Biomarker responsiveness in Norwegian Sea mussels, Mytilus edulis, exposed at low temperatures to aqueous fractions of crude oil alone and combined with dispersant.

Biological effects of aqueous fractions of a crude oil, alone or in combination with dispersant, were investigated in mussels, Mytilus edulis, exposed at three temperatures (5, 10 and 15 °C). Polycyclic aromatic hydrocarbons (PAHs) tissue concentrations were determined, together with genotoxicity, oxidative stress and general stress biomarkers and the Integrated Biological Response (IBR) index. The bioaccumulation of individual PAHs varied depending on the exposure temperature, with relevant bioaccumulation of phenantrene and fluoranthene at 5 °C and heavier (e.g. 5-rings) PAHs at 15 °C. The values and response profiles of each particular biomarker varied with exposure time, concentration of the oil aqueous fraction and dispersant addition, as well as with exposure temperature. Indeed, PAH bioaccumulation and biomarker responsiveness exhibited specific recognizable patterns in mussels exposed at low temperatures. Thus, genotoxicity was recorded early and transient at 5 °C and delayed but unremitting at 10-15 °C. Catalase activity presented a temperature-dependent response profile similar to the genotoxicity biomarker; however, glutathione-S-transferase responsiveness was more intricate. Lysosomal membrane stability in digestive cells decreased more markedly at 5 °C than at higher temperatures and the histological appearance of the digestive gland tissue was temperature-specific, which was interpreted as the combined effects of PAH toxicity and cold stress. It can be concluded that the profile and level of the biological effects are definitely different at low temperatures naturally occurring in the Arctic/Subarctic region (e.g. 5 °C) than at higher temperatures closer to the thermal optimum of this species (10-15 °C).

Integrated assessment of biological responses to pollution in wild mussels (Mytilus edulis) from subarctic and arctic areas in the Norwegian sea.

North Atlantic and Arctic Oceans contain large amount of undiscovered oil and gas reserves. Therefore threat of oil spills and its hazardous ecological consequences are of great importance to the marine environment. Although mussels (Mytilus sp.) respond clearly to contaminants, biomarkers have shown variability linked to biological and environmental changes. In order to help avoiding misinterpretation of biological responses the aim of this study was to reveal the effect of natural variability in the responsiveness to pollution of a battery of cell and tissue-level biomarkers in mussels. Mussels were collected in relatively non-impacted and potentially impacted sites at ports and the vicinity of a waste water treatment plant in Trondheim and Tromsø in autumn of 2016. Although the battery of biomarkers used herein proved to be useful to discriminate impacted and non-impacted mussel populations, some confounding factors altering the biological responses were identified. Geographical/latitudinal factors seemed to be critical regarding the reproductive cycle, reserve material storage and the prevalence of parasites such as Gymnophallus cf. Bursicola trematodes. Mussels from the reference site in Tromsø displayed general stress responses at different levels, which could be influenced by the pathogenic effect of the Gymnophallus cf. Bursicola trematode and by a more advanced gametogenic developmental stage compared to the mussels from Trondheim, which could lead to misinterpretation of the reasons behind the measured stress levels in those mussels. Despite these confounding effects, the use of integrative tools such as IBR index helped to discriminate mussel populations from chemically impacted and non-impacted sites. Overall, this work serves as an anchor point both as a reference of the baseline level values of the analyzed endpoints in the studied geographical area and time of the year, and as an indication of the potential extent of the environmental confounding factors in monitoring programs causing stress on the analyzed mussel populations.

The influence of temperature in sea urchin embryo toxicity of crude and bunker oils alone and mixed with dispersant.

This investigation deals with how temperature influences oil toxicity, alone or combined with dispersant (D). Larval lengthening, abnormalities, developmental disruption, and genotoxicity were determined in sea urchin embryos for assessing toxicity of low-energy water accommodated fractions (LEWAF) of three oils (NNA crude oil, marine gas oil -MGO-, and IFO 180 fuel oil) produced at 5-25 °C. PAH levels were similar amongst LEWAFs but PAH profiles varied with oil and production temperature. The sum of PAHs was higher in oil-dispersant LEWAFs than in oil LEWAFs, most remarkably at low production temperatures in the cases of NNA and MGO. Genotoxicity, enhanced after dispersant application, varied depending on the LEWAF production temperature in a different way for each oil. Impaired lengthening, abnormalities and developmental disruption were recorded, the severity of the effects varying with oil, dispersant application and LEWAF production temperature. Toxicity, only partially attributed to individual PAHs, was higher at lower LEWAF production temperatures.

