Getting fat and stressed: Effects of dietary intake of titanium dioxide nanoparticles in the liver of turbot Scophthalmus maximus.

The extensive use of nanomaterials, including titanium dioxide nanoparticles (TiO2 NPs), raises concerns about their persistence in ecosystems. Protecting aquatic ecosystems and ensuring healthy and safe aquaculture products requires the assessment of the potential impacts of NPs on organisms. Here, we study the effects of a sublethal concentration of citrate-coated TiO2 NPs of two different primary sizes over time in flatfish turbot, Scophthalmus maximus (Linnaeus, 1758). Bioaccumulation, histology and gene expression were assessed in the liver to address morphophysiological responses to citrate-coated TiO2 NPs. Our analyses demonstrated a variable abundance of lipid droplets (LDs) in hepatocytes dependent on TiO2 NPs size, an increase in turbot exposed to smaller TiO2 NPs and a depletion with larger TiO2 NPs. The expression patterns of genes related to oxidative and immune responses and lipid metabolism (nrf2, nfκb1, and cpt1a) were dependent on the presence of TiO2 NPs and time of exposure supporting the variance in hepatic LDs distribution over time with the different NPs. The citrate coating is proposed as the likely catalyst for such effects. Thus, our findings highlight the need to scrutinize the risks associated with exposure to NPs with distinct properties, such as primary size, coatings, and crystalline forms, in aquatic organisms.

Single-cell ICP-MS for studying the association of inorganic nanoparticles with cell lines derived from aquaculture species.

The current research deals with the use of single-cell inductively coupled plasma-mass spectrometry (scICP-MS) for the assessment of titanium dioxide nanoparticle (TiO2 NP) and silver nanoparticle (Ag NP) associations in cell lines derived from aquaculture species (sea bass, sea bream, and clams). The optimization studies have considered the avoidance of high dissolved background, multi-cell peak coincidence, and possible spectral interferences. Optimum operating conditions were found when using a dwell time of 50 µs for silver and 100 µs for titanium. The assessment of associated TiO2 NPs by scICP-MS required the use of ammonia as a reaction gas (flow rate at 0.75 mL min-1) for interference-free titanium determinations (measurements at an m/z ratio of 131 from the 48Ti(NH)(NH3)4 adduct). The influence of other parameters such as the number of washing cycles and the cell concentration on accurate determinations by scICP-MS was also fully investigated. Cell exposure trials were performed using PVP-Ag NPs (15 and 100 nm, nominal diameter) and citrate-TiO2 NPs (5, 25, and 45 nm, nominal diameter) at nominal concentrations of 10 and 50 µg mL-1 for citrate-TiO2 NPs and 5.0 and 50 µg mL-1 for PVP-Ag NPs. Results have shown that citrate-TiO2 NPs interact with the outer cell membranes, being quite low in the number of citrate-TiO2 NPs that enters the cells (the high degree of aggregation is the main factor which leads to the aggregates being in the extracellular medium). In contrast, PVP-Ag NPs have been found to enter the cells.

Proteomics reveals multiple effects of titanium dioxide and silver nanoparticles in the metabolism of turbot, Scophthalmus maximus.

