Sex and sex-related differences in gamete development progression impinge on biomarker responsiveness in sentinel mussels.

In marine pollution monitoring, the biomarkers recorded in sentinel organisms are influenced by natural confounding factors that may jeopardise their interpretation. Among these confounding factors, little is known about the influence of sex along the annual reproductive cycle. The present investigation aims at contributing to understand how sex and sex-related differences in gamete development progression impinge on biomarker baseline values and on biomarker responsiveness to pollution in sentinel mussels. Mussels (Mytilus galloprovincialis) were collected from a relatively clean locality and from a chronically polluted site in the Basque Coast (Bay of Biscay) in January, April, August and November. Sex and gametogenesis stages were determined for each mussel. Tissue concentration of metals and PAHs was analysed. A battery of biomarkers was investigated: cytochrome c oxidase, pyruvate kinase and phosphoenolpyruvate carboxykinase enzyme activities; levels of protein carbonyls, malondialdehyde and 4-hydroxy-2-nonenal; lysosomal enlargement and membrane stability; intracellular neutral lipid accumulation; cell type composition and thinning of the digestive gland epithelium; and survival-in-air. Sex- and reproductive stage-related differences were found in bioaccumulation and in the values and responsiveness of most of the biomarkers. However, the patterns of sex-related differences were not consistent across all biomarkers. The differences in the biomarker responses between females and males also depended on the season, reflecting the progression of the gametogenesis cycle. Thus, selecting mussels of one specific sex does not seem to be a crucial requisite to carry out biomarker-based monitoring; yet, it is highly recommended to identify sex condition and gamete developmental stage of each mussel to test for the potentially confounding effects of sex, reproductive status and sex-related variability along the reproductive cycle.

Enhanced discrimination of basophilic cells on mussel digestive gland tissue sections by means of toluidine-eosin staining.

Changes in the cell type composition of the digestive gland epithelium constitute a common and recognized biological response to stress in mussels. Usually, these changes are identified as alterations in the relative proportion of basophilic cells, determined in tissue sections stained with hematoxylin-eosin (H&E) and measured in terms of volume density of basophilic cells (VvBAS) after stereological quantification. However, the identification and discrimination of basophilic cells may be a difficult issue, even for a trained operator, especially when, in circumstances of environmental stress, basophilic cells lose their basophilia and the perinuclear area of digestive cells gains basophilia. Thus, the present study was aimed at exploring the best available practices (BAPs) to identify and discriminate basophilic cells on tissue sections of mussel digestive gland. In a first step, a thorough screening of potentially suitable staining methods was carried out; the final selection included several trichrome staining methods and some of their variants, as well as toluidine-based stains. Next, the sample processing (fixation/dehydration steps) was optimized. Toluidine-eosin (T&E) staining after fixation in 4% formaldehyde at 4 °C for 24 h was considered the BAP to identify and discriminate basophilic cells in the digestive gland of mussels. Using the mussel Mytilus galloprovincialis as a target organism, this approach was successfully applied to quantify VvBAS values after automated image analysis and compared with the conventional H&E staining in different field and laboratory tests. It is worth noting that VvBAS values were always higher after T&E staining than after H&E staining, apparently because discrimination of basophilic cells was enhanced. Thus, until more data are available, any comparison with VvBAS values obtained in previous studies using H&E staining must be done cautiously. Finally, the T&E staining was successfully used to discriminate basophilic cells in tissue sections of other marine molluscs of ecotoxicological interest, including Mytilus edulis, Mytilus trossulus, Crassostrea gigas and Littorina littorea.

The influence of short-term experimental fasting on biomarker responsiveness in oil WAF exposed mussels.