Variability and distribution of parasites, pathologies and their effect on wild mussels (Mytilus sp) in different environments along a wide latitudinal span in the Northern Atlantic and Arctic Oceans.

Histopathological examination in mussels can provide useful information for the diagnosis of ecosystem health status. The distribution of parasites in mussels can be conditioned by several environmental factors, including mussels collecting sites or the presence/absence of other species necessary to complete the complex life cycle of certain parasites. Thus, these variables could not only govern the parasitic burden of mussels but also the presence of pathologies associated to parasitism. The aim of this study was to identify the histopathological alterations which could be indicative of a health status distress along a wide latitudinal span in the Northern Atlantic and Arctic Oceans in mussels of two size-classes sampled in clean and impacted sites. A latitudinal gradient is clearly observed in gamete developmental stages as northern and southern mussels presented different conditions at the same period. Furthermore, mussels of the same size in different latitudes presented differences in the reproductive cycle and the appearance of related pathologies, which probably meant the age of individuals was different. In addition, specific parasitic profiles ruled by latitudinal conditions and the settlement of mussels in the shore (horizontal/vertical) have been demonstrated to be significantly influential in the health condition of mussels. Furthermore, the present work provides the first histological description of Gymnophallus cf. bursicola parasite causing a considerable host response in Tromsø and Iceland plus the report of grave histopathological status that included high prevalence of granulocytomas in Scotland and Germany.

Toxicity to sea urchin embryos of crude and bunker oils weathered under ice alone and mixed with dispersant.

A multi-index approach (larval lenghthening and malformations, developmental disruption, and genotoxicity) was applied using sea-urchin embryos as test-organisms. PAH levels measured in the under-ice weathered aqueous fraction (UIWAF) were lower than in the low-energy water accommodated fraction (LEWAF) and similar amongst UIWAFs of different oils. UIWAFs and LEWAFs caused toxic effects, more markedly in UIWAFs, that could not be attributed to measured individual PAHs or to their mixture. Conversely, UIWAF was less genotoxic than LEWAF, most likely because naphthalene concentrations were also lower. In agreement, NAN LEWAF, the most genotoxic, exhibited the highest naphthalene levels. Dispersant addition produced less consistent changes in PAH levels and embryo toxicity in UIWAFs than in LEWAFs, and did not modify LEWAF genotoxicity. Overall, under ice weathering resulted in lowered waterborne PAHs and genotoxicity but augmented embryo toxicity, not modified by dispersant application.

Txikispora philomaios n. sp., n. g., a micro-eukaryotic pathogen of amphipods, reveals parasitism and hidden diversity in Class Filasterea.

This study provides a morphological, ultrastructural, and phylogenetic characterization of a novel micro-eukaryotic parasite (2.3-2.6 µm) infecting amphipod genera Echinogammarus and Orchestia. Longitudinal studies across two years revealed that infection prevalence peaked in late April and May, reaching 64% in Echinogammarus sp. and 15% in Orchestia sp., but was seldom detected during the rest of the year. The parasite infected predominantly hemolymph, connective tissue, tegument, and gonad, although hepatopancreas and nervous tissue were affected in heavier infections, eliciting melanization and granuloma formation. Cell division occurred inside walled parasitic cysts, often within host hemocytes, resulting in hemolymph congestion. Small subunit (18S) rRNA gene phylogenies including related environmental sequences placed the novel parasite as a highly divergent lineage within Class Filasterea, which together with Choanoflagellatea represent the closest protistan relatives of Metazoa. We describe the new parasite as Txikispora philomaios n. sp. n. g., the first confirmed parasitic filasterean lineage, which otherwise comprises four free-living flagellates and a rarely observed endosymbiont of snails. Lineage-specific PCR probing of other hosts and surrounding environments only detected T. philomaios in the platyhelminth Procerodes sp. We expand the known diversity of Filasterea by targeted searches of metagenomic datasets, resulting in 13 previously unknown lineages from environmental samples.