Titanium dioxide (TiO2) and silver (Ag) NPs are among the most used engineered inorganic nanoparticles (NPs); however, their potential effects to marine demersal fish species, are not fully understood. Therefore, this study aimed to assess the proteomic alterations induced by sub-lethal concentrations citrate-coated 25 nm ("P25") TiO2 or polyvinylpyrrolidone (PVP) coated 15 nm Ag NPs to turbot, Scophthalmus maximus. Juvenile fish were exposed to the NPs through daily feeding for 14 days. The tested concentrations were 0, 0.75 or 1.5 mg of each NPs per kg of fish per day. The determination of NPs, Titanium and Ag levels (sp-ICP-MS/ICP-MS) and histological alterations (Transmission Electron Microscopy) supported proteomic analysis performed in the liver and kidney. Proteomic sample preparation procedure (SP3) was followed by LC-MS/MS. Label-free MS quantification methods were employed to assess differences in protein expression. Functional analysis was performed using STRING web-tool. KEGG Gene Ontology suggested terms were discussed and potential biomarkers of exposure were proposed. Overall, data shows that liver accumulated more elements than kidney, presented more histological alterations (lipid droplets counts and size) and proteomic alterations. The Differentially Expressed Proteins (DEPs) were higher in Ag NPs trial. The functional analysis revealed that both NPs caused enrichment of proteins related to generic processes (metabolic pathways). Ag NPs also affected protein synthesis and nucleic acid transcription, among other processes. Proteins related to thyroid hormone transport (Serpina7) and calcium ion binding (FAT2) were suggested as biomarkers of TiO2 NPs in liver. For Ag NPs, in kidney (and at a lower degree in liver) proteins related with metabolic activity, metabolism of exogenous substances and oxidative stress (e.g.: NADH dehydrogenase and Cytochrome P450) were suggested as potential biomarkers. Data suggests adverse effects in turbot after medium/long-term exposures and the need for additional studies to validate specific biological applications of these NPs.

Estimates of recent and historical effective population size in turbot, seabream, seabass and carp selective breeding programmes.

BACKGROUND: The high fecundity of fish species allows intense selection to be practised and therefore leads to fast genetic gains. Based on this, numerous selective breeding programmes have been started in Europe in the last decades, but in general, little is known about how the base populations of breeders have been built. Such knowledge is important because base populations can be created from very few individuals, which can lead to small effective population sizes and associated reductions in genetic variability. In this study, we used genomic information that was recently made available for turbot (Scophthalmus maximus), gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax) and common carp (Cyprinus carpio) to obtain accurate estimates of the effective size for commercial populations. METHODS: Restriction-site associated DNA sequencing data were used to estimate current and historical effective population sizes. We used a novel method that considers the linkage disequilibrium spectrum for the whole range of genetic distances between all pairs of single nucleotide polymorphisms (SNPs), and thus accounts for potential fluctuations in population size over time. RESULTS: Our results show that the current effective population size for these populations is small (equal to or less than 50 fish), potentially putting the sustainability of the breeding programmes at risk. We have also detected important drops in effective population size about five to nine generations ago, most likely as a result of domestication and the start of selective breeding programmes for these species in Europe. CONCLUSIONS: Our findings highlight the need to broaden the genetic composition of the base populations from which selection programmes start, and suggest that measures designed to increase effective population size within all farmed populations analysed here should be implemented in order to manage genetic variability and ensure the sustainability of the breeding programmes.

A genome-wide association study, supported by a new chromosome-level genome assembly, suggests sox2 as a main driver of the undifferentiatiated ZZ/ZW sex determination of turbot (Scophthalmus maximus).

BACKGROUND: Understanding sex determination (SD) across taxa is a major challenge for evolutionary biology. The new genomic tools are paving the way to identify genomic features underlying SD in fish, a group frequently showing limited sex chromosome differentiation and high SD evolutionary turnover. Turbot (Scophthalmus maximus) is a commercially important flatfish with an undifferentiated ZW/ZZ SD system and remarkable sexual dimorphism. Here we describe a new long-read turbot genome assembly used to disentangle the genetic architecture of turbot SD by combining genomics and classical genetics approaches. RESULTS: The new turbot genome assembly consists of 145 contigs (N50 = 22.9 Mb), 27 of them representing >95% of its estimated genome size. A genome wide association study (GWAS) identified a ~ 6.8 Mb region on chromosome 12 associated with sex in 69.4% of the 36 families analyzed. The highest associated markers flanked sox2, the only gene in the region showing differential expression between sexes before gonad differentiation. A single SNP showed consistent differences between Z and W chromosomes. The analysis of a broad sample of families suggested the presence of additional genetic and/or environmental factors on turbot SD. CONCLUSIONS: The new chromosome-level turbot genome assembly, one of the most contiguous fish assemblies to date, facilitated the identification of sox2 as a consistent candidate gene putatively driving SD in this species. This chromosome SD system barely showed any signs of differentiation, and other factors beyond the main QTL seem to control SD in a certain proportion of families.

