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Polycyclic aromatic hydrocarbons (PAHs) are among the most toxic and bioavailable components found in petroleum and represent a high risk to aquatic organisms. The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other planar aromatic hydrocarbons, including certain PAHs. Ahr acts as a xenosensor and modulates the transcription of biotransformation genes in vertebrates, such as cytochrome P450 1A (cyp1a). Atlantic cod (Gadus morhua) possesses two Ahr proteins, Ahr1a and Ahr2a, which diverge in their primary structure, tissue-specific expression, ligand affinities, and transactivation profiles. Here, a luciferase reporter gene assay was used to assess the sensitivity of the Atlantic cod Ahrs to 31 polycyclic aromatic compounds (PACs), including two- to five-ring native PAHs, a sulfur-containing heterocyclic PAC, as well as several methylated, methoxylated, and hydroxylated congeners. Notably, most parent compounds, including naphthalene, phenanthrene, and partly, chrysene, did not act as agonists for the Ahrs, while hydroxylated and/or alkylated versions of these PAHs were potent agonists. Importantly, the greater potencies of substituted PAH derivatives and their ubiquitous occurrence in nature emphasize that more knowledge on the toxicity of these environmentally and toxicologically relevant compounds is imperative. publishedVersion

The research objective was to study the presence of DNA damages in haddock exposed to petrogenic or pyrogenic polyaromatic hydrocarbons (PAHs) from different sources: 1) extracts of oil produced water (PW), dominated by 2-ring PAHs; 2) distillation fractions of crude oil (representing oil-based drilling mud), dominated by 3-ring PAHs; 3) heavy pyrogenic PAHs, mixture of 4/5/6-ring PAHs. The biological effect of the different PAH sources was studied by feeding juvenile haddock with low doses of PAHs (0.3–0.7 mg PAH/kg fish/ day) for two months, followed by a two-months recovery. In addition to the oral exposure, a group of fish was exposed to 12 single compounds of PAHs (4/5/6-ring) via intraperitoneal injection. The main endpoint was the analysis of hepatic and intestinal DNA adducts. In addition, PAH burden in liver, bile metabolites, gene and protein expression of CYP1A, GST activity, lipid peroxidation, skeletal deformities and histopathology of livers were evaluated. Juvenile haddock responded quickly to both intraperitoneal injection and oral exposure of 4/5/6-ring PAHs. High levels of DNA adducts were detected in livers three days after the dose of the single compound exposure. Fish had also high levels of DNA adducts in liver after being fed with extracts dominated by 2-ring PAHs (a PW exposure scenario) and 3-ring PAHs (simulating an oil exposure scenario). Elevated levels of DNA adducts were observed in the liver of all exposed groups after the 2 months of recovery. High levels of DNA adduct were found also in the intestines of individuals exposed to oil or heavy PAHs, but not in the PW or control groups. This suggests that the intestinal barrier is very important for detoxification of orally exposures of PAHs. publishedVersion

The present study was conducted during a time of drought to assess the concentration of herbicides and their potential for accumulation in marine biota found in the near shore marine environment of an urban setting (Camps Bay, Cape Town, South Africa). The purpose was to establish whether raw sewage containing selected persistent chemicals that are released through a local marine outfall would be sufficiently diluted by the ocean to prevent impact on the near-shore marine environment of the suburb Camps Bay. Samples of seawater, sediment, seaweed, and selected marine organisms present in the near shore environment, such as limpets (Cymbula granatina), mussels (Mytilus galloprovincialis), and sea urchins (Parechinus angulosus), were analysed for five indicator herbicides, namely atrazine, alachlor, simazine, metolachlor, and butachlor, with gas chromatography coupled with a mass spectrometer. The concentration of the compounds detected ranged from below the limit of detection (LOD) to 4.2 ng/L in seawater,LOD to 45.3 ng/g dry weight (dw) in sediments,LOD to 157.8 ng/g dw in marine organisms, and between 12.3 and 87.0 ng/g dw in seaweed. Results of ecological risk assessment for acute and chronic risk, calculated following the US Environmental Protection Agency method, showed a possible threat to the near shore environment from sewage released by the marine outfall, as the herbicide contamination in the biota posed a low to high ecotoxicological risk for marine organisms. The hazard quotients and carcinogenic risk caused by herbicide pollution in Camps Bay seawater were above the acceptable level indicating that these chemicals pose adverse health effects, should an average sized human (70 kg) consume any of the marine species analysed herein on a daily basis (daily consumption of 54 g), over a lifetime period (life expectancy of 70 years). The study also indicated the extensive use of these herbicides for cosmetic and ornamental purposes in gardening, or for weed control in an urban setting, thus, it is apparent that consumers and municipalities need to be educated about their inadvertent use and safe disposal of these compounds. The use of these herbicides to control weeds within the area and discharge of sewage to Camps Bay must be checked through adequate control of the trade, handling of herbicides, and enforcement of appropriate sanctions and development of suitable regulations.

