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This study aimed to develop a multidisciplinary approach to assess the ecological status of six moderate-sized French estuaries. For each estuary, we gathered geographical information, hydrobiological data, chemistry of pollutants and fish biology, including integration of proteomics and transcriptomics data. This integrative study covered the entire hydrological system studied, from the watershed to the estuary, and considered all the anthropogenic factors that can impact this environment. To reach this goal, European flounder (Platichthys flesus) were collected from six estuaries in September, which ensures a minimum residence time of five months within an estuary. Geographical metrics are used to characterize land use in each watershed. The concentrations of nitrite, nitrate, organic pollutants, and trace elements were measured in water, sediments and biota. All of these environmental parameters allowed to set up a typology of estuaries. Classical fish biomarkers, coupled with molecular data from transcriptomics and shotgun proteomics, highlighted the flounder's responses to stressors in its environment. We analysed the protein abundances and gene expression levels in the liver of fish from the different estuaries. We showed clear positive deregulation of proteins associated with xenobiotic detoxification in a system characterized by a large population density and industrial activity, as well as in a predominantly agricultural catchment area (mostly cultures of vegetables and pig breeding) mainly impacted by pesticides. Fish from the latter estuary also displayed strong deregulation of the urea cycle, most probably related to high nitrogen load. Proteomic and transcriptomic data also revealed a deregulation of proteins and genes related to the response to hypoxia, and a probable endocrine disruption in some estuaries. Coupling these data allowed the precise identification of the main stressors interacting within each hydrosystem.

The objective of this study was to develop an integrative approach in ecotoxicology (from biomarkers to population genetics) to assess the ecological status of fish populations. Flounders (Platichthys flesus) collected after the spawning season in the heavily polluted Seine estuary were compared with the moderately polluted Bay of Douarnenez. The muscle energetic reserves were highly depleted in Seine vs. Douarnenez fish. The Seine fish displaying a reduced capacity to manage the oxidative stress and a higher energetic metabolism. An increase in the content of muscle membrane phospholipids (sphingomyelin, phosphatidylserine, free sterols) was detected in the Seine vs. Douarnenez fish. The data integration allowed to hypothesize relationships between membrane phospholipids, xenobiotic metabolism, bioenergetics, and antioxidant defence. The genetic diversity considering neutral markers was maintained in the heavily polluted Seine population compared with the Douarnenez population. Finally, we suggest that the high physiological cost of tolerance to toxicants in the Seine flounder population could compromise its capacity to respond in the future to an additional stressor like warming waters in shallow depth. Thus, this population could be submitted to an ecological risk.

European flounder in Rhine and Meuse, about a seafish going upstream European flounder (Platichthys flesus) is a catadromous fish. In the River Rhine flounder is abundant in the lower reaches, the estuaria serve as nursery grounds. Upstream in the German Rhine its abundance decreases upstream of Bonn. Occasional reports prove it does reach the upper part of the Rhine, close to the Swiss border, a 800 km trip from the sea. In the Meuse flounder historically swam up to Liège (Belgium), 300 km from the North sea. Nowadays multiple weirs block its passage in this river. Combining datasets from throughout the entire Rhine catchment improves insight in the species distribution in freshwater. Flounder populations are under stress, not only caused by migration barriers, but also through pollution, climate change and invasive species. De bot staat bekend als zeevis en als een soort van brakke estuaria. De verbazing is dan ook vaak groot wanneer je deze platvis ver stroomopwaarts in de rivier tegenkomt. Er zijn anekdotes dat de soort tot in Zwitserland de Rijn op kan zwemmen. Wat heeft een zeevis daar te zoeken? Om te weten of bot echt zover stroomopwaarts het zoete water op gaat, zijn databestanden uit het gehele stroomgebied van Rijn en Maas verzameld.

Summer growth reduction in juvenile flatfish has been observed in various temperate coastal areas, suggesting a general mechanism. One possible mechanism that might explain this phenomenon is related to trophic limitation. After the spring phytoplankton bloom macrozoobenthic infauna becomes less active above the sediment, thereby affecting the time spent by predatory flatfish on searching for prey and hence, causing a reduction in food intake and in growth. Here, our aim is to gather evidence to substantiate this so-called “summer growth reduction” hypothesis by analyzing summer growth for 0-group flounder Platichthys flesus at the Balgzand intertidal area in the western Dutch Wadden Sea, under the prediction that flounder, as a more epibenthic predator, would suffer less or not at all from summer growth reduction in contrast to 0-group plaice Pleuronectes platessa, a more benthic feeder. Summer growth was studied for three contrasting years with respect to preceding winter water temperature conditions (cold, average and warm year) to exclude possible irreversible non-genetic adaptation of growth to water temperature conditions. Growth performance was analyzed by combining information on individual growth based on otolith daily ring analysis with predictions of maximum growth (= under optimal food conditions) based on a Dynamic Energy Budget model. In line with expectations and in contrast to 0-group plaice, no trend in growth performance over time was found suggesting that 0-group flounder showed no growth reduction after summer, providing further basis for a future testing of the trophic limitation hypothesis