Mussels are widely used in toxicological experimentation; however, experimental setups are not standardized yet. Although there is evidence of changes in biomarker values during food digestion and depending on the mussel nutritive status, the mode of feeding differs among toxicological experiments. Typically, mussels are fed with different diets in different long-term experiments, while fasting is the most common approach for short-term studies. Consequently, comparisons among experiments and reliable interpretations of biomarker results are often unfeasible. The present investigation aimed at determining the influence of fasting (against feeding with Isochrysis galbana) on biomarkers and their responsiveness in mussels exposed for 96 h to the water accommodated fraction (WAF) of a heavy fuel oil (0%, 6.25%, 12.5% and 25% WAF in sea water). PAH tissue levels in digestive gland and a battery of biomarkers were compared. WAF exposure led to decrease of cytochrome-C-oxidase activity, modulated glutathione-S-transferase activity, augmented lipid peroxidation, inhibited acetyl cholinesterase (AChE) activity, and led to lysosomal enlargement (VvLYS and S/VLYS) and membrane destabilisation, lipofuscin accumulation, and histopathological alterations (VvBAS, MLR/MET and CTD ratio) in the digestive gland epithelium; and were integrated as IBR/n (biological response index). Overall, no significant changes were recorded in AChE activity, S/VLYS and CTD ratio in any experimental treatment, while all the other biomarkers showed significant changes depending on the fasting/feeding condition, the exposure to WAF and/or their interaction. As a result, the integrated biomarker index IBR/n was higher at increasing WAF exposure levels both in fasted and fed mussels albeit the response was more marked in the latter. The response profiles were qualitatively similar between fasted and fed mussels but quantitatively more pronounced in fed mussels, especially upon exposure to the highest concentration (25% WAF). Therefore, it is highly recommended that mussels are also supplied with food during short-term, like during long-term toxicological experiments. This practice would avoid the interference of fasting with biological responses elicited by the tested chemicals and allow for reliable comparison with data obtained in long-term experiments and monitoring programmes.

Multifunctional PLA-PHB/cellulose nanocrystal films: processing, structural and thermal properties.

Cellulose nanocrystals (CNCs) synthesized from microcrystalline cellulose by acid hydrolysis were added into poly(lactic acid)-poly(hydroxybutyrate) (PLA-PHB) blends to improve the final properties of the multifunctional systems. CNC were also modified with a surfactant (CNCs) to increase the interfacial adhesion in the systems maintaining the thermal stability. Firstly, masterbatch pellets were obtained for each formulation to improve the dispersion of the cellulose structures in the PLA-PHB and then nanocomposite films were processed. The thermal stability as well as the morphological and structural properties of nanocomposites was investigated. While PHB increased the PLA crystallinity due to its nucleation effect, well dispersed CNC and CNCs not only increased the crystallinity but also improved the processability, the thermal stability and the interaction between both polymers especially in the case of the modified CNCs based PLA-PHB formulation. Likewise, CNCs were better dispersed in PLA-CNCs and PLA-PHB-CNCs, than CNC.

Contact mechanics at nanometric scale using nanoindentation technique for brittle and ductile materials.

In the last years, Nanoindentation or Instrumented Indentation Technique has become a powerful tool to study the mechanical properties at micro/nanometric scale (commonly known as hardness, elastic modulus and the stress-strain curve). In this review, the different contact mechanisms (elastic and elasto-plastic) are discussed, the recent patents for each mechanism (elastic and elasto-plastic) are summarized in detail, and the basic equations employed to know the mechanical behaviour for brittle and ductile materials are described.

Electrochemical deposition of ZnO nanostructures. mechanism of growth.

The growth kinetics of ZnO nanorods were studied by means of an electrochemical deposition process on ITO electrodes submerged in an aqueous solution of 5 mM ZCl+0.1 M KCl at 80 degrees C. The stages of the growth kinetics showed a mechanism of formation of nano-spheres which with time were transformed into hexagonal crystals of ZnO. Cathodic current densities of 0.1 to 1 mAcm2 formed hexagonal columns of 800 nm thickness while stronger currents formed a high-density layer of cylindrical nanorods 80 nm in diameter. An amorphous barrier layer was found between the surface of the electrode and the base of the nano-columns. The conditions assayed showed the possibility of adjusting the morphological characteristics of the layer in order to obtain different properties in different applications.