Influence of dispersant application on the toxicity to sea urchin embryos of crude and bunker oils representative of prospective oil spill threats in Arctic and Sub-Arctic seas.

This study deals with the toxicity assessment of crude and bunker oils representative of prospective oil spill threats in Arctic and Sub-Arctic seas (NNA: Naphthenic North-Atlantic crude oil; MGO: Marine Gas Oil; IFO: Intermediate Fuel Oil 180), alone or in combination with a third-generation dispersant (Finasol OSR52®). Early life stages of sea urchin, Paracentrotus lividus, were selected for toxicity testing of oil low-energy water accommodated fractions. A multi-index approach, including larval size increase and malformation, and developmental disruption as endpoints, was sensitive to discriminate from slight to severe toxicity caused by the tested aqueous fractions. IFO (heavy bunker oil) was more toxic than NNA (light crude oil), with MGO (light bunker oil) in between. The dispersant was toxic and further on it enhanced oil toxicity. Toxic units revealed that identified PAHs were not the main cause for toxicity, most likely exerted by individual or combined toxic action of non-measured compounds.

Biological responses and toxicopathic effects elicited in Solea senegalensis juveniles by waterborne exposure to benzo[a]pyrene.

Polycyclic aromatic hydrocarbons (PAHs) are priority contaminants in coastal and estuarine ecosystems under anthropogenic pressure. Although PAHs tend to accumulate in the sediment, toxicity for benthic flat fish such as soles may be caused by PAHs released from the sediment to the water column. Within this context, the present investigation aims at recognizing toxicopathic effects elicited after waterborne exposure to benzo[a]pyrene B[a]P, a model individual PAH compound, in juvenile Solea senegalensis. Sole juveniles were exposed to various concentrations of waterborne B[a]P for 3 and 7 days. Brain, liver, gills and gonad were the target tissues selected to determine biochemical and lysosomal biomarkers, and histopathology. Biological responses and toxicopathic effects were consistent with B[a]P concentration and exposure time. From day 3, hepatic catalase inhibition indicated potential oxidative effects of B[a]P. At day 7, contaminant exposure produced hepatic glutathione-S-transferase induction at low concentrations and inhibition at higher levels, evidencing a bell-shaped response. A clear gradient in lysosomal membrane destabilisation was observed in relation with B[a]P concentrations. Histopathological lesions were more frequent at day 7 and at higher contaminant levels. It seems that environmentally relevant waterborne concentrations of B[a]P (1000 ng/l) would suffice to cause toxicopathic effects on sole juveniles in relatively short exposure times. In agreement, the Integrative Biological Response index (IBR/n) indicated a dose-dependent decline in health condition upon exposure to B[a]P (IBR/nHighB[a]P > IBR/nMidB[a]P > IBR/nLowB[a]P > IBR/nDMSO > IBR/nControl). Overall, changes in antioxidant enzymes activity, lysosomal biomarkers and gill and liver histopathology are responsive early-warning signs of health disturbance in sole juveniles exposed to waterborne PAHs.

Cosmopolitan Distribution of Endozoicomonas-Like Organisms and Other Intracellular Microcolonies of Bacteria Causing Infection in Marine Mollusks.

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia-like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU_2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae, the genera Mycoplasma and Francisella, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens.

Biological responses and toxicopathic effects elicited in Solea senegalensis juveniles on exposure to contaminated sediments under laboratory conditions.