Fish telemetry studies give unique information about fish movements (migrations), but there are still several methodological constraints. A critical step is the choice of an appropriate tag and tagging method, essential to guaranteeing that the tagged fish is representative of the general wild population. The effects of two tagging methods with acoustic transmitters on the behavior, survival, condition, and growth of the European flounder Platichthys flesus (L.) were evaluated in tank experiments. Two 33-day assays were conducted: one with adult flounder to test intracoelomic (internal) and external tagging with Vemco V7-2 L dummy tags, and a second assay with juveniles using only external tags. Intracoelomic implantation resulted in very low survival rate (10%), fish lethargy, and a lack of response to food. External tagging did not affect behavior but had negative effects on the final condition of adult flounder, and on the specific growth rates (SGR) of smaller-sized fish. The external tagging method is the most appropriate for P. flesus because of the technique’s simplicity, expedience, and fewer negative effects upon the fish.

The Baltic Sea is a frontrunner in ecosystem analysis and assessment, which have been used also in multispecies fisheries management advice. In the Baltic Sea, the multispecies assessment and management advice have been focused on the pelagic interactions between cod (Gadus morhua), sprat (Sprattus sprattus) and herring (Clupea harengus), by virtue of their well-known ecology. However, the fish in-teractions occurring in the benthic habitat are largely unknown. This study investi-gates, for the first time, the feeding interactions between the most important de-mersal fish species, cod and flounder (Platichthys flesus), in three areas of the eastern Baltic Sea. In this study I use stomach data from 2015 and 2016, collected in the ICES subdivisions 25 – 28 by the Swedish University of Agricultural Sci-ences, Department of Aquatic Resources. The diet of cod differs between the areas but, overall, shows an ontogenetic shift with a decrease of benthic prey and an in-crease of fish preys with size. In the coastal area the amount of benthic prey is al-ways > 50% irrespective of predator size, while in the offshore areas the amount of fish prey increase > 50% with increasing cod size. Conversely, the diet of flounder is relatively constant between sizes and areas. Cluster analyses revealed similarity between the diet of flounder and small-medium size cod in the offshore areas. A significant diet overlap was found between cod < 30 cm and flounder > 20 cm in the offshore area in SD 25, which is mainly driven by similar benthic prey, espe-cially Saduria entomon, in the diet of both predators. These results point to a food competition between cod and flounder, likely augmented by recent increased abundance of Baltic flounder stocks. This competition could decrease the availa-bility of benthic prey for cod, which, in turn, can lead to low condition factor, a re-duction of cod growth and ultimately accentuate the negative effects of hypoxia on cod. Because of all of these reasons

Se describe la comunidad de peces del río Masma y sus principales afluentes a partir del inventario demográfico de las especies (trucha común y reo, anguila, salmón atlántico, lamprea, platija y mugílidos) y a partir del análisis del hábitat (calidad del agua, morfometría del cauce, vegetación y presión pesquera). Se realizaron 22 estaciones de muestreo por pesca eléctrica durante tres años. Los resultados informan de un hábitat de buena calidad para los salmónidos, levemente alterado por la acción humana y con una abundancia de peces intermedia (densidad: 3 847 ind/ha; biomasa: 127.1 kg/ha), con existencias y producciones mucho más altas en los afluentes que en el curso principal. La especie más abundante fue la trucha común (68% de los ejemplares de la muestra), escaseando los individuos de las clases de edad superiores a 4+. Se concluye que el origen del desequilibrio demográfico que se detecta está en la elevada presión de los pescadores tanto en tramos libres como en vedados y acotados. La alta presión pesquera sobre la trucha común es en parte atribuible a que la talla mínima pescable que se aplica en la zona incluye clases de edad no reproductivas (1+). Se expone una propuesta de mejora de la gestión basada en conservar el hábitat en su estado actual, junto con el cambio de las tallas mínimas de captura y la reubicación de las zonas de pesca, con objeto de favorecer la regeneración natural de las poblaciones de salmónidos y lograr a medio plazo estabilizar las clases de edad, The fish populations in the Masma River and its main affluents were described on the basis of the study of the demographic characteristics of the species (brown trout, European eel, Atlantic salmon, sea lamprey, European flounder and Mugilidae) and the habitat analysis (water quality, morphometry of the river bed, vegetation and fishing effort). An electrofishing survey was carried out in 22 sampling points during three years. The expected high quality of the habitat for salmonids was shown in a slightly altered environment (intermediate stocks, 3 847 ind/ha, 127.1 kg/ha), being the abundances and productions much higher in the affluents than in the main river. Brown trout was the most abundant species (68 % of the individuals of the sample), with low presence of individuals of age 4+ and upper classes. The unbalanced age distribution is originated by the overfishing pressure in the area. The high fishing pressure on brown trout is partly attributable to the fishing minimum size, that encompasses non reproductive classes (1+). A management improvement is proposed, based on the preservation of the current state of the habitat, changes in the minimum catching length and relocation of fishing areas, to achieve and enhance natural regeneration of salmonids and to stabilize age classes in the medium term

