Stable isotope ratios of carbon, nitrogen and sulphur and mercury concentrations as descriptors of trophic ecology and contamination sources of Mediterranean whales.

The Mediterranean Sea remains a complex system for mercury (Hg) cycling and accumulation in marine vertebrates. The extremely high levels these animals present demand for an urgent understanding of such processes and the development of new analytical techniques that go beyond the simple contamination monitoring. It was often proposed that prey selection or habitat use may affect Hg contamination in animals; however, it was never possible to measure which factor influences more rates and pathways of contamination. In this paper, we directly integrate toxicological information (Hg levels) and ecological tracers (stable isotopes of C, N and S) into a common data analysis framework (isotopic niches), with the aim of quantifying the influence of species' trophic behaviour on Hg contamination. The analysis was conducted on skin biopsies of fin whales Balaenoptera physalus, long-finned pilot whales Globicephala melas and sperm whales Physeter microcephalus. Their different trophic modes and residency in the area make them model species for the analysis of Hg accumulation along NWMS food webs. We measured Total Hg (T-Hg) concentrations through absorbance spectrometry with the DMA80 Milestone. Carbon, nitrogen and sulphur isotope compositions were measured via mass spectrometry in an IRMS coupled to an Elemental Analyser (EA) Isoprime. Comparison of ecological and contamination niches allowed to explain Hg accumulation in Mediterranean marine predators. Factors such as food web complexity, trophic position, hunting distribution or habitat use (e.g., foraging depth) did not influence Hg exposure. It is rather the selection of prey type, which determines the range of potential Hg sources and as a consequence the rates of accumulation in whales' tissues. A generalist piscivorous species such as the pilot whales will bioaccumulate more Hg than specialised sperm whales feeding mostly on cephalopods.

Strontium in fin whale baleen: A potential tracer of mysticete movements across the oceans?

Strontium is a metal broadly distributed in oceanic waters, where its concentrations follow gradients mainly driven by oceanographic and biological factors. Studies on terrestrial vertebrates show that Sr can accumulate in mammalian hair in amounts mainly related to the external environment, a property that has been scarcely investigated in aquatic mammals. Cetaceans are marine mammals whose skin is generally hairless, but the species belonging to the mysticete group feed through a filtering apparatus made of keratinous baleen plates that, like hair, grow continuously. During their annual latitudinal migrations, mysticetes cross water masses with variable chemo-physical characteristics that may be reflected in these tissues. In the present study, baleen plates were sampled from 10 fin whales obtained from NW Spain (N = 5) and SW Iceland (N = 5) to investigate Sr concentrations along the plates growth axis. Samples were taken longitudinally at regular 1 cm-intervals on each plate. Sr concentrations, determined through mass spectrometry, ranged from 5 to 40 mg kg-1 and increased from proximal to distal positions along plates. These results suggest a progressive adsorption of Sr on the plate surface, a process that also occurs in mammalian hair. Increasing trends were similar in the two regions but overall concentrations were significantly higher in NW Spain, reflecting different Sr baseline concentrations in the two areas and indicating isolation between the two whale populations. Some oscillations in Sr longitudinal trends were also detected, likely indicating that whales migrate across water masses with different Sr baselines. These results suggest that Sr concentrations in keratinous tissues of marine mammals can be used as ecological tracers of their migrations and habitat use.

Metal Levels in Whales from the Gulf of Maine: A One Environmental Health approach.

One Environmental Health has emerged as an important area of research that considers the interconnectedness of human, animal and ecosystem health with a focus on toxicology. The great whales in the Gulf of Maine are important species for ecosystem health, for the economies of the Eastern seaboard of the United States, and as sentinels for human health. The Gulf of Maine is an area with heavy coastal development, industry, and marine traffic, all of which contribute chronic exposures to environmental chemicals that can bioaccumulate in tissues and may gradually diminish an individual whale's or a population's fitness. We biopsied whales for three seasons (2010-2012) and measured the levels of 25 metals and selenium in skin biopsies collected from three species: humpback whales (Megaptera novaeangliae), fin whales (Balaenoptera physalus), and a minke whale (Balaenoptera acutorostrata). We established baseline levels for humpback and fin whales. Comparisons with similar species from other regions indicate humpback whales have elevated levels of aluminum, chromium, iron, magnesium, nickel and zinc. Contextualizing the data with a One Environmental Health approach finds these levels to be of potential concern for whale health. While much remains to understand what threats these metal levels may pose to the fitness and survival of these whale populations, these data serve as a useful and pertinent start to understanding the threat of pollution.

Bone as a surrogate tissue to monitor metals in baleen whales.

