal and exposure time to provide a robust biomarker of contaminant effect. This supports our previous observation of analyses of flounders from the southern North Sea and Dutch estuaries and would indicate that caging studies with ‘ultra-clean’ animals would be required for any monitoring studies.

In the small intestines of flounders adapted to sea-water or to fresh water, intracellular chloride activity is maintained above equilibrium activity. In sea-water adapted animals this accumulation is inhibited by piretanide whereas fresh-water adapted animals are insensitive to the drug. This indicates different chloride accumulation mechanisms.

The statistical procedure of analysing for trends over time in contaminant concentrations in fish provided by the International Council for the Exploration of the Sea (ICES) 1. is univariate and 2. analyses data on only one species at a time from a given sample area, even if data on more than one species are available. In this study temporal variations in contaminants in Atlantic cod ( Gadus morhua ) and European flounder ( Platichthys flesus ) from the Belgian coast are examined at the multispecies level rather than the single species level. The ICES univariate procedure 1. has been extended to the analysis of combined data for the two (or more) species and 2. is supplemented by the multivariate analysis of covariance procedure. Not only did the two species respond differently to variations in contaminant levels with time but even within a species contaminant levels did not vary in the same direction.