In opdracht van Rijkswaterstaat zijn in 2015 door IMARES werkzaamheden uitgevoerd in het kader van het Joint Assessment and Monitoring Program van de OSPARCOM. In dit project worden botten van drie locaties verzameld door IMARES en onderzocht op biologische parameters, waaronder visziekten, en gehalten milieukritische stoffen, zowel organische contaminanten als metalen. Tot en met 2013 was het chemisch onderzoek gericht op gezonde mannelijke botten, in lijn met de nieuwe OSPAR/JAMP-richtlijn worden vanaf 2014 gezonde vrouwelijke exemplaren onderzocht. De werkzaamheden zijn volgens protocol uitgevoerd. In 2015 is het chemisch onderzoek naar milieukritische stoffen in bot uitgevoerd op de locaties Kustzone Noordwijk, Westerschelde en Eems-Dollard. Het visziekte-onderzoek is uitgevoerd op de locaties Westerschelde en Eems-Dollard. Dit jaar zijn voor de tweede keer de componenten heptachloor, HBCD en de perfluorverbindingen gemeten in bot, vanwege Richtlijn 2013/39/EU die 12 augustus 2013 is gepubliceerd en rechtsgeldig wordt vanaf 2018. De verzamelde gegevens en analyse-uitkomsten zijn in tabelvorm als bijlagen achter in dit rapport bijgevoegd en worden als DIF-files aangeleverd voor opslag in DONAR.

The reproductive success for pelagic spawning Baltic flounders is strongly linked to the hydrodynamics in the spawning areas. Egg survival is dependent upon the ability to achieve neutral buoyancy at a depth interval where temperature and oxygen concentrations are favourable for egg development. The main focus of this thesis was to compare egg survival probabilities of pelagic eggs from Baltic flounder in the Bornholm, Gdansk and Gotland basins, prior to and after the saline water inflow in December 2014. The results showed greatly enhanced survival probabilities in Bornholm basin 2015 (p<0.01), as egg survival increased from 47% in 2014 to 100% the following year. In Gdansk basin the situation was similar, and survival probability increased from 13% to 100% (p<0.01). In Gotland basin no difference in survival probability was identified, although the dominant cause of mortality shifted from sedimentation, i.e. due to low salinity conditions in 2014, to oxygen deficiency in 2015 (p<0,01)., BONUS INSPIRE-project, the joint Baltic Sea research and development programme (Art 185), funded jointly by the European Union’s Seventh Programme for research, technological development and demonstration and the Swedish Research Council Formas

In opdracht van Rijkswaterstaat zijn in 2014 door IMARES werkzaamheden uitgevoerd in het kader van het Joint Assessment and Monitoring Program van de OSPARCOM. De werkzaamheden bestonden uit het verzamelen van monsters bot waarvan biologische parameters werden bepaald. Tevens werden in deze botten milieukritische stoffen geanalyseerd. De verzamelde gegevens en analyse-uitkomsten worden aangeleverd in dit rapport.