Whole-sediment toxicity assays contribute to elucidating the intricate association between the presence of contaminants in sediments and their toxicopathic effects in benthic fish. In the present study, Solea senegalensis juveniles were exposed under laboratory conditions to contaminated whole-sediments for 7 and 28 days. Sediments were obtained from a low to moderately polluted estuary, a highly polluted harbour and from the mixture of both field-collected sediments. Biometry data were recorded. Liver, brain, gills, and gonads were dissected out and processed to determine markers of oxidative stress, neurotoxicity and lysosomal biomarkers, and histopathology. Analyses of sediment granulometry and chemical profiles indicated different degrees of toxicity and suggested a distinct release of pollutants from each sediment in relation with their physicochemical properties. Interestingly, biological responses were in agreement with contaminant levels reported in source sediments. The most distinct toxicopathic effects were detected upon exposure to the harbour's sediment and particularly on day 28. Overall, enhanced hepatic glutathione-S-transferase activity and lysosomal enlargement were detected in all experimental groups, demonstrating a toxic effect from all sediments whilst catalase inhibition, lysosomal membrane destabilisation, changes in lysosomal content and liver histopathology were most pronounced in soles exposed to the harbour's sediment. The Integrative Biomarker Response index (IBR/n) evidenced that exposure to the three sediments caused an impact of diverse magnitude in sole health (IBR/nHarbour > IBR/nMixture > IBR/nEstuary). The magnitude of biological responses essentially depended on the presence of contaminants in source sediments, which seemed to be altered by the conditions imposed by whole-sediment toxicity assays.

Effects of elevated temperatures and cadmium exposure on stress biomarkers at different biological complexity levels in Eisenia fetida earthworms.

Several ecotoxicological studies assessed metal toxicity upon soil biota and other communities but were mainly focused on the study of a single chemical and usually under optimal conditions of temperature. Meanwhile an increasing global warming is leading to new scenarios by combining different stress factors; chemical stress and thermal stress. Presently, this study aims to assess the joint effects produced by cadmium and elevated temperature on earthworms different levels of biological complexity. Eisenia fetida earthworms were maintained at 19 °C and 26 °C and simultaneously exposed to four Cd concentrations (1.25, 2.5, 25 and 125 mg Cd/Kg soil) for 14 (Short term exposure) and 56 days (reproduction test). Endpoints were addressed at different levels of biological complexity: reproductive impairment (cocoons and juvenile productions), Cd tissue accumulation, mortality of adults, weight loss and cytotoxic effects (coelomocyte viability). In the Short term exposure, increase in temperature produced a larger accumulation of Cd. Hence, earthworms exposed to 125 mg Cd/kg soil under heat stress (26 °C) showed a two fold higher Cd accumulation comparing to those at 19 °C. Earthworms exposed to moderate-high concentrations of Cd (2.5-125 mg Cd/kg) and maintained at 26 °C showed severe weight loss and high mortality rates. The neutral red uptake capacity of coelomocytes extruded from earthworms exposed to the highest Cd concentration decreased after 14 d at 19 °C, and more markedly at 26 °C. The reproduction impairment (decreased number of cocoons) was enhanced after exposure to concentrations higher than 2.5 mg Cd/kg at 26 °C, and after exposure to 125 mg Cd/kg at 19 °C. Earthworm reproduction capability is highly vulnerable to the effect of toxicants at elevated temperatures and sublethal concentrations.

Ultrastructure, phylogeny and histopathology of two novel haplosporidians parasitising amphipods, and importance of crustaceans as hosts.

This study provides morphological, ultrastructural and phylogenetic characterization of 2 novel species of Haplosporidia (Haplosporidium echinogammari n. sp. and H. orchestiae n. sp.) infecting amphipods of the genera Echinogammarus and Orchestia collected in southwestern England. Both parasites infect the connective tissues associated with the digestive gland and the tegument, and eventually infect other organs causing disruption of host tissues with associated motor impairment and fitness reduction. Prevalence of infection varied with host species, provenance and season, being as high as 75% for individuals of E. marinus infected with H. echinogammari in June (n = 50). Although no spores were found in any of the infected amphipods examined (n = 82), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the 2 new species. Phylogenetic analysis of the new species based on the small subunit (SSU) rDNA gene placed H. echinogammari close to H. diporeiae in haplosporidian lineage C, and H. orchestiae in a novel branch within Haplosporidium. Genetic diversity of the haplosporidians infecting these and other amphipod species was evaluated and compared to morphological and ultrastructural changes to host tissues. The phylogenetic relationship of haplosporidian infections in other crustacean hosts is discussed after inclusion into the analysis of 25 novel SSU rDNA sequences obtained from crabs, isopods and crayfish.