The aim of the present study was to investigate effects of defined mixtures of polycyclic aromatic hydrocarbons (PAHs) and perfluoroalkyl substances (PFASs), at low, environmentally relevant (1× = L), or high (20× = H) doses, on biological responses in Atlantic cod (Gadus morhua). To this end, farmed juvenile cod were exposed at day 0 and day 7 via intraperitoneal (i.p.) injections, in a two-week in vivo experiment. In total, there were 10 groups of fish (n = 21-22): two control groups, four separate exposure groups of PAH and PFAS mixtures (L, H), and four groups combining PAH and PFAS mixtures (L/L, H/L, L/H, H/H). Body burden analyses confirmed a dose-dependent accumulation of PFASs in cod liver and PAH metabolites in bile. The hepatosomatic index (HSI) was significantly reduced for three of the combined PAH/PFAS exposure groups (L-PAH/H-PFAS, H-PAH/L-PFAS, H-PAH/H-PFAS). Analysis of the hepatic proteome identified that pathways related to lipid degradation were significantly affected by PFAS exposure, including upregulation of enzymes in fatty acid degradation pathways, such as fatty acid β-oxidation. The increased abundances of enzymes in lipid catabolic pathways paralleled with decreasing levels of triacylglycerols (TGs) in the H-PFAS exposure group, suggest that PFAS increase lipid catabolism in Atlantic cod. Markers of oxidative stress, including catalase and glutathione S-transferase activities were also induced by PFAS exposure. Only minor and non-significant differences between exposure groups and control were found for cyp1a and acox1 gene expressions, vitellogenin concentrations in plasma, Cyp1a protein synthesis and DNA fragmentation. In summary, our combined proteomics and lipidomics analyses indicate that PFAS may disrupt lipid homeostasis in Atlantic cod., This study was funded by Research Council of Norway through the dCod 1.0: decoding the systems toxicology of Atlantic cod project (Center for Digital Life Norway project no. 248840) and the iCod 2.0: Integrative environmental genomics of Atlantic cod project(project no. 244564). The authors wish to thank Frederike Keitel-Gröner for contributing to sampling, and Essa Ashan Khan for contributing to sampling and manuscript information. We also thank Hui-Shan Tung and Pål Olsvik for contributing to liver RT-qPCR preparation and analysis at the Institute of Marine Research (IMR). We further thank Inge Jonassen and Ketil Hylland for valuable input proteomics analysis, and on experiment and manuscript design, respectively.

Background The expansion of offshore oil exploration increases the risk of marine species being exposed to oil pollution in currently pristine areas. The adverse effects of oil exposure through toxic properties of polycyclic aromatic hydrocarbons (PAHs) have been well studied in Atlantic cod (Gadus morhua). Nevertheless, the fate of conjugated metabolites in the intestinal tract and their effect on the diversity of intestinal microbial community in fish is less understood. Here, we investigated the intestinal microbial community composition of Atlantic cod after 28 days of exposure to crude oil (concentration range 0.0–0.1 mg/L). Results Analysis of PAH metabolites in bile samples confirmed that uptake and biotransformation of oil compounds occurred as a result of the exposure. Various evidence for altered microbial communities was found in fish exposed to high (0.1 mg/L) and medium (0.05 mg/L) concentrations of oil when compared to fish exposed to low oil concentration (0.01 mg/L) or no oil (control). First, altered banding patterns were observed on denaturing gradient gel electrophoresis for samples pooled from each treatment group. Secondly, based on 16S rRNA sequences, higher levels of oil exposure were associated with a loss of overall diversity of the gut microbial communities. Furthermore, 8 operational taxonomic units (OTUs) were found to have significantly different relative abundances in samples from fishes exposed to high and medium oil concentrations when compared to samples from the control group and low oil concentration. Among these, only one OTU, a Deferribacterales, had increased relative abundance in samples from fish exposed to high oil concentration. Conclusions The results presented herein contribute to a better understanding of the effects of oil contamination on the gut microbial community changes in fish and highlight the importance of further studies into the area. Our findings suggest that increased relative abundance of bacteria belonging to the order Deferribacterales may be indicative of exposure to oil at concentrations higher than 0.05 mg/L. Electronic supplementary material The online version of this article (10.1186/s12866-018-1171-2) contains supplementary material, which is available to authorized users.

Background: Microorganisms are widespread in all environments, including in and on animal bodies. The gut microbiome has an essential influence on fish health, and is affected by several persistent and harmful organic and inorganic contaminants. Considering the shifts in gut microbiota composition observed in those studies, we hypothesized that certain microbial groups in the gut can serve as indicators of pollution. To test this hypothesis, we explored the possibility of identifying key microbial players that indicate environmental contamination. Methods: Published 16S rRNA gene amplicon sequencing data generated from the gut microbiota of Atlantic cod caught in geographically different Norwegian waters were used for bacterial diversity comparison. Results: Different microbiomes were identified between the northern Norway and southern Norway samples. Several bacterial genera previously identified as polycyclic aromatic hydrocarbon degraders were present only in the samples collected in the southern Norway area, suggesting fish contamination with oil-related compounds. Conclusions: The results contribute to the identification of bacterial taxa present in the Atlantic cod gut that indicate fish exposure to contaminants in the marine environment.