Fiskars morfologi, det vill säga dess utseende och form, kan variera beroende på hur de utvecklas och fortplantar sig. Morfometriska analyser används för olika typer av studier då det ger information om grupper av individer med samma reproduktions-takt, tillväxt och överlevnad samt fenotypiska grupper. Skrubbskädda, Platichthys flesus, delas in i två olika lektyper; utsjölekande och kustlekande, där de utsjöle-kande lägger pelagiska ägg och de kustlekande lägger demersala ägg. Morfomet-riska analyser kan vara en bra metod att skilja dessa lektyper åt i förvaltningen. I denna studie fotograferades skrubbskäddor och morfometriska analyser utfördes i samarbete med BONUS-INSPIRE för att avgöra om dessa två lektyper skiljer sig åt i morfologi. Skillnader mellan de olika lektyperna hittades för längden på stjärtfe-nan, bröstfenan och huvudet samt avståndet mellan anal- och bukfena och fiskens bredd. Skillnader kunde även ses mellan de olika könen samt mellan de olika fångst-områdena. De kustlekande skrubbskäddorna lägger mer energi på reproduktiv till-växt och har därför bredare buk pga. större gonader, de har kortare huvud, bröst- och stjärtfena vilket indikerar att lite energi läggs på kroppstillväxt. Utsjölekande skrubbskäddor lägger istället mer energi på somatisk tillväxt vilket förklarar att de är relativt längre men smalare kropp än de kustlekande. Honor har bredare buk än hanar då de har större gonader, tarmar och lever. Individerna klassades utifrån deras morfologi i en diskriminantanalys som visade att 76 av 85 individer klassades till rätt lektyp när man endast såg till deras morfologi, det ger en felmarginal på 10,5%., The morphology of fishes could vary depending on how they develop and repro-duce. Morphometric analysis is used in many different types of studies because it gives information about groups of individuals with the same rate of development, growth and survival and phenotypic groups. The European flounder, Platichthys flesus, can be categorized into two different types of groups depending on their spawning strategies; deep water spawning (offshore spawning) with pelagic eggs and shallow water spawning (costal spawning) with demersal eggs. Morphometric analysis could be a good method to use when trying to separate these two spawning types apart in an efficient and economic way. In this study the flounder was photo-graphed and morphometric analysis was conducted in cooperation with BONUS INSPIRE to see if the two different spawning types differed in morphology. Differ-ences between the different spawning types was found on the length of the pectoral fin, the cadual fin and the head. Differences was also found for the distance between the insertion of the anal fin and the insertion of pelvic the fin, differences was also seen in body depth. There was also morphometric differences when looking at the different types of sex and the different catch areas. The costal spawning flounder focuses more on reproductive growth and has therefore a wider body depth because their gonads are larger. They have a shorter head, pectoral fin and cadual fin which indicate that more energy is used for body growth. Offshore spawning flounder fo-cuses more on somatic growth which explain their longer and slimmer body com-pared to the costal spawning flounder. Females have a larger abdomen than males because their gonads, liver and intestine are larger. With a discriminant analysis all the individuals where classified into spawning type based only on their morphology, 76 of 85 individuals where classified into correct spawning type which gives an error of 10,5 %.

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, as well 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 sites were also related with local features, such as the extension of the continental platform and adaptations to transport and retention mechanisms.

The habitat quality of European shallow-water nurseries was studied for 3 common flatfish species based on juvenile growth conditions. Field growth of 0-group plaice Pleuronectes platessa, flounder Platichthys flesus and sole Solea solea, from both published and unpublished studies, was compared with maximum growth predicted by a bioenergetics model based on the dynamic energy budget theory. In plaice and flounder, realized growth ratio decreased consistently during the growing season in most of the nurseries analyzed, indicating a widespread pattern of declining conditions. In sole, growth performance was not maximal, but as opposed to the other species, no clear temporal trend in realized growth ratio was observed. A latitudinal comparison of realized growth ratio over the various nurseries indicated clear positive trends for plaice and flounder, with better growth conditions at northern latitudes. In sole, despite some variability, the same trend was found during part of the summer. In the absence of clear gradients in benthic prey biomass, we hypothesize that increased food limitation in southern locations is most likely caused by interspecific competition reducing maximum individual intake rates. These results suggest that, in the context of global warming, habitat quality of southern European nurseries for juvenile growth may be particularly affected by the combined interaction of food and thermal constraints.

De in dit rapport beschreven werkzaamheden zijn in 2010 door IMARES uitgevoerd op basis van een opdracht van Rijkswaterstaat in het kader van het Joint Assessment and Monitoring Program van de OSPARCOM. De opdracht hield in het verkrijgen van biologische gegevens van bot (visziekten). De benodigde monsters bot werden verzameld door IMARES. Tevens werd materiaal van bot verzameld voor chemisch onderzoek en geanalyseerd.

De benodigde monsters bot werden verzameld door IMARES ter verkrijging van biologische gegevens van bot (visziekten). Tevens werd materiaal van bot verzameld voor chemisch onderzoek en geanalyseerd.

Our understanding of turbot and flounder ecology in the Baltic Sea is insufficient for sound management decisions. This thesis aims to fill some gaps in current knowledge by providing information of the ecology of turbot and flounder within their juvenile habitat, and to relate these findings to issues assumed relevant for recruitment variation. Main focus is on turbot due to its relatively low abundance and high variability in recruitment. The distribution of both species was studied on different scales, as was environmental effects on food consumption in 0-group turbot. The 0-group turbot display a relatively restricted spatial distribution compared to flounder. This is possibly due to a more specialized diet, which may make them more vulnerable to habitat degradation, especially eutrophication as a strong negative correlation was found with the organic content in the sediment. The species show high temporal and spatial overlap when settling in July-September, with peak abundances in August, and at depths <1 m. Both species display sedentary behavior within the nursery ground. Compared to flounder, turbot was more mobile potentially due to its restricted diet calling for extended searches. For turbot, feeding conditions appear to vary between size groups, which potentially could cause variations in survival between years through size-selective mortality. But, the predation may be low in central Baltic Sea as the abundance of the main predator, brown shrimp are comparatively low during flatfish occupancy. A significant positive relationship was found between the recruitment of turbot and flounder, which suggests that no inter-specific interactions during the juvenile stage affect recruitment. This co-variation also suggests that the recruitment of the species is determined by the same phenomena, potentially by large scale abiotic factors during the egg- and larval stage. For turbot, additional variability is potentially generated during the juvenile stage due to, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 3: Submitted. Paper 4: Submitted.