Influence of season-depending ecological variables on biomarker baseline levels in mussels (Mytilus trossulus) from two Baltic Sea subregions.

For reliable mussel monitoring programmes based on biomarkers, regionally relevant reference values and their natural variability need to be known. The Baltic Sea exhibits high inter-regional and seasonal variability in physical factors such as salinity, temperature and primary production. The aim of this pilot study is to depict the effects of season-related environmental factors in a selected battery of biomarkers in two environmentally different subregions of the Baltic Sea to help establishing reference data for biochemical, cellular and tissue-level biomarkers. In order to achieve that, mussels were collected from reference sites in Kiel (Germany) and Tvärminne (Finland) during three seasons: summer and autumn 2016, and spring 2017. Finally, in order to characterize the ecological situation, analysis of the chemical tissue burden was performed and chlorophyll­a and particulate organic carbon concentration and temperature changes were analyzed at each sampling locality using satellite remote sensing images. An integrated biomarker response index was performed to summarize the biomarker responses of each locality and season. The biochemical endpoints showed seasonal variability regulated by temperature, food supply and reproductive cycle, while among the cellular endpoints only lipofuscin accumulation and lysosomal structural changes showed slight seasonal variation. Seasonal changes in tissue level biomarkers were observed only at the northern Baltic Sea site Tvärminne, dictated by the demanding energetic trade-off caused by reproduction. In conclusion, the characterization of the ecological variables and physico-chemical conditions at each site, is crucial to perform a reliable assessment of the effects of a hypothetical pollution scenario in the Baltic Sea. Moreover, reference levels of biomarkers and their responses to natural environmental conditions must be established.

Food-type may jeopardize biomarker interpretation in mussels used in aquatic toxicological experimentation.

To assess the influence of food type on biomarkers, mussels (Mytilus galloprovincialis) were maintained under laboratory conditions and fed using 4 different microalgae diets ad libitum for 1 week: (a) Isochrysis galbana; (b) Tetraselmis chuii; (c) a mixture of I. galbana and T. chuii; and (d) a commercial food (Microalgae Composed Diet, Acuinuga). Different microalgae were shown to present different distribution and fate in the midgut. I. galbana (≈4 µm Ø) readily reached digestive cells to be intracellularly digested. T. chuii (≈10 µm Ø and hardly digestible) was retained in stomach and digestive ducts for long times and extracellularly digested. Based on these findings, it appeared likely that the presence of large amounts of microalgal enzymes and metabolites might interfere with biochemical determinations of mussel's biomarkers and/or that the diet-induced alterations of mussels' digestion could modulate lysosomal and tissue-level biomarkers. To test these hypotheses, a battery of common biochemical, cytological and tissue-level biomarkers were determined in the gills (including activities of pyruvate kinase, phosphoenolpyruvate carboxykinase and cytochrome c oxidase) and the digestive gland of the mussels (including protein, lipid, free glucose and glycogen total content, lysosomal structural changes and membrane stability, intracellular accumulation of neutral lipids and lipofuscins, changes in cell type composition and epithelial thinning, as well as altered tissue integrity). The type of food was concluded to be a major factor influencing biomarkers in short-term experiments though not all the microalgae affected biomarkers and their responsiveness in the same way. T. chuii seemed to alter the nutritional status, oxidative stress and digestion processes, thus interfering with a variety of biomarkers. On the other hand, the massive presence of I. galbana within digestive cells hampered the measurement of cytochemical biomarkers and rendered less reliable the results of biochemical biomarkers (as these could be attributed to both the mussel and the microalgae). Research to optimize dietary food type, composition, regime and rations for toxicological experimentation is urgently needed. Meanwhile, a detailed description of the food type and feeding conditions should be always provided when reporting aquatic toxicological experiments with mussels, as a necessary prerequisite to compare and interpret the biological responses elicited by pollutants.