Disentangling Genetic Variation for Resistance and Endurance to Scuticociliatosis in Turbot Using Pedigree and Genomic Information.

Selective breeding for improving host responses to infectious pathogens is a promising option for disease control. In fact, disease resilience, the ability of a host to survive or cope with infectious challenge, has become a highly desirable breeding goal. However, resilience is a complex trait composed of two different host defence mechanisms, namely resistance (the ability of a host to avoid becoming infected or diseased) and endurance (the ability of an infected host to survive the infection). While both could be targeted for genetic improvement, it is currently unknown how they contribute to survival, as reliable estimates of genetic parameters for both traits obtained simultaneously are scarce. A difficulty lies in obtaining endurance phenotypes for genetic analyses. In this study, we present the results from an innovative challenge test carried out in turbot whose design allowed disentangling the genetic basis of resistance and endurance to Philasterides dicentrarchi, a parasite causing scuticociliatosis that leads to substantial economic losses in the aquaculture industry. A noticeable characteristic of the parasite is that it causes visual signs that can be used for disentangling resistance and endurance. Our results showed the existence of genetic variation for both traits (heritability = 0.26 and 0.12 for resistance and endurance, respectively) and for the composite trait resilience (heritability = 0.15). The genetic correlation between resistance and resilience was very high (0.90) indicating that both are at a large extent the same trait, but no significant genetic correlation was found between resistance and endurance. A total of 18,125 SNPs obtained from 2b-RAD sequencing enabled genome-wide association analyses for detecting QTLs controlling the three traits. A candidate QTL region on linkage group 19 that explains 33% of the additive genetic variance was identified for resilience. The region contains relevant genes related to immune response and defence mechanisms. Although no significant associations were found for resistance, the pattern of association was the same as for resilience. For endurance, one significant association was found on linkage group 2. The accuracy of genomic breeding values was also explored for resilience, showing that it increased by 12% when compared with the accuracy of pedigree-based breeding values. To our knowledge, this is the first study in turbot disentangling the genetic basis of resistance and endurance to scuticociliatosis.

Genetic differences in host infectivity affect disease spread and survival in epidemics.

Survival during an epidemic is partly determined by host genetics. While quantitative genetic studies typically consider survival as an indicator for disease resistance (an individual's propensity to avoid becoming infected or diseased), mortality rates of populations undergoing an epidemic are also affected by endurance (the propensity of diseased individual to survive the infection) and infectivity (i.e. the propensity of an infected individual to transmit disease). Few studies have demonstrated genetic variation in disease endurance, and no study has demonstrated genetic variation in host infectivity, despite strong evidence for considerable phenotypic variation in this trait. Here we propose an experimental design and statistical models for estimating genetic diversity in all three host traits. Using an infection model in fish we provide, for the first time, direct evidence for genetic variation in host infectivity, in addition to variation in resistance and endurance. We also demonstrate how genetic differences in these three traits contribute to survival. Our results imply that animals can evolve different disease response types affecting epidemic survival rates, with important implications for understanding and controlling epidemics.

Differential gene expression and SNP association between fast- and slow-growing turbot (Scophthalmus maximus).

Growth is among the most important traits for animal breeding. Understanding the mechanisms underlying growth differences between individuals can contribute to improving growth rates through more efficient breeding schemes. Here, we report a transcriptomic study in muscle and brain of fast- and slow-growing turbot (Scophthalmus maximus), a relevant flatfish in European and Asian aquaculture. Gene expression and allelic association between the two groups were explored. Up-regulation of the anaerobic glycolytic pathway in the muscle of fast-growing fish was observed, indicating a higher metabolic rate of white muscle. Brain expression differences were smaller and not associated with major growth-related genes, but with regulation of feeding-related sensory pathways. Further, SNP variants showing frequency differences between fast- and slow-growing fish pointed to genomic regions likely involved in growth regulation, and three of them were individually validated through SNP typing. Although different mechanisms appear to explain growth differences among families, general mechanisms seem also to be involved, and thus, results provide a set of useful candidate genes and markers to be evaluated for more efficient growth breeding programs and to perform comparative genomic studies of growth in fish and vertebrates.

Integrative Transcriptome, Genome and Quantitative Trait Loci Resources Identify Single Nucleotide Polymorphisms in Candidate Genes for Growth Traits in Turbot.

Growth traits represent a main goal in aquaculture breeding programs and may be related to adaptive variation in wild fisheries. Integrating quantitative trait loci (QTL) mapping and next generation sequencing can greatly help to identify variation in candidate genes, which can result in marker-assisted selection and better genetic structure information. Turbot is a commercially important flatfish in Europe and China, with available genomic information on QTLs and genome mapping. Muscle and liver RNA-seq from 18 individuals was carried out to obtain gene sequences and markers functionally related to growth, resulting in a total of 20,447 genes and 85,344 single nucleotide polymorphisms (SNPs). Many growth-related genes and SNPs were identified and placed in the turbot genome and genetic map to explore their co-localization with growth-QTL markers. Forty-five SNPs on growth-related genes were selected based on QTL co-localization and relevant function for growth traits. Forty-three SNPs were technically feasible and validated in a wild Atlantic population, where 91% were polymorphic. The integration of functional and structural genomic resources in turbot provides a practical approach for QTL mining in this species. Validated SNPs represent a useful set of growth-related gene markers for future association, functional and population studies in this flatfish species.

Gene expression profiles of spleen, liver, and head kidney in turbot (Scophthalmus maximus) along the infection process with Philasterides dicentrarchi using an immune-enriched oligo-microarray.

We evaluated the expression profiles of turbot in spleen, liver, and head kidney across five temporal points of the Philasterides dicentrarchi infection process using an 8x15K Agilent oligo-microarray. The microarray included 2,176 different fivefold replicated gene probes designed from a turbot 3' sequenced EST database. We were able to identify 221 differentially expressed (DE) genes (8.1% of the whole microarray), 113 in spleen, 83 in liver, and 90 in head kidney, in at least 1 of the 5 temporal points sampled for each organ. Most of these genes could be annotated (83.0%) and functionally categorized using GO terms (69.1%) after the additional sequencing of DE genes from the 5' end. Many DE genes were related to innate and acquired immune functions. A high proportion of DE genes were organ-specific (70.6%), although their associated GO functions showed notable similarities in the three organs. The most striking difference in functional distribution was observed between the up- and downregulated gene groups. Upregulated genes were mostly associated to immune functions, while downregulated ones mainly involved metabolism-related genes. Genetic response appeared clustered in a few groups of genes with similar expression profiles along the temporal series. The information obtained will aid to understand the turbot immune response and will specifically be valuable to develop strategies of defense to P. dicentrarchi to achieve more resistant broodstocks for turbot industry.

QTL detection for Aeromonas salmonicida resistance related traits in turbot (Scophthalmus maximus).

BACKGROUND: Interactions between fish and pathogens, that may be harmless under natural conditions, often result in serious diseases in aquaculture systems. This is especially important due to the fact that the strains used in aquaculture are derived from wild strains that may not have had enough time to adapt to new disease pressures. The turbot is one of the most promising European aquaculture species. Furunculosis, caused by the bacterium Aeromonas salmonicida, produces important losses to turbot industry. An appealing solution is to achieve more robust broodstock, which can prevent or diminish the devastating effects of epizooties. Genomics strategies have been developed in turbot to look for candidate genes for resistance to furunculosis and a genetic map with appropriate density to screen for genomic associations has been also constructed. In the present study, a genome scan for QTL affecting resistance and survival to A. salmonicida in four turbot families was carried out. The objectives were to identify consistent QTL using different statistical approaches (linear regression and maximum likelihood) and to locate the tightest associated markers for their application in genetic breeding strategies. RESULTS: Significant QTL for resistance were identified by the linear regression method in three linkage groups (LGs 4, 6 and 9) and for survival in two LGs (6 and 9). The maximum likelihood methodology identified QTL in three LGs (5, 6 and 9) for both traits. Significant association between disease traits and genotypes was detected for several markers, some of them explaining up to 17% of the phenotypic variance. We also identified candidate genes located in the detected QTL using data from previously mapped markers. CONCLUSIONS: Several regions controlling resistance to A. salmonicida in turbot have been detected. The observed concordance between different statistical methods at particular linkage groups gives consistency to our results. The detected associated markers could be useful for genetic breeding strategies. A finer mapping will be necessary at the detected QTL intervals to narrow associations and around the closely associated markers to look for candidate genes through comparative genomics or positional cloning strategies. The identification of associated variants at specific genes will be essential, together with the QTL associations detected in this study, for future marker assisted selection programs.

Detection of growth-related QTL in turbot (Scophthalmus maximus).

BACKGROUND: The turbot (Scophthalmus maximus) is a highly appreciated European aquaculture species. Growth related traits constitute the main goal of the ongoing genetic breeding programs of this species. The recent construction of a consensus linkage map in this species has allowed the selection of a panel of 100 homogeneously distributed markers covering the 26 linkage groups (LG) suitable for QTL search. In this study we addressed the detection of QTL with effect on body weight, length and Fulton's condition factor. RESULTS: Eight families from two genetic breeding programs comprising 814 individuals were used to search for growth related QTL using the panel of microsatellites available for QTL screening. Two different approaches, maximum likelihood and regression interval mapping, were used in order to search for QTL. Up to eleven significant QTL were detected with both methods in at least one family: four for weight on LGs 5, 14, 15 and 16; five for length on LGs 5, 6, 12, 14 and 15; and two for Fulton's condition factor on LGs 3 and 16. In these LGs an association analysis was performed to ascertain the microsatellite marker with the highest apparent effect on the trait, in order to test the possibility of using them for marker assisted selection. CONCLUSIONS: The use of regression interval mapping and maximum likelihood methods for QTL detection provided consistent results in many cases, although the high variation observed for traits mean among families made it difficult to evaluate QTL effects. Finer mapping of detected QTL, looking for tightly linked markers to the causative mutation, and comparative genomics are suggested to deepen in the analysis of QTL in turbot so they can be applied in marker assisted selection programs.

Gene expression profiles of the spleen, liver, and head kidney in turbot (Scophthalmus maximus) along the infection process with Aeromonas salmonicida using an immune-enriched oligo-microarray.

We evaluated the expression profiles of turbot in the spleen, liver, and head kidney across five temporal points of the Aeromonas salmonicida infection process using an 8 × 15 K Agilent oligo-microarray. The microarray included 2,176 different fivefold replicated gene probes designed from a turbot 3' sequenced EST database. We were able to identify 471 differentially expressed (DE) genes (17.3% of the whole microarray), 223 in the spleen, 246 in the liver, and 125 in the head kidney, in at least one of the five temporal points sampled for each organ. Most of these genes could be annotated (83.0%) and functionally categorized using Gene Ontology terms (69.1%) after the additional sequencing of DE genes from the 5' end. Many DE genes were related to innate and acquired immune functions in accordance to previous studies with this pathogen in other fish species. A high proportion of DE genes were organ specific (77.1%), but their associated GO functions were rather similar in the three organs. The most striking difference in functional distribution was observed between the up- and down-regulated gene groups. Up-regulated genes were mostly associated to key immune functions while down-regulated ones mainly involved metabolism- and transport-related genes. Genetic response appeared clustered in groups of genes with similar expression profiles along the temporal series. The spleen showed the most clustering while the liver and head kidney displayed a higher diversification. The information obtained will aid to understand the turbot immune response and will specifically be valuable to develop strategies of defense to A. salmonicida to achieve more resistant broodstocks for turbot industry.

Design and performance of a turbot (Scophthalmus maximus) oligo-microarray based on ESTs from immune tissues.

An expressed sequence tag database from immune tissues was used to design the first high-density turbot (Scophthalmus maximus) oligo-microarray with the aim of identifying candidate genes for tolerance to pathogens. Specific oligonucleotides (60 mers) were successfully designed for 2,716 out of 3,482 unique sequences of the database. An Agilent custom oligo-microarray 8 x 15 k (five replicates/gene; eight microarrays/slide) was constructed. The performance of the microarray and the sources of variation along microarray analysis were examined on spleen pools of controls and Aeromonas salmonicida-challenged fish at 3 days postinfection. Only 48 out of 2,716 probes did not show signal of hybridization on the 32 microarrays employed, thus demonstrating the consistency of the bioinformatic applications of our database. An asymmetric hierarchical design was employed to ascertain the noise associated with biological and technical (RNA extraction, labeling, hybridization, slide, and dye bias) factors using 1C and 2C labeling approaches. The high correlation coefficient between replicates at most factors tested demonstrated the high reproducibility of the signal. An analysis of random-effects variance revealed that technical variation was mostly negligible, and biological variation represented the main factor, even using pooled samples. One-color approach performed at least as well as 2C, suggesting their usefulness due to its higher design flexibility and lower cost. A relevant proportion of genes turn out to be differentially labeled depending on fluorophore, which alerts for the likely need of swapping replication in 2C experiments. A set of differentially expressed genes and enriched functions related to immune/defense response were detected at 3 days postchallenging.

Identification of the major sex-determining region of turbot (Scophthalmus maximus).

Sex determination in fish is a labile character in evolutionary terms. The sex-determining (SD) master gene can differ even between closely related fish species. This group is an interesting model for studying the evolution of the SD region and the gonadal differentiation pathway. The turbot (Scophthalmus maximus) is a flatfish of great commercial value, where a strong sexual dimorphism exists for growth rate. Following a QTL and marker association approach in five families and a natural population, we identified the main SD region of turbot at the proximal end of linkage group (LG) 5, close to the SmaUSC-E30 marker. The refined map of this region suggested that this marker would be 2.6 cM and 1.4 Mb from the putative SD gene. This region appeared mostly undifferentiated between males and females, and no relevant recombination frequency differences were detected between sexes. Comparative genomics of LG5 marker sequences against five model species showed no similarity of this chromosome to the sex chromosomes of medaka, stickleback, and fugu, but suggested a similarity to a sex-associated QTL from Oreochromis spp. The segregation analysis of the closest markers to the SD region demonstrated a ZW/ZZ model of sex determination in turbot. A small proportion of families did not fit perfectly with this model, which suggests that other minor genetic and/or environmental factors are involved in sex determination in this species.

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens.

BACKGROUND: The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. RESULTS: A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value < or = 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. CONCLUSION: A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.

Dietary uptake of dioxins (PCDD/PCDFs) and dioxin-like PCBs in Spanish aquacultured turbot (Psetta maxima).

The human population is exposed to dioxins (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) mainly through diet; bioaccumulation and biomagnification in aquatic environment results in fishery products and by-products being an important vector to humans. The determination of PCDD/Fs and dl-PCBs in fillets of young turbots (Psetta maxima) (0-2 years) from aquaculture plant (Galicia, Spain) (N = 21) and in feeding stuffs were carried out, and dietary accumulation values and lipid-normalized biomagnification factors (BMF) relating concentration in fish and in feed were calculated. Levels found in feeding stuffs (0.5 pg TEQ-PCDD/F/g and 1.6 pg TEQ-dl-PCB/g), and turbots (0.13-0.27 pg TEQ-PCDD/F/g fresh weight and 0.35-1.2 pg TEQ-dl-PCB/g fresh weight) were below maximum permitted levels set by EC. Levels of toxic compounds in feeding stuff are reflected in fish fillets; predominant isomers are 2,3,7,8-TCDF, OCDD, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF and 1,2,3,4,6,7,8-HpCDD, and PCBs 118, 105, 156 and 167. Relevant compounds accounting for total toxicity are the same congeners in feeding stuff and turbots: 2,3,4,7,8- PeCDF; 2,3,7,8-TCDF; 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD, and PCB 126. Higher accumulation efficiency values were obtained for dl-PCBs (30-46%); tetra- and penta-chloro substituted PCDD/Fs showed the highest values (27-34%) of the PCDD/F group. Biomagnification was shown for these compounds (BMF around 1.5).