The isolation of the dominant isoform of haemoglobin from Atlantic cod caught in southwest Norway is reported and its X-ray crystal structure is presented., The crystal structure of haemoglobin from Atlantic cod has been solved to 2.54 Å resolution. The structure consists of two tetramers in the crystallographic asymmetric unit. The structure of haemoglobin obtained from one individual cod suggests polymorphism in the tetrameric assembly.

Fixed wavelength fluorescence (FF) and synchronous fluorescence scanning (SFS) of fish bile are commonly used methods to analyze for exposure to polycyclic aromatic hydrocarbons (PAHs) from petrogenic and pyrogenic sources. A range of conjugated oxidation products from petrogenic PAHs are normally accumulated in the bile. Therefore their detection is important. In the present study, phenanthrene and naphthalene metabolites, formed in vivo in Atlantic cod (Gadus morhua), were used to study the response of these compounds in both FF and SFS analyses. The selected synthetic metabolites were (−)-(1R,2R)-1,2-dihydrophenanthrene-1,2-diol and (−)-(1R,2R)-1,2-dihydronaphthalene-1,2-diol. The study findings showed that the recommended excitation and emission wavelengths for FF analysis do not comprise the maximum emission wavelengths for these metabolites, providing an incorrect estimation of the PAH exposure. A method developed in our laboratory for the synthesis of (−)-(1R,2R)-1,2-dihydrophenanthrene-1,2-diol is also described.

The use of angiotensin-I converting enzyme (ACE) inhibitors is a common strategy for treating kidney disease. Several amino acid sequences with ACE inhibiting activity are identified in filet and rest raw material from various species of fish, and fish protein hydrolysates could be of interest for possible treatment or prevention of kidney disease. Therefore, we hypothesized that protein hydrolysates from rest raw material from herring and salmon contained ACE inhibiting motifs, and could beneficially affect typical markers for kidney function in an obesity rat model prone to developing renal failure. We identified 81 and 49 peptide sequences with known ACE inhibiting activity in herring and salmon protein hydrolysates from rest raw material, respectively. To investigate the effects of fish protein hydrolysates on markers of kidney function, obese Zucker fa/fa rats consumed diets with 25% of protein from herring (HER) or salmon (SAL) protein hydrolysate from rest raw material and 75% of protein from casein/whey, or 100% protein from casein/whey (CAS) for 4 weeks. Rats fed HER or SAL diets had lower urine concentrations (relative to creatinine) of protein, cystatin C and glucose when compared to rats fed CAS diets, with no differences between groups for serum concentrations of protein, creatinine and cystatin C. To conclude, protein hydrolysates from herring and salmon rest raw material contained several peptide sequences with known ACE inhibiting activities, and resulted in lower urine concentrations of proteins, cystatin C and glucose when fed to obese Zucker rats.

Occurrence of polycyclic aromatic hydrocarbon (PAH) contamination in the marine environment represents a risk to marine life and humans. In this study, plasma samples from Atlantic cod (Gadus morhua) were analysed by shotgun mass spectrometry to investigate the plasma proteome in response to exposure to single PAHs (naphthalene or chrysene) and their corresponding metabolites (dihydrodiols). In total, 369 proteins were identified and ranked according to their relative abundance. The levels of 12 proteins were found significantly altered in PAH exposed fish and are proposed as new biomarker candidates. Eleven proteins were upregulated, primarily immunoglobulin components, and one protein was downregulated (antifreeze protein type IV.) The uniformity of the upregulated proteins suggests a triggered immune response in the exposed fish. Overall, the results provide valuable knowledge for future studies of the Atlantic cod plasma proteome and generate grounds for establishing new plasma protein biomarkers for environmental monitoring of PAH related exposure.

PAH metabolites present in bile are well-known biological markers of exposure in fish, and their investigation is recommended by the ICES (International Council for the Exploration of the Sea) and the OSPAR convention (Convention for the Protection of the Marine Environment of the North-East Atlantic) for monitoring purposes. Development of analytical strategies for fish bile is encouraged by the need for more sensitive and informative markers (e.g., capable of tracking the PAH composition of contamination sources) and strengthened by recent results in both fish genomics and proteomics. Herein, the study of the Atlantic cod bile proteome is presented. Preliminary testing for discovering new sensitive markers in the form of expressed proteins affected by PAH exposure (i.e., PAH-protein adducts) is reported. Protein markers were identified using LC-MS/MS analysis, as single biological indicators. Through multivariate analyses, the overall proteome was revealed to be a sensitive multi-biological marker of exposure to PAHs.

Environmental monitoring requires cost-effective and efficient methods for detecting potential effects of pollution, and valve gape behaviour has been used with this purpose for a range of contaminants in freshwater and marine bivalves. The current study investigated the use of a new method for measuring valve behaviour responses in mussels (Mytilus edulis) exposed to dispersed crude oil (DCO). Results confirmed that valve gape is a sensitive parameter; at the high DCO concentration (0.25mgL-1) the mean valve gape was reduced from 49 to 31%, and mussels increased shell movement (measured as distance travelled) or spent more time closed to avoid contact with the oil. At the low DCO concentration (0.015mgL-1) the distance travelled parameter was the most sensitive endpoint. Results also demonstrated that valve gape behaviour is a valid endpoint when monitoring mussels for exposure to DCO.