Metals are massively deposited in the marine environment through direct emissions or atmospheric dry and wet depositions, a process since long enhanced by human activities. Metal contamination in the marine organisms has been increasingly investigated, but most research focuses on few tissues, elements and species considered indicative. Baleen whales have been scarcely studied in this respect. Here we contribute to the fragmented knowledge on this field examining the concentrations of zinc, copper, lead, titanium and strontium in the bone of fin whales (Balaenoptera physalus) from NW Spain and W Iceland. Bone was selected because it is a tissue commonly available in archival historic collections, and it is therefore useful to examine long-term trends in metal pollution. We tested differences between populations and we investigated age- and sex-related accumulation trends, as well as the occurrence of placental transfer. Sr concentrations and Pb accumulation rates with age were significantly higher in individuals from NW Spain than in those from W Iceland. Placental transfer occurred, at different levels, for all metals: as a result fetuses showed significantly higher Cu, Pb and Zn concentrations than adults. After birth, only Zn and Pb concentrations significantly increased with age. Through this study we contributed to fill some gaps in the knowledge regarding metal contamination in marine mammals, and we concluded that bone can be a suitable surrogate tissue to monitor a number of trace elements, provided that dissimilarities in tissue-specific deposition are taken into account when comparing concentrations from different tissues.

Fin whales and microplastics: The Mediterranean Sea and the Sea of Cortez scenarios.

The impact that microplastics have on baleen whales is a question that remains largely unexplored. This study examined the interaction between free-ranging fin whales (Balaenoptera physalus) and microplastics by comparing populations living in two semi-enclosed basins, the Mediterranean Sea and the Sea of Cortez (Gulf of California, Mexico). The results indicate that a considerable abundance of microplastics and plastic additives exists in the neustonic samples from Pelagos Sanctuary of the Mediterranean Sea, and that pelagic areas containing high densities of microplastics overlap with whale feeding grounds, suggesting that whales are exposed to microplastics during foraging; this was confirmed by the observation of a temporal increase in toxicological stress in whales. Given the abundance of microplastics in the Mediterranean environment, along with the high concentrations of Persistent Bioaccumulative and Toxic (PBT) chemicals, plastic additives and biomarker responses detected in the biopsies of Mediterranean whales as compared to those in whales inhabiting the Sea of Cortez, we believe that exposure to microplastics because of direct ingestion and consumption of contaminated prey poses a major threat to the health of fin whales in the Mediterranean Sea.

Chromium Is Elevated in Fin Whale (Balaenoptera physalus) Skin Tissue and Is Genotoxic to Fin Whale Skin Cells.

Hexavalent chromium (Cr(VI)) is present in the marine environment and is a known carcinogen and reproductive toxicant. Cr(VI) is the form of chromium that is well absorbed through the cell membrane. It is also the most prevalent form in seawater. We measured the total Cr levels in skin biopsies obtained from healthy free-ranging fin whales from the Gulf of Maine and found elevated levels relative to marine mammals in other parts of the world. The levels in fin whale biopsies ranged from 1.71 to 19.6 µg/g with an average level of 10.07 µg/g. We also measured the cytotoxicity and genotoxicity of Cr(VI) in fin whale skin cells. We found that particulate and soluble Cr(VI) are both cytotoxic and genotoxic to fin whale skin cells in a concentration-dependent manner. The concentration range used in our cell culture studies used environmentally relevant concentrations based on the biopsy measurements. These data suggest that Cr(VI) may be a concern for whales in the Gulf of Maine.

The fin whale, a marine top consumer, exposes strengths and weaknesses of the use of fluoride as ecological tracer.

Fluoride is retained in bone tissues of animals and its availability in the environment varies between regions according to natural and anthropogenic sources. These properties suggest this element as a suitable tracer of origin, distribution or movements of animals. In marine environments, krill builds-up fluoride concentrations that are transferred to its predators. In this study we examine the ability of bone fluoride concentrations to discriminate two separate populations of a krill consumer, the fin whale. Background levels of the sampling areas (Western Iceland and North-Western Spain) were determined through the analysis of krill samples. As expected, due to the high load of volcanic-derived fluoride in Icelandic waters, krill from W Iceland showed much higher fluoride concentrations than that from NW Spain. Concentrations in whales' bone were correlated with sex and age, increasing linearly with age in females and showing significantly lower values and a different age-related pattern of accumulation in males. Fluoride concentrations in whales' bone were much higher than in krill, indicating accumulation of the element but, rather unexpectedly, the area of origin had no influence on concentrations. This apparent contradiction may be explained either by the integration in bone of food consumed in other areas, or by the activation of homeostatic responses at very high levels of fluoride exposure. It is concluded that fluoride can be a useful tracer only if age and sex data are integrated into the analysis, year-round information on diet is available and/or the investigated population is exposed to mild levels of this element.

Large filter feeding marine organisms as indicators of microplastic in the pelagic environment: the case studies of the Mediterranean basking shark (Cetorhinus maximus) and fin whale (Balaenoptera physalus).