Since 1975, juvenile flatfish (plaice and flounder) populations have been monitored at the Balgzand intertidal and, based on this data series (1975-2007), the interannual patterns in predation pressure were quantified. Temporal patterns in abundance have changed greatly, especially for plaice. Up to the early 1980s, 3 year classes (0-, I- and II-group) were present and growing up in the area, but from the late 1980s onwards, densities of the I- and II-group plaice dropped from a few hundred individuals per 1000 m(2) to levels close to zero. It appears that the Balgzand intertidal has lost its nursery function for I- and II-group plaice, although feeding conditions have remained the same or even slightly increased since the late 1970s. The absence of I- and II-group flatfish in the intertidal cannot be explained at present; however, processes operating offshore are most likely involved. As a consequence, the annual predation pressure by juvenile flatfish upon the intertidal macrozoobenthos dropped by 94%, declining from an average of approximately 5 to 0.25 g ash free dry mass m(-2). Such a decline in top-down predation may not only have directly influenced the macrozoobenthic community, but may also have indirectly affected food availability and accessibility for other top predators (e.g. shorebirds).

To investigate feeding habits of juvenile flounder (Platichthys flesus) and turbot (Psetta maxima) in relation to habitat characteristics a field survey with push net sampling was conducted in nursery areas with different ecological characteristics in the northern Baltic proper. Sampling sites were stratified to cover several different habitat types defined by substrate and wave exposure. Apart from flatfishes and epifauna, samples of macrofauna, meiofauna and hyperbenthic planktons were collected from each site together with data on vegetation, depth, salinity, temperature and turbidity. The diet differed between species where flounder diet was dominated by chironomids, copepods and oligochaetes while turbot apart from chironomids had a high incidence of amphipods, gobies and mysids. In both species there was a shift in diet with size, although this shift was influenced by the habitat. Among the environmental variables investigated, wave exposure was found to significantly influence flounder diet. Food preference in the most exposed areas was dominated by oligochaetes and copepods instead of chironomids, which dominated in sheltered areas. This study shows that habitat characteristics can have a major influence on feeding habits of juvenile flatfish., authorCount :2

De in dit rapport beschreven werkzaamheden zijn in 2009 door IMARES uitgevoerd op basis van een opdracht van Rijkswaterstaat in het kader van het Joint Assessment and Monitoring Program van de OSPARCOM. De opdracht hield in het verkrijgen van biologische gegevens van bot (visziekten). De benodigde monsters bot werden verzameld door IMARES. Tevens werd materiaal van bot verzameld voor chemisch onderzoek en geanalyseerd.

Ingegaan wordt op de bijzondere levenswijze van de bot en de kansen en bedreigingen voor deze vissoort in de zoete en zoute wateren van Nederland (Kennisdocument op http://edepot.wur.nl/10782)

Survival and growth of the earliest life-stages is considered a key factor in determining the abundance of many marine fish species. For flatfishes, the availability of high quality nursery areas is essential for successful recruitment. Regarding the Baltic Sea, there are large gaps in knowledge on factors that influence the distribution of flatfishes during this sensitive stage. To identify the characteristics of important nursery areas in the Baltic for flounder (Platichthys flesus) and turbot (Psetta maxima), a field survey with push net sampling was conducted in the northern Baltic proper during autumn 2006. The sampling stations were stratified to cover several different habitat types defined by substrate and wave exposure. Apart from density of young-of-the-year (YOY) flatfishes, a number of ecological characteristics of the habitat were recorded. Physical habitat variables included substrate type, salinity, depth, turbidity, vegetation and habitat structure. Variables describing biotic processes, such as prey availability and abundance of competitors, were also sampled. The relationships between the spatial distribution of species and these ecological characteristics were fitted to presence/absence data of juvenile flatfish using generalized additive models (GAM). The best habitat descriptors for flounder in order of contribution were: substrate, habitat structure, salinity, wave exposure and occurrence of filamentous algae. Positive effects of increasing wave exposure, salinity and structure were detected while a high cover of filamentous algae had a negative effect. Sand and gravel were preferred over soft and stony substrates. For turbot the best habitat descriptors in order of contribution were: occurrence of filamentous algae, substrate and turbidity. Turbot showed a preference for areas with a low cover of filamentous algae, high turbidity and sandy substrate. Prey availability and abundance of competitors were not included in the models, indicating tha

De in dit rapport beschreven werkzaamheden zijn door Wageningen IMARES uitgevoerd op basis van een opdracht van Rijkswaterstaat in het kader van het Joint Assessment and Monitoring Program van de OSPARCOM. De opdracht hield in het verkrijgen van biologische gegevens van bot. De benodigde monsters bot werden verzameld door IMARES. Tevens werd materiaal van bot verzameld voor chemisch onderzoek en geanalyseerd. De opdracht is gebaseerd op het werkdocument “Werkplan monitoring visziekten en chemische stoffen in botten, 2008”, van 7 mei 2008.