International audience; The monitoring of the presence of polycyclic aromatic hydrocarbons (PAH) in the aquatic environment is a worldwide activity since some of these compounds are well-established carcinogens and mutagens. Contaminants in this class are in fact regarded as priority hazardous substances for environmental pollution (Water Framework Directive 2000/60/EC). In this study, Atlantic cod (Gadus morhua) was selected to assess in vivo effects of two PAH and their first metabolic products, namely, the corresponding trans-dihydrodiols, using biological markers. Fish were exposed for 1 wk to a single PAH (naphthalene or chrysene) and its synthetic metabolites ((1R,2R)-1,2-dihydronaphthalene-1,2-diol and (1R,2R)-1,2-dihydrochrysene-1,2-diol) by intraperitoneal injection in a continuous seawater flow system. After exposure, PAH metabolism including PAH metabolites in bile and ethoxyresorufin O-deethylase (EROD) activity, oxidative stress glutathione S-transferases (GST) and catalase (CAT) activities, and genotoxicity such as DNA adducts were evaluated, as well as general health conditions including condition index (CI), hepatosomatic index (HSI), and gonadosomatic index (GSI). PAH metabolite values were low and not significantly different when measured with the fixed-wavelength fluorescence screening method, while the gas chromatography-mass spectroscopy (GC-MS) method showed an apparent dose response in fish exposed to naphthalene. DNA adduct levels ≥0.16 × 10(-8) relative adduct level (RAL) were detected. It should be noted that 0.16 × 10(-8) RAL is considered the maximal acceptable background level for this species. The other biomarkers activities of catalase, GST, and EROD did not display a particular compound- or dose-related response. The GSI values were significantly lower in some chrysene- and in both naphthalene- and naphthalene diol-exposed groups compared to control.

Recent research has shown that fish residual materials contain a range of components with interesting biological activity. Therefore, there is a great potential in the marine bioprocess industry to utilize these by-products as starting material for generating more valuable products. The aim of the present study was to search for bioactive peptides (in particular small natural bioactive peptides with molecular weight lower than 10 kDa) in Atlantic herring (Clupea harengus L.) by-products such as skin and more general residual materials. By such means a range of peptides with claimed interesting biological activities was found. Herein the activity of the detected bioactive peptides and strategies for isolating peptide fragments containing the bioactive motif is discussed. Identification of bioactive peptides in crude peptide/protein sources (skin and residual materials) was performed directly using a combination of mass spectrometry (Orbitrap), bioinformatics and database search. This method was a good angle of approach in order to map the potential in new species and species that have been very little studied.

Biological markers of produced water (PW) exposure were studied in Atlantic cod (Gadus morhua) in both laboratory and field experiments, using authentic PW from a North Sea oil field. In the laboratory study, the PW exposure yielded significantly elevated levels of metabolites of polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APs) in bile even at the lowest exposure dose (0.125% PW). Other biomarkers (hepatic CYP1A induction and DNA adduct formation) responded at 0.25% and 0.5% PW concentrations. In the field study, bile metabolite markers and hepatic CYP1A were clearly increased in fish caged close to the PW outfall. Induction of plasma vitellogenin was not found in laboratory or field exposures, suggesting that the levels of oestrogen agonists (such as APs) might not have been sufficient to elicit induction, under the present conditions. The applicability of the biomarkers for use in water column biomonitoring programs is discussed.

Polar marine surface waters are characterized by high levels of dissolved oxygen, seasonally intense UV irradiance and high levels of dissolved organic carbon. Therefore, the Arctic sea-ice habitat is regarded as a strongly pro-oxidant environment, even though its significant ice cover protects the ice-associated ( = sympagic ) fauna from direct irradiation to a large extent. In order to investigate the level of resistance to oxyradical stress, we sampled the sympagic amphipod species Gammarus wilkitzkii during both winter and summer conditions, as well as exposed specimens to simulated levels of near-natural and elevated levels of UV irradiation. Results showed that this amphipod species possessed a much stronger antioxidant capacity during summer than during winter. Also, the experimental UV exposure showed a depletion in antioxidant defences, indicating a negative effect of UV exposure on the total oxyradical scavenging capacity. Another sympagic organism, Onisimus nanseni , was sampled during summer conditions. When compared to G. wilkitzkii , the species showed even higher antioxidant scavenging capacity.

