Population structure, habitat use and conservation of short-finned pilot whales Globicephala macrorhynchus in the Archipelago of Madeira

This thesis provides information on the grouping structure, survival, abundance, dive characteristics and habitat preferences of short-finned pilot whales occurring in the oceanic archipelago of Madeira (Portugal, NE Atlantic), based on data collected between 2001-2011, and contributes for its conservation. Photo-identification methods and genetic analyses demonstrated that there is a large degree of variability in site fidelity, including resident, regular visitor and transient whales, and that they may not be genetically isolated. It is proposed that the pilot whales encountered in Madeira belong to a single population encompassing several clans, possibly three clans of island-associated (i.e. resident and regular visitor) whales and others of transients, each containing two to three matrilineal pods. Mark-recapture methods estimated that the island-associated community is composed of less than 150 individuals and that their survival rate is within the range of other long-lived cetacean species, and that around 300 whales of different residency patterns uses the southern area of the island of Madeira from mid-summer to mid-autumn. No significant trend was observed between years. Time-depth recorders deployed in adult whales during daytime revealed that they spend over ¾ of their time at the surface, that they have a low diving rate, and that transient whales also forage during their passage. The analyses of visual data collected from nautical and aerial line-transect surveys indicate a core/preferred habitat area in the south-east of the island of Madeira. That area is used for resting, socializing, foraging, breeding, calving and birthing. Thus, that area should be considered as an important habitat for this species, at least seasonally (during autumn) when the species is more abundant, and included in conservation plans. No direct threat needing urgent measures was identified, although the impact of some activities like whale-watching or marine traffic should be assessed.

Development of a novel microextraction by packed sorbent-based approach followed by ultrahigh pressure liquid chromatography as a powerful technique for quantification phenolic constituents of biological interest in wines

A novel analytical approach, based on a miniaturized extraction technique, the microextraction by packed sorbent (MEPS), followed by ultrahigh pressure liquid chromatography (UHPLC) separation combined with a photodiode array (PDA) detection, has been developed and validated for the quantitative determination of sixteen biologically active phenolic constituents of wine. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (linearity, sensitivity, selectivity, precision, accuracy), experiments are included to assess the effect of the important experimental parameters on the MEPS performance such as the type of sorbent material (C2, C8, C18, SIL, and M1), number of extraction cycles (extract-discard), elution volume, sample volume, and ethanol content, were studied. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (250 μL) in five extraction cycle and in a short time period (about 5 min for the entire sample preparation step). The wine bioactive phenolics were eluted by 250 μL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a HSS T3 analytical column (100 mm × 2.1 mm, 1.8 μm particle size) using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and methanol (eluent B) in the gradient elution mode (10 min of total analysis). The method gave satisfactory results in terms of linearity with r2-values > 0.9986 within the established concentration range. The LOD varied from 85 ng mL−1 (ferulic acid) to 0.32 μg mL−1 ((+)-catechin), whereas the LOQ values from 0.028 μg mL−1 (ferulic acid) to 1.08 μg mL−1 ((+)-catechin). Typical recoveries ranged between 81.1 and 99.6% for red wines and between 77.1 and 99.3% for white wines, with relative standard deviations (RSD) no larger than 10%. The extraction yields of the MEPSC8/UHPLC–PDA methodology were found between 78.1 (syringic acid) and 99.6% (o-coumaric acid) for red wines and between 76.2 and 99.1% for white wines. The inter-day precision, expressed as the relative standard deviation (RSD%), varied between 0.2% (p-coumaric and o-coumaric acids) and 7.5% (gentisic acid) while the intra-day precision between 0.2% (o-coumaric and cinnamic acids) and 4.7% (gallic acid and (−)-epicatechin). On the basis of analytical validation, it is shown that the MEPSC8/UHPLC–PDA methodology proves to be an improved, reliable, and ultra-fast approach for wine bioactive phenolics analysis, because of its capability for determining simultaneously in a single chromatographic run several bioactive metabolites with high sensitivity, selectivity and resolving power within only 10 min. Preliminary studies have been carried out on 34 real whole wine samples, in order to assess the performance of the described procedure. The new approach offers decreased sample preparation and analysis time, and moreover is cheaper, more environmentally friendly and easier to perform as compared to traditional methodologies.