Prospective biomonitor and sentinel bivalve species for pollution monitoring and ecosystem health disturbance assessment in mangrove-lined Nicaraguan coasts.

This research aims at contributing to the use of Polymesoda arctata, Anadara tuberculosa, and Larkinia grandis as prospective biomonitors and sentinels, surrogate of Crassostrea rhizophorae for pollution biomonitoring in mangrove-lined coastal systems. Localities were selected along the Nicaraguan coastline in the rainy and dry seasons during 2012-2013: A. tuberculosa and L. grandis were collected in the Pacific, and P. arctata in the Caribbean. The tissue concentration of metals, polycyclic aromatic hydrocarbons (PAHs) and persistent organic pollutants (POPs) were integrated into pollution indices (chemical pollution index -CPI- and pollution load index -PLI-) and biological endpoints (flesh-condition, reproduction, histopathology and stress-on-stress) were determined as biomarkers of ecosystem health disturbance. In the Caribbean, contaminant tissue concentration was low in P. arctata, with some exceptions. Ag, As, Cd, Hg, Ni and V were mainly recorded during dry season, and PAHs and POPs (HCHs, DDTs, AHTN, PCBs and BDE85) during rainy season. Metals and PAHs were not a major threat in the study area; in contrast, high levels of HCHs and DDTs and low-to-moderate levels of musk fragrances and PBDEs were recorded. Minor differences were found in biological parameters albeit during the rainy season the LT50 values were low and seemingly associated to high PLI and CPI values. In the Pacific, the main pollutants recorded in A. tuberculosa and L. grandis were HCHs, DDTs, AHTN and PDBEs in rainy season and Cd in dry season. Although basic research is needed to understand the general biology, ecology and diseases in these Pacific species, biological endpoints comparable to those used in other sentinel bivalves are seemingly suitable biomarkers of health disturbance. Overall, Caribbean P. arctata and Pacific A. tuberculosa and L. grandis seem to be potential target species for pollution monitoring and ecosystem health disturbance assessment in mangrove-lined Nicaraguan coastal systems. Their use together with C. rhizophorae would provide opportunities for common approaches to be applied in inter-ocean countries of the Mesoamerican region.

Interactive effects of osmotic stress and burrowing activity on protein metabolism and muscle capacity in the soft shell clam Mya arenaria.

Bioturbators such as sediment-dwelling marine bivalves are ecosystem engineers that enhance sediment-water exchange and benthic-pelagic coupling. In shallow coastal areas, bivalves are exposed to frequent disturbance and salinity stress that might negatively affect their activity and physiological performance; however, the mechanisms underlying these effects are not fully understood. We investigated the effects of osmotic stress (low and fluctuating salinity) and repeated burrowing on aerobic and contractile capacity of the foot muscle (assessed by the activity of succinate dehydrogenase and myosin ATPase) as well as the levels of organic osmolytes (free amino acids) and biochemical markers of protein synthesis and proteolysis in key osmoregulatory and energy storing tissues (gills and hepatopancreas, respectively) in a common bioturbator, the soft shell clam Mya arenaria. Osmotic stress and exhaustive exercise altered the foot muscle capacity of soft shell clams and had a strong impact on protein and amino acid homeostasis in tissues not directly involved in locomotion. Acclimation to constant low salinity (5 practical salinity units) depleted the whole-body free amino acid pool and affected protein synthesis but not protein breakdown in the gill. In contrast, fluctuating (5-15) salinity increased protein breakdown rate, suppressed protein synthesis, caused oxidative damage to proteins in the gill and selectively depleted whole-body glycine pool. Clams acclimated to normal salinity (15) increased the aerobic capacity of the foot muscle upon repeated burrowing, whereas acclimation to low and fluctuating salinity reduced this adaptive muscle plasticity. Under the normal and low salinity conditions, exhaustive exercise induced protein conservation pathways (indicated by suppression of protein synthesis and catabolism), but this effect was disrupted by fluctuating salinity. These findings indicate that exhaustive exercise and osmotic stress interactively affect whole-body protein homeostasis and functional capacity of the foot muscle in soft shell clams which might contribute to reduced burrowing activity of bivalve bioturbators in osmotically challenging environments such as estuaries and shallow coastal zones.