Ciphergen ProteinChip Technology™ is a proteomic tool, used for the discovery of new and sensitive biomarkers. This approach was used to evaluate the protein profile of crabs exposed to various pollutants. Two different exposure experiments were performed: spider crabs ( Hyas araneus ) were exposed for 3 weeks to diallyl phatalate (DAP), bisphenol A (BisA) and polybrominated diphenyl ether (PBDE-47), while shore crabs ( Carcinus maeanas ) were exposed to crude oil, crude oil spiked with alkylphenols (APs) and 4-nonylphenol (NP). Gender and species-related protein pattern alterations were observed and compared to controls. Results showed different responses to pollutants by the two species. Major disruption in protein peak expression was observed in samples exposed to mixtures of pollutants, i.e. oil spiked with APs. Compared to shore crab, spider crab species showed a lower degree of response in terms of number of altered protein peaks following exposure. In general, female individuals of both species showed a larger number of significantly altered proteins compared to males. Data analysis by non-metric multi-dimensional scaling (MDS) was performed. Bi-dimesional-MDS plots revealed a good separation of groups for both spider and shore crabs. In some cases, a good discrimination can also be observed between the two genders within each treatment. Results highlight the potential of crabs as sentinel organisms for the aquatic environment. The results indicate that SELDI-ToF technology is a powerful tool to discover protein expression signatures for different pollutants and sex dependent responses.

The overall objective of this study was to compare the expression of plasma proteins in juvenile cod and turbot after a 3 week exposure to two different chemicals known to be estrogenic: 4-nonylphenol (NP, 29 microg/L) and bisphenol A (BPA, 59 microg/L). ProteinChip) array technology in combination with surfaced enhanced laser desorption ionisation-time of flight (SELDI-TOF) was used to investigate general responses in plasma proteins. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) was used to analyse two specific biomarkers of estrogenic exposure, vitellogenin (Vtg) and zona radiata protein (Zrp) in plasma. Both methods revealed clear species specific responses. In cod, 67% of significantly altered proteins showed the same response (up or down regulated) in NP and BPA exposed animals (males and females combined). The rest were either specific to NP (10%), BPA (19%) or they showed opposite responses to the two chemicals (4%). In contrast, only 20% of significantly altered proteins were common for NP and BPA exposed turbot: 60% were altered only in NP and 17% only in BPA. Furthermore, in BPA exposed cod, 77% of the responses were common for male and females, whereas turbot showed only 21% similarity for the two genders. However, NP exposed male and female turbot showed 88% similarity in responses. As gender was not determined in NP exposed cod, gender specific responses could not be determined. ELISA results supported that cod responded clearly to both chemicals as a large increase was observed in Vtg and Zrp levels. Turbot responded strongly to NP, but seemed only slightly affected by BPA. Overall, the results indicated that cod are more sensitive or respond with less specificity to estrogenic chemicals than turbot. The relatively large degree of common responses in NP and BPA exposed cod may indicate that in cod BPA have similar mode of action as NP. Generally, the results show the potential of SELDI-TOF as a tool for comparing multiple responses, and for identifying exposure as well as gender specific responses.

The development of rapid and sensitive diagnostic tools to assess the effect of stressors on organisms is a principal objective of environmental proteomics. This study is focused on evaluating the potential of using surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) to assess stress in Atlantic salmon (Salmo salar). Plasma and mucus samples were taken from fish that had previously been maintained in a range of high density conditions, together with control fish maintained under low density conditions. Samples were collected during the post-density stress period for protein profile analysis. The mass spectra were analysed to evaluate reproducibility and to search for condition specific changes in protein expression. Multivariate analysis of the peak relative intensity data indicated a segregation of the data into three entities in accordance with the density level fish had been subjected to during the density stress period. This segregation was seen in both plasma and mucus data.

The aim of this study was to evaluate the susceptibility to pollutant mediated oxidative stress of the Mediterranean mussel Mytilus galloprovincialis in the Venice lagoon (Italy). In June 2003, mussels from a farm were transplanted to eight sites in the lagoon for five weeks. Oxidative stress responses were measured by: (i) total oxyradical scavenging capacity (TOSC) assay, for an overall evaluation of the oxidative stress response capability; (ii) catalase (CAT), as a key enzyme involved in the antioxidant defence system; (iii) malondialdehyde (MDA), as an indicator of lipid peroxidation, to evaluate an oxidative damage; (iv) metallothioneins (MTs), as they play a role in the antioxidant defence. The TOSC analysis revealed a reduced capability to eliminate: (i) peroxyl radical in mussels transplanted at Palude della Rosa, Valle Millecampi and Chioggia; (ii) hydroxyl radical at Campalto and Valle Millecampi; (iii) peroxynitrite at Valle Millecampi. Inhibition in CAT activity, observed in all the monitored sites, confirms the presence of an oxidative pressure in transplanted mussels. In addition, Pearson correlation analysis was performed in order to observe possible links between the various parameters. The PCA was a powerful tool to discriminate impacted sites, suggesting that the mussels transplanted throughout the Venice lagoon were subjected to different levels of oxidative pressure. Furthermore, it provided an easy and useful tool to summarize the obtained results.

In this study, a spatial and temporal survey at three sites located in the “canals” of the Venice historic centre (Italy) and at a reference site was undertaken to evaluate stress effects on mussels sampled in the Venice urban area, where raw sewage is discharged without treatment directly into the water. A battery of biomarkers (metallothionein, micronuclei, condition index and survival in air) was used to evaluate the stress condition of the animals. At the same time the alkali-labile phosphate assay (ALP) was performed in mussel' hemolymph with the aim to find an estrogenic effect biomarker in this mussel species. Biomarker results showed an impairment of the general health condition in the mussels coming from the urban area, in agreement with the chemical analysis. Significantly higher level of the ALP was found in male mussels sampled in April in the urban area, in comparison with the ones from the reference site ( P 0.001 ). Finally, the PCA proved an easy and useful tool to summarize the obtained results, also able to classify the data to indicate a pollution gradient in the Venice urban area.

The Venice Lagoon has been the focus of many environmental studies due to its heavy contaminant load derived from a variety of sources, including industrial activity, oil tanker traffic, and waste runoff from the mainland. In recent years, there has been increasing concern about water quality in the urban areas of Venice related to the discharge of untreated sewage directly into canals, adding to the pollutant load already existing in these areas. One way of gauging the impact of these chemicals is monitoring the local fauna. In the search for good indicators of water quality in the Venice urban area, two physiological indices for mussels-survival in air and condition index-have been evaluated. In 2002, a seasonal study was undertaken; mussels (Mytilus galloprovincialis) were collected in three sites located in the canals of the city and a clean reference site. At the same time, two transplantation experiments were performed, deploying farmed mussels for 1,2, 4, and 12 weeks in the historic centre of Venice and also at a reference site. Data from survival in air test and the condition index of native and transplanted mussels are reported. The results suggest an impact of the complex mixture of pollutants on mussel health status. Both native mussels and those transplanted to the urban area showed reduced survivability in air and decreased condition index values, indicating a less healthy status in animals from the canals of the city. Data are discussed in relation to pollutant bioaccumulation.

Increased research efforts are currently focusing on Atlantic cod (Gadus morhua) and its significance for monitoring the contaminant situation in marine environments. Polycyclic aromatic hydrocarbons (PAHs) are well known toxic and carcinogenic compounds, thus continuous monitoring is required to ensure ecosystem sustainability and human food safety. A sensitive biomarker of PAH exposure in humans is the detection of PAH metabolites bound to albumin in blood. The potential of a similar PAH-albumin biomarker in Atlantic cod was therefore investigated by a desktop bioinformatic study followed by liquid chromatography mass spectrometry/mass spectrometry analysis of plasma from 16 fish. For the first time, an albumin-like protein in plasma of Atlantic cod is described, and the biomarker potential based on PAH-albumin adduct detection is discussed. Due to the detected low abundance of the albumin-like protein, it was found unlikely to be applicable as a new biomarker tool for evaluation of PAH exposure.

Histopathological characteristics of specific organs express condition, and represent time-integrated impacts on the organism stemming from alterations at lower levels of biological organisation. As integrative parameters, histochemical investigations have proved to be sensitive tools to detect effects of chemical compounds. The objective of this study was to determine changes in the tissues of mussels collected at a PAH contaminated site compared to a reference site using histopathological and histochemical parameters: lipofuscin (LF) accumulation in mussel digestive gland, and lysosomal membrane stability (LMS), and using additional information provided by body burden analysis to compare the sensitivity of these parameters. The histochemical measurements for both LF and LMS gave a clear indication of a high level of stress in animals from the PAH contaminated site. This LF accumulation in lysosomes is the result of peroxidation of autophagocytosed proteins associated with protein aggregates and oxidatively damaged organelles. These measurements were able to detect the effects of PAHs, and showed a strong relationship with the body burden results.

Within the BEEP project (Biological Effects of Environmental Pollution in Marine Ecosystems) the Work Package 1 was addressed to the development of new and more sensitive biomarkers of exposure in several sentinel organisms. Within this framework, common mesocosm exposures of organic pollutants relevant for marine ecosystems were conducted in the facilities of Akvamiljo a/s (Stavanger, Norway). In the first experiment, Atlantic cod (Gadus morhua), turbot (Scophthalmus maximus) and shore crab (Carcinus maenas) were exposed to nonylphenol, North Sea crude oil and a combination of crude oil and alkylated phenols. Mussels (Mytilus edulis) were exposed to North Sea crude oil and a combination of crude oil, alkylated phenols and PAHs. In the second experiment, Atlantic cod, turbot, mussel and spider crab (Hyas araneus) were exposed to the plasticizers bisphenol A and diallyl phatalate and the brominated flame retardant BDE-47. The main purpose of the present study was to provide the 30 participating Institutes with samples which had been exposed to defined contaminant concentrations in a controlled laboratory exposure for 3 weeks. This paper describes the mesocosm experimental design, the transplantation and treatment of the organisms, and the contaminant exposures.

Biomarkers of exposure & effect:: validationBiomarker: MNExposure/effect represented: genetic damageStudy type (in vitro, animals, humans): fishMode of exposure (if in vivo) (acute, chronic, root of exposure):chronicTissue/biological material/sample size:4*103 erythrocytes/animalMethod of analysis: MN testSensitivity (LOD): low baseline frequencyIntra-individual variation: large range of variabilityInter-individual variation:0-2 MN frequencyDose-response: 2.95 MN/1000 cells compared to 1MN/1000 cells in the control, p

Residual biological effects of the 1991 HAVEN oil spill off the Ligurian (Arenzano) coast were assessed in this study. Samples of the fish species Boops boops, Mullus barbatus, and Uranoscupus scaber were collected from two polluted sites near the HAVEN wreck and from an uncontaminated area. In addition to this, mussels were caged along the coast affected by the HAVEN disaster. The physiological status of fish and mussels was assessed using a battery of stress and exposure biomarkers. The PAH content of mussel and fish tissues was also analyzed. Significant biological responses were observed in lysosomal membrane stability, neutral lipid and lipofuscin accumulation and micronucleus frequency for mussels caged at two sites close to the HAVEN wreck. Chemical analyses indicated, however, that these effects are not caused by aromatic hydrocarbons. For this reason, we suggest that the aftermath of the HAVEN disaster contributes very little to coastal ecosystem pollution. This was also confirmed by the few biological effects observed in fish specimens (Boops boops) collected from surface waters. Nevertheless, it is important to point out that benthic fish displayed a stress syndrome potentially caused by aromatic hydrocarbons released from the oil tanker, as witnessed by an enhanced EROD activity and increased lipofuscin and neutral lipid lysosomal contents.

Oxidative stress related investigations to monitor the impact of the pollutant discharges into the Venice lagoon (Italy) originating from anthropogenic activities (raw sewage water, agricultural and industrial effluents, oil tanker traffic), on marine organisms have classically been carried out by analyzing specific, single antioxidants (i.e. catalase, superoxide dismutase). In this paper, two studies are reported where the total oxyradical scavenging capacity assay (TOSC) was selected and measured toward peroxyl, and hydroxyl free radicals, and peroxynitrite in mussels (Mytilus galloprovincialis) transplanted into the Venice city and throughout the lagoon to measure the biological effects of anthropogenic activities. In the first experiment, mussels from a clean site (farm) were transplanted to the urban area of Venice for 0, 1, 2 and 4 weeks; cytosolic TOSC toward peroxyl and hydroxyl free radicals, and peroxynitrite revealed that the transplantation process caused a stress (handling stress, anoxia, oxidative burst) resulting in a reduction of TOSC in both control and urban sites, therefore, preventing clear interpretation of the data after one week. At week 2, a significant TOSC reduction (P < 0.05) toward peroxyl and hydroxyl radicals in the urban site revealed that mussels experienced oxidative pressure exerted by pollutants. Most TOSC values returned to initial levels at week 4; however, TOSC induction was noticed in the control group toward peroxyl and hydroxyl radicals while in the exposed group it was not indicating an inhibition of the oxidative metabolism. In the second experiment, mussels were deployed at seven different sites throughout the lagoon. After five weeks of exposure, significant TOSC reduction was measured (P < 0.05) toward peroxyl for Palude della Rosa, Chioggia and Valle Millecampi, toward hydroxyl radicals for Valle Millecampi and Campalto and toward peroxynitrite for Valle Millecampi. Although these data indicate a depletion of the low molecular weight scavengers, additional biomarkers are needed to draw a conclusion on the health of the mussels. TOSC was proved to be an interesting health index parameter to measure pollution impact in a transplantation study provided that the mussels are exposed for two weeks and a control is run in parallel.

The clam Chamelea gallina is fairly common along the western coastline of the Adriatic Sea, where it is subjected to intense fishing. To evaluate possible causes of stress in clam populations, the effects of acute mechanical disturbance on haemocyte functionality were analysed in both laboratory and field experiments. Among the various sources of stress that clams undergo during commercial fishing by hydraulic dredges, water pressure and mechanical sorting were considered, and three increasing levels of stress were applied. A reduction in immunosurveillance was highlighted, concentrations of circulating haemocytes, phagocytic and acid phosphatase activity indices decreased with increasing mechanical stress. The opposite trend shown by the beta-glucuronidase activity index is discussed. The response of clam haemocytes, detected on seasonal bases in two sites, often exhibited peculiar patterns, mostly depending on the reproductive cycle, and were probably influenced by different features of the sea bottom. Although recovery after stress was observed in laboratory experiments, some considerations are made on detrimental effects experienced in the field by under-sized clams, which are first fished and then thrown back into the sea.

The impact of experimental dredging was assessed on the venus clam Chamelea gallina from seabeds off the Lagoon of Venice. Commercial-sized clams were collected using high water pressure and mechanical sorting (HP), as in professional fishing, and low water pressure without sorting (LP). To highlight the effects of acute stress due to the different approaches in harvesting clams, various biological responses were evaluated at both organism and cell level. Filtration and respiration rates showed opposite behaviour, the former significantly decreasing, the latter increasing, when mechanical stress was higher. Consequently, lower values of scope for growth in HP clams were observed. The survival in air test exhibited significantly lower median survival time values (LT50) in more severely stressed animals. The haematocrit value and phagocytic index significantly decreased in HP samples, revealing a detrimental effect on mechanisms of immunological defence. Acid phosphatase and β-glucuronidase activity was also affected by increasing mechanical stress, even though an opposite trend was shown in the two investigated enzymes. Lastly, higher damage levels were detected in shells of HP clams.

A morphological and enzymatic characterization of Chamelea gallina haemocytes was carried out as a prerequisite for further studies on venus clam immunobiology. Two main types of circulating haemocytes were identified (1) hyalinocytes (79.2%), agranular cells with a central nucleus surrounded by a little cytoplasm, and (2) granulocytes (16.5%), smaller granular cells with smaller nuclei. Small cells with a strongly basophilic nucleus and a thin layer of peripheral cytoplasm, probably undifferentiated blast cells (4.3%), were also observed. Both granulocytes and hyalinocytes can assume a spreading or round morphology. The enzymatic activities of haemocytes were also investigated. Some of the granulocytes and hyalinocytes were positive for hydrolytic enzymes, suggesting a role for these cells in phagocytosis; no oxidative enzymes were detected in C. gallina haemocytes. Granulocytes and hyalinocytes can easily adhere to the substratum and exhibit a low phagocytosis activity towards foreign particles (6.3%), whereas the fraction of cells containing ingested material significantly increased after pre-incubation of test particles with cell-free haemolymph, which suggests the presence of opsonin(s) in the haemolymph.

The Venus clam Chamelea gallina is fairly common along the western coasts of the Adriatic and is subjected to intense fishing. Since over the last 20 years extensive hypoxic and anoxic conditions have repeatedly damaged this natural resource, we decided to study the effects of anoxic stress on the functionality of clam haemocytes and the consequences on immune responses. Clams, exposed to air, close their valves and tissues become anoxic and metabolism processes switch to anaerobiosis. In these conditions, a significant decrease in the haematocrit value and in the percentage of acid phosphatase-positive haemocytes was observed, while the number of cells with b-glucuronidase significantly increased after day 1. The above indices generally returned to control values when clams were re-immersed in seawater after 1 day of treatment. Clams exposed to air for 2 days and then re-immersed, attempted to recover in the subsequent 3 days. Animals had fully recovered on day 4. Three-day-exposed clams did not recover. Phagocytic and adhesion indices decreased significantly after the first day of air exposure. The change in frequency of three types of circulating cells (spreading, round, apoptotic) was also monitored. 2002 Elsevier Science Inc. All rights reserved.

The effects of Cu and Cd on functional responses of hemocytes in Tapes philippinarum, a Veneridae clam widespread in the lagoon of Venice, were evaluated. Bivalves were exposed for 7 days to various concentrations of CuCl(2) x 2H(2)O (0, 10, 60, and 110 microg/L) and CdCl(2) x H(2)O (0, 150, 300, and 450 microg/L). Hemocytes were collected from exposed clams, and the effects of Cu and Cd on phagocytosis, Neutral Red retention capacity, superoxide dismutase, and cytochrome oxidase activities were investigated. Hemocytes from animals exposed to Cu showed a significant decrease in phagocytic activity, whereas no inhibition was observed in cells from Cd-exposed animals. Diffusion of Neutral Red into the cytosol, an index of lysosomal membrane alteration, was enhanced by high concentrations of both metals. Different results in biochemical parameters were observed. Exposure to 60 and 110 microg Cu/L caused a significant reduction in hemocyte superoxide dismutase activity, whereas no changes resulted after Cd exposure. Increased cytochrome oxidase activity was observed in hemocytes from mollusks exposed to 60 microg Cu/L and 300 microg Cd/L; no significant differences were found in cells from bivalves exposed to 110 microg Cu/L and 450 microg Cd/L. These results suggest a relationship between heavy metal exposure and alterations in functional responses of hemocytes in T. philippinarum and indicate that the type of observed effects vary with the nature and concentration of heavy metals. Moreover, the data obtained in the analyzed clam support the possibility of using it as sentinel organism in biomonitoring studies, even if used biomarkers will be further evaluated in field conditions.

This study is focused on evaluating biological effects on the target species Chamelea gallina as a result of repeated exploitation of clam beds by hydraulic dredging. A biomarker approach was adopted and two physiological indices were applied: adenylate energy charge (AEC) (biochemical level) and reburrowing behaviour (organism level). The aim was to investigate the biological response to various types of fishing, i.e., commercial systems using water at high pressures (HP), and experimental systems at lower pressures (LP) in the field, and to evaluate the response of clams to mechanical stress in the laboratory. Field results showed that HP-treated clams exhibited significantly lower levels of AEC compared with LP treatments. A similar trend was shown by reburrowing behaviour; HP-treated clams reburrowed less. Laboratory results were less clear: a very low level of AEC was measured in both control and treated clams. These poor conditions in foot muscle did not indicate worsening at organism level, as no dead or dying clams were recorded throughout the experiment. However, repeated mechanical stress reduced the percentage of reburrowing clams, which suggested that harvesting may affect reburrowing behaviour.