The impact of microplastics (plastic fragments smaller than 5 mm) on large filter feeding marine organisms such as baleen whales and sharks are largely unknown. These species potentially are ingesting micro-litter by filter feeding activity. Here we present the case studies of the Mediterranean fin whale (Balaenoptera physalus) and basking shark (Cetorhinus maximus) exploring the toxicological effects of microplastics in these species measuring the levels of phthalates in both species. The results show higher concentration of MEHP in the muscle of basking shark in comparison to fin whale blubber. These species can be proposed as indicators of microplastics in the pelagic environment in the implementation of Descriptor 8 and 10 of the EU Marine Strategy Framework Directive (MSFD).

Discrimination of stable isotopes in fin whale tissues and application to diet assessment in cetaceans.

RATIONALE: In stable isotope research, the use of accurate, species-specific diet-tissue discrimination factors (i.e., Δ(13)C and Δ(15)N) is central to the estimation of trophic position relative to primary consumers and to the identification of the dietary sources of an individual. Previous research suggested that the diet of fin whales from the waters off northwestern Spain is overwhelmingly based on krill, thus permitting reliable calculation of discrimination values in this wild population. METHODS: After confirming that the stable isotope ratios (δ(13)C and δ(15)N values) in muscle from 65 aged fin whales remained constant through age classes (4-65 years), the signatures were determined in muscle, bone protein, skin, liver, kidney, baleen plates and brain, as well as food (krill), from a subset of individuals to calculate discrimination factors. Signatures were determined by means of elemental analysis isotope ratio mass spectrometry (EA-IRMS) using a ThermoFinnigan Flash 1112. RESULTS: The isotopic values remained constant regardless of age. The mean Δ(15)N values between krill and whale tissues ranged from 2.04 in bone protein to 4.27‰ in brain, and those of Δ(13)C ranged from 1.28 in skin to 3.11‰ in bone protein. This variation was consistent with that found in other groups of mammals, and is attributed to variation in tissue composition and physiology. CONCLUSIONS: Because discrimination factors are relatively constant between taxonomically close species, the results here obtained may be reliably extrapolated to other cetaceans to improve dietary reconstructions. The skin discrimination factors are of particular relevance to monitoring diet through biopsies or other non-destructive sampling methods. The large difference in bone protein discrimination factors from those of other tissues should be taken into consideration when bone collagen is used to determine trophic level or to assess diet in paleodietary isotopic reconstructions.

Fetal and early post-natal mineralization of the tympanic bulla in fin whales may reveal a Hitherto undiscovered evolutionary trait.

The evolution of the cetacean skeleton followed a path that differentiated this group from other terrestrial mammals about 50 million years ago [1], and debate is still going on about the relationships between Cetacea and Artiodactyla [2], [3], [4]. Some skeletal traits of the basilosaurids (the more advanced forms of Archaeocetes), such as the expansion of the peribullary air sinuses, dental modification and vertebral size uniformity [5] are maintained and further emphasized also in contemporary odontocetes and mysticetes. Using Dual-Energy X-Ray Absorptiometry here we report that the deposition of bone mineral in fetal and newborn specimens of the fin whale Balaenoptera physalus is remarkably higher in the bulla tympanica than in the adjacent basal skull or in the rest of the skeleton. Ossification of the tympanic bulla in fetal Artiodactyla (bovine, hippopotamus) is minimal, becomes sensible after birth and then progresses during growth, contrarily to the precocious mineralization that we observed in fin whales. Given the importance of the ear bones for the precise identification of phylogenetic relationship in therian evolution [6], this feature may indicate a specific evolutionary trait of fin whales and possibly other cetacean species or families. Early mineralization of the tympanic bulla allows immediate sound conduction in the aquatic medium and consequently holds potential importance for mother-calf relationship and postnatal survival.

Methoxylated polybrominated diphenyl ethers (MeO-PBDEs) are major contributors to the persistent organobromine load in sub-Arctic and Arctic marine mammals, 1986-2009.

A selection of MeO-BDE and BDE congeners were analyzed in pooled blubber samples of pilot whale (Globicephala melas), ringed seal (Phoca hispida), minke whale (Balaenoptera acutorostrata), fin whale (Balaenoptera physalus), harbor porpoise (Phocoena phocoena), hooded seal (Cystophora cristata), and Atlantic white-sided dolphin (Lagenorhynchus acutus), covering a time period of more than 20 years (1986-2009). The analytes were extracted and cleaned-up using open column extraction and multi-layer silica gel column chromatography. The analysis was performed using both low resolution and high resolution GC-MS. MeO-PBDE concentrations relative to total PBDE concentrations varied greatly between sampling periods and species. The highest MeO-PBDE levels were found in the toothed whale species pilot whale and white-sided dolphin, often exceeding the concentration of the most abundant PBDE, BDE-47. The lowest MeO-PBDE levels were found in fin whales and ringed seals. The main MeO-BDE congeners were 6-MeO-BDE47 and 2'-MeO-BDE68. A weak correlation only between BDE47 and its methoxylated analog 6-MeO-BDE47 was found and is indicative of a natural source for MeO-PBDEs.

Organochlorine pesticides and polychlorinated biphenyls in fin