Bioaccumulation, tissue and cell distribution, biomarkers and toxicopathic effects of CdS quantum dots in mussels, Mytilus galloprovincialis.

The bioaccumulation, cell, tissue distribution, and biological effects of 5 nm glutathione-capped CdS quantum dots (CdS QDs) in mussels was compared to bulk and aqueous Cd forms through a two-tier experimental approach. In the 1st tier, mussels were exposed for 3 d to 0.05, 0.5 and 5 mg Cd/l (QDs, bulk, aqueous), bioaccumulation, distribution and lysosomal responses were investigated. In the 2nd tier, mussels were exposed for 21 d to the same forms at the lowest effective concentration selected after Tier 1 (0.05 mg Cd/l), biomarkers and toxicopathic effects were investigated. Accumulation was comparable in QDs and aqueous Cd exposed mussels after 3 d. After 21 d, QDs exposed mussels accumulated less than mussels exposed to aqueous Cd and localised in the endo-lysosomal system and released to the alveoli lumen (21 d) after exposure to QDs and aqueous Cd. Intracellular levels of Cd increased on exposure to QDs and aqueous Cd, and to a lesser extent to bulk, and accompanied by the up-regulation of metallothionein 10 (1 d) and 20 (1, 21 d). Lysosomal membrane destabilisation depended on Cd2+ released by all forms but was marked after exposure to aqueous Cd (1 d). Toxicopathic effects (vacuolisation, loss of digestive cells and haemocytic infiltration) were evident after exposure to QDs (1 d) and aqueous Cd (21 d). Toxicity most likely depended on the ionic load resulting from Cd2+ release from the different forms of Cd; yet nanoparticle-specific effects of QDs cannot be disregarded.

Effects of dietary Pb and Cd and their combination on lysosomal and tissue-level biomarkers and histopathology in digestive gland of the land snail, Cantareus apertus (Born, 1778).

The present study was aimed at determining cell and tissue-level biomarkers and histopathological alterations in the green garden snail, Cantareus apertus (Born, 1778), exposed to different nominal dietary concentrations of Pb (25, 100 and 2500 mg Pb/kg), Cd (5, 10 and 100 mg Cd/kg) and their combination (25 mg Pb + 5 mg Cd/Kg, 100 mg Pb + 10 mg Cd/kg and 2500 mg Pb + 100 mg Cd/ kg) for 1 and 8 weeks. Lead and Cd exerted histopathological effects on the digestive gland in a dose-dependent manner and related to lysosomal and tissue-level biomarkers. The biological responses observed included digestive cell vacuolisation and numerical atrophy, calcium cell hydropic degeneration, excretory cell hypertrophy, inflammatory responses, blood vessel congestion, and disruption of the blood vessel wall and the interstitial connective tissue. Lysosomal enlargement and transient intracellular accumulation of neutral lipids and lipofuscins were also observed, together with alterations in the cell type composition and thinning of the digestive gland epithelium and with diverticular distortion. This response profile fits well with the biological effects reported after metal exposure in gastropods from other regions, as well as with data obtained in parallel studies dealing with metal bioaccumulation and intralysosomal accumulation, mortality, feeding, growth, oxidative stress and neurotoxicity exerted elicited by Pb, Cd and their mixture in green garden snails under the present experimental conditions. Consequently, C. apertus seems to be a suitable model species for the biomarker-based assessment of the biological effects of Pb and Cd, alone or in combinations, thus providing a challenging opportunity to advance in identifying suitable sentinel species for metal pollution biomonitoring and ecosystem health assessment in soil ecosystems in Northern Africa.

Chemical contamination assessment in mangrove-lined Caribbean coastal systems using the oyster Crassostrea rhizophorae as biomonitor species.