We found significant population structure and isolation by distance among samples of flounder (Platichthys flesus) in the Baltic, Kattegat and Skagerrak seas using microsatellite genetic markers. This pattern was almost entirely due to a difference between flounder that have demersal spawning in the northern Baltic, as compared to pelagic spawners in the southern Baltic and on the west coast of Sweden. Among demersal spawners we found neither genetic differentiation nor any isolation by distance among sampling sites. We speculate that demersal flounder are descendants of a population that colonized the Baltic previous to pelagic spawners. The demersal flounder may thus have had longer time to adapt to the low salinity in the Baltic, and accordingly display egg characteristics that make it possible to reproduce at the low salinity levels in the northern Baltic. Among pelagic spawners significant isolation by distance was detected. Pelagic spawners have previously been shown to display clinal variation in egg size, which allows them to float also at the moderate salinity levels up to the region north of the island Bornholm. Management units for harvesting should ideally be based on true biological populations, and for the commercially important flounder up to 15 different management stocks in the Baltic have been suggested. We could not find a population genetic foundation for such a high number of management units, and our data suggest three management units: the northern Baltic (demersal populations), southern Baltic with the Oresund straits and the most northwestern sampling sites (Skagerrak, Kattegat and North Sea).

De werkzaamheden bestonden uit het verzamelen van monsters bot waarvan biologische parameters werden bepaald. Tevens werden in deze botten milieukritische stoffen geanalyseerd. In 2007 is het chemisch onderzoek op de locaties Westerschelde, Waddenzee en Eems+Dollard uitgevoerd. De verzamelde gegevens en analyse+uitkomsten worden aangeleverd in dit rapport.

The flounder (Platichthys flesus) is a flatfish that inhabits marine coastal environments, especially estuaries. It is an alternative quantitative biological indicator to the common marine mussel (Mytilus spp.), which is currently used as a sentinel species to monitor chemical contamination in numerous monitoring programmes. Findings from two decades of monitoring cadmium (Cd) and mercury (Hg) using both sentinel species in the Seine Estuary (France) are reported. For comparison, time-series of water concentrations for the same two metals at the mouth of the River Seine are given. Cd concentrations in the liver of the fish and in the soft tissue of mussels show similar temporal trends, consistent with the major temporal variations of Cd concentrations recorded in river water and with changes in industrial discharge of Cd (phosphogypsum waste) within the Seine Estuary. On the other hand, Hg concentrations in the muscles of flounders show temporal variations with no link to that observed in mussels or fluvial Hg contributions, which are in fact nearly covariant. It is concluded that optimization of the use of flounders as sentinel organisms for monitoring temporal trends of metal contamination in estuarine environments requires in-depth knowledge of its ecology within the area studied. An adapted sampling strategy based on this knowledge should provide results that are easier to interpret.

Screening of a flounder ovary cDNA library with a rainbow trout p53 probe led to the isolation of a p53-unrelated cDNA encoding an unknown 161 amino acid protein. In view of its apparent molecular weight and yet unknown function, the deduced protein was named Xp18. Corresponding orthologous cDNAs or expressed sequence tags have been identified in several species including human, rodents, bovine, chicken and zebrafish and a related cDNA has also been isolated in the fruit fly. Deduced amino acid sequences appeared to be extremely well conserved throughout vertebrate evolution. Structure predictions suggested that Xp 18 may correspond to an integral protein comprising four transmembrane domains. The charged C-termini of all known vertebrate Xp18-like proteins displayed a characteristic KXKXX motif which is considered as an endoplasmic reticulum targeting sequence. Gene expression, as shown by Northern blot and quantitative reverse transcription-polymerase chain reaction analysis, was significantly higher in the ovary and to a lesser extent in the brain. Xp18 transcripts were also detected by in situ hybridization in most of the circumventricular regions of the brain of adult flounders. The gene encoding the human protein is located on chromosome Xq22.1, a genome region involved in numerous genetic diseases including premature ovarian failure.

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The research presented in this thesis focused on the mechanistic aspects of the toxic and biochemical effects of PCBs in flounder (Platichthys flesus), with the aim to provide a scientific basis for the suggested involvement of PCBs in the aetiology of diseases observed in flounder. Therefore, the first goal was to study the biochemical effects of PCBs in flounder by investigating the inducibility of cytochrome P4501A and associated EROD activity and glutathione-S-transferase activity upon administration to PCBs and TCDD in vivo. Secondly, the mechanism of action of PCBs in flounder was investigated by identifyingg the hepatic microsomal Ali receptor pathway. In addition, the potency of individual PCB congeners and a commercial PCB mixture to specifically inhibit the catalytic CYP1A activity was studied using in vitro techniques. Finally, the endocrine disrupting effect of PCBs was studied by analysing the retinoid and thyroid hormone levels in flounder after acute and chronic exposure to either a commercial PCB mixture or contaminated harbour sludge.summary of resultsShort-time exposure of flounder to the technical PCB mixture Clophen A50 showed minor effects on total hepatic cytochrome P450 concentrations and only a relatively slight induction in EROD activity, indicating that flounder is not very sensitive towards i.p. and oral administration of Clophen A50 ( chapter 3 and 5). The most potent PCB congener (CB-126), in terms of mammalian derived TCDD toxic equivalents (TEQ), was found to be more potent as inducer of CYP1A protein and activity in flounder, but not to the extend as was expected based on TCDD TEQs ( chapter 5). On the other hand, high induction of the hepatic microsomal CYP1A content and associated EROD activity were observed in flounder orally exposed to TCDD ( chapter 4). Interestingly, TCDD induced EROD activity in flounder could be inhibited by co-treatment with Clophen A50. In contrast, administration of flounder with a combination of TCDD and C

RIVM rapport:Veldonderzoeken uitgevoerd in diverse verontreinigde kustwateren in zowel Europa als de Verenigde Staten laten een duidelijke relatie tussen vervuiling en een toegenomen prevalentie van tumoren en infectieziekten bij vissen zien. Een van de chemische verbindingen in de veelheid van xenobiotica die gevonden kunnen worden in vervuilde wateren en sedimenten is de organotinverbinding tributyltin (TBT), hoofdzakelijk afkomstig van verf waarmee scheepshuiden worden behandeld om ongewenste aangroei van onder andere algen en schelpdieren tegen te gaan. Dit rapport beschrijft een onderzoek waarin botten (Platichthys flesus) via het water werden blootgesteld aan bis(tri-n-butyltin)oxide (TBTO) onder gecontroleerde laboratoriumomstandigheden. De mogelijke histopathologische effecten aan diverse organen (kieuw, huid, ogen, lever, buiknier, ovarium/testikel, milt en maagdarmkanaal) werden onderzocht en tevens werd er een morfometrisch onderzoek van de thymus (zwezerik) uitgevoerd om de doelorganen van TBTO bij de bot vast te stellen. Daarnaast werd het functioneren van zowel de specifieke- als aspecifieke afweer onderzocht door gebruik te maken van ex vivo / in vitro immuun functietesten. Blootstelling van botten aan TBTO gehaltes in dezelfde orde van grootte als de maximaal gemeten TBT gehaltes in de veldsituatie (experiment: 17.3 mug TBT ; veld: 7.2 mug TBT) veroorzaakte sterfte na 7-12 dagen, een vermindering van de conditiefactor, kieuwlaesies en een significante onderdrukking van de aspecifieke weerstand. Er werden geen duidelijke effecten waargenomen op het relatieve volume van de thymus en op het specifieke immuunsysteem na blootstelling aan TBTO., Field studies in various polluted coastal areas in Europe and the United States of America clearly indicate a relation between pollution and the increase in prevalence of tumours and infectious diseases in fish. One of the chemicals of interest in the myriad of xenobiotics found in polluted waters and sediments is the organotin compound tributyltin (TBT), originating mainly from antifouling paints used on the hulls of ships. This report describes a study in which flounders (Platichthys flesus) were exposed to bis(tri-n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. The possible histopathological effects on several organs (gill, skin, eye, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined and morphometric analysis of the thymus was performed to assess the target organ(s) for TBTO in this fish species. Also the function of the non specific and specific resistance was studied using ex vivo/ in vitro immune function tests. Exposure of flounder to TBTO, in concentrations which are in the same order of magnitude as maximum TBT levels measured in the field (experiment: 17.3 mug TBT ; field: 7.2 mug TBT), caused mortality after 7-12 days, decreased the condition factor, resulted in gill lesions, and induced significant reduction of the non specific resistance. No marked effects on the relative thymus volume, or the specific immune system were noted after exposure to TBTO.

RIVM rapport:De stand van zaken betreffende de ontwikkeling van immuunfunctie testen voor de bot, Platichthys flesus is beschreven. Epidemiologische veldstudies hebben aangetoond dat deze platvis species een hoge prevalentie aan infectieuze ziekten heeft, hetgeen gecorreleerd kan worden met de aanwezigheid van contaminanten in het aquatische milieu. Om een mogelijk causaal verband vast te kunnen stellen en om de bot als indicator species te kunnen gebruiken, zijn immuunfunctie-testen ontwikkeld. Het rapport beschrijft de ontwikkeling en toepassing van assays voor het bepalen van niet-specifieke cytotoxie, lymfocytenstimulatie en de immunohistochemische identificatie van leukocyt populaties in de bot. Deze assays, tezamen met andere testen die momenteel in ontwikkeling zijn, zullen gebruikt worden om de immuunstatus van de bot te 'peilen' in immunotoxicologische experimenten., The state of the art concerning the development of immune function tests in flounder, Platichthys flesus has been described. It is known from epidemiological field surveys that this flatfish species has a high prevalence of infectious diseases which is correlated with aquatic pollution. In order to provide more evidence for a possible causal link and to use flounder as an indicator species, immune function tests were developed. This report describes the development and application of assays to measure non-specific cytotoxicity, lymphocyte stimulation and immunohistochemical identification of leukocyte populations of flounder. These assays, as well as others which are developed at the moment, will be applied to monitor the immune status of flounder in immunotoxicological experiments.

RIVM rapport:Dit rapport beschrijft het histopathologisch onderzoek van levers van platvis (bot, Platichthys flesus) afkomstig van de Nederlandse kustwateren en estuaria welke bemonsterd zijn in de periode 1985-89. Bemonstering vond plaats op een 10-tal lokaties waarbij 210 levers zijn verzameld met waarneembare knobbels (diameter > 2 mm); daarnaast zijn 315 ogenschijnlijke normale levers onderzocht. Van de 210 levers met waarneembare afwijkingen bleek 67% inderdaad tumoren te bevatten, de meeste waren goedaardig (hepatocellulair adenoom). Bij 13.6% bleek sprake van een kwaadaardig tumor (hepatocellulair carcinoom). De meeste overige afwijkingen bleken te bestaan uit zgn. "foci of cellular alteration" (fca). welke wel worden beschouwd als voorstadia van tumoren. Levertumoren komen het meest frequent voor bij bot langs de Hollandse en Zeeuwse kust, prevalenties kunnen hier oplopen tot 30% in vissen van 6 jaar en ouder. Verder werd de tumorziekte vaker bij vrouwelijke dan bij mannelijke botten waargenomen. Histopathologisch onderzoek van de ogenschijnlijk normale levers gaf niettemin een scala van veranderingen te zien, waaronder hepatocellulair adenoom, fca's, ontstekingen, vervalshaardjes, regeneratieve haardjes,hydropische degeneratie van lever- en galgangcellen en fibrillair veranderde levercellen waarvan de betekenis onduidelijk is. Daarnaast werd stapeling (vacuolisatie) van glycogeen en vet gezien, alsmede zgn. melanomacrofagen centra. De laatste twee veranderingen vallen binnen het normale beeld, maar werden gekwantificeerd (semikwantitatief). Uit de resultaten kan worden geconcludeerd dat een geringe fractie van de tumoren zou worden gemist als het onderzoek zich zou beperken tot alleen de zichtbare afwijkende levers. De verspreiding van de fca's kwam goed overeen met die van de tumoren hetgeen de hypothese ondersteund dat deze veranderingen tot hetzelfde proces behoren. Van de overige veranderingen en parameters vertoonde alleen de hydropische galgangdegeneratie ene over, This paper reports the detailed histopathological analysis of livers of flounder (Platichthys flesus) sampled in Dutch coastal and estuarine waters during 1985-89. In conjunction with an epidemiological study of grossly identifiable diseases at 10 sampling locations, 210 livers showing gross nodular lesions (diameter > 2 mm) and 315 livers showing no gross pathology were collected. Of the 210 livers with grossly observable nodules, 67% were diagnosed as having neoplasia. The majority of these neoplasms were identified as hepatocellular adenoma ; 13.1% were diagnosed as hepatocellular carcinoma. Most of the remaining modules were diagnosed as foci of cellular alteration, which are considered to be pre-neoplastic lesions. Routine histopathological examination of livers showing no gross pathology resulted in the identification of a range of lesions including hepatocellular adenoma, foci of cellular alteration, inflammatory lesions, focal necrosis, regenerative foci, hydropic vacuolated lesions of bile duct cells and hepatocytes, and fibrillar hepatocytes of unknown significance. In addition, indices were used to quantify the presence of storage vacuoles (glycogen and lipid) and the density of melanomacrophage centres. The results indicate that a small proportion of neoplasms would be missed if only livers with grossly detectable nodules were subjected to histological examination. The prevalence of foci of cellular alteration at the different sampling sites showed a good correspondence with that of neoplasia, providing support for the hypothesis that these conditions represent stages of the same process. Of the other lesions and quantitative indices, only bile duct vacuolization had a spatial distribution similar to that of neoplasia. The findings indicate that the occurrence of liver neoplasms in flounder represents a promising tool for monitoring exposure to potential carcinogens. However, because of the long latency period associated with tumour development, there wi

Six populations of flounder (Platichthys flesus ) from the Mediterranean and Atlantic coasts were analysed by enzymatic electrophoresis at 21 loci. The results confirm the separation of the species in three genetic groups. Differences between these genetic groups have also been observed.