This paper aims to contribute to the use of mangrove cupped oyster, Crassostrea rhizophorae, as a biomonitor species for chemical contamination assessment in mangrove-lined Caribbean coastal systems. Sampling was carried out in eight localities (three in Nicaragua and five in Colombia) with different types and levels of contamination. Oysters were collected during the rainy and dry seasons of 2012-2013 and the tissue concentrations of metals, polycyclic aromatic hydrocarbons (PAHs), and persistent organic pollutants (POPs) were determined. Low tissue concentrations of metals (except Hg) and PAHs; moderate-to-high tissue concentrations of Hg, hexachlorocyclohexanes (HCHs), and dichlorodiphenyl-trichloroethanes (DDTs); detectable levels of chlorpyrifos, polychlorinated biphenyls (PCBs) (mainly CB28, CB118, CB138 and CB 153) and brominated diphenyl ethers 85 (BDE85); and negligible levels of musks were recorded in Nicaraguan oysters. A distinct profile of POPs was identified in Colombia, where the tissue concentrations of PCBs and synthetic musk fragrances were low to moderate, and Ag, As, Cd, Pb, and PAHs ranged from moderate to extremely high. Overall, the values recorded for HCHs, DDTs and PCBs in Nicaraguan mangrove cupped oysters greatly exceeded the reference values in tissues of C. rhizophorae from the Wider Caribbean Region, whereas only the levels of PCBs were occasionally surpassed in Colombia. Different contaminant profiles were distinguished between oysters from Nicaragua and Colombia in radar plots constructed using the main groups of contaminants (metals, PAHs, musks, PCBs, and organochlorine pesticides (OCPs)). Likewise, integrated pollution indices revealed differences in the levels of contaminants. Moreover, the profiles and levels in oyster tissues also varied with season. Thus, principal component analysis clearly discriminated Nicaraguan and Colombian localities and, especially in Colombia, seasonal trends in chemical contamination and differences amongst localities were evidenced. The geographical and environmental disparity of the studied scenarios may represent to a large extent the diversity of mangrove-lined Caribbean coastal systems and therefore the present results support the use of C. rhizophorae as suitable biomonitor species at Caribbean regional scale, where seasonal variability is a major factor controlling pollutant mobility and bioavailability.

Green garden snail, Cantareus apertus, as biomonitor and sentinel for integrative metal pollution assessment in roadside soils.

The present investigation was conceived to study, in a small scale field study, the potential of the green garden snail, Cantareus apertus, as biomonitor and sentinel for integrative metal pollution assessment in soils. For this purpose, we investigated the association between the trace metal (Cd, Pb, As, Fe, Cr, Cu, Ni, and Zn) concentrations in soil, plants (Trifolium repens), and C. apertus depending on the distance (20, 150, and 700 m) from a main roadside in Tunisia as well as between metal concentrations and biomarkers of oxidative stress, oxidative damage, and neurotoxicity in C. apertus. Results revealed a clear association between the concentration of metals such as Ni, Cu, and Zn in snail digestive gland, both amongst them and with oxidative stress and neurotoxicity biomarkers recorded in the same organ. Interestingly, Ni, Pb, and Zn occurred at the highest concentration in soil, plant, and snails and the association appeared related to the immediacy of the roadside and the concentration of these three metals tended to decrease with distance from the roadside in the soil-plant-snail system. Conversely, Cd and Cu were bioaccumulated in plants and snails but their concentrations in soil were not high and did not show a decline in concentration with distance from the roadside. After PCA analysis, PC-01 (56% of the variance) represented metal bioaccumulation and associated toxic effects in snails in the presence of high levels of metal pollution (nearby the roadside) while PC-02 (35% of the variance) represented stress induced by moderate levels of metal pollution (at intermediate distances from the roadside). The four studied sites were clearly discriminated one from each other, depending on how they are affected by traffic pollution. In summary, this field study reveals that (a) C. apertus can be used as biomonitor for metal pollution in roadside soils and as sentinel for pollution effects assessment based on biochemical biomarkers; and (b) that oxidative stress and neurotoxicity biomarkers endow with a powerful biological tool for metal pollution biomonitoring in soils, especially in combination with chemical analysis of the soil-plant-snail transfer system. Moreover, this study provides some baseline data for future impact assessments concerning trace metal pollution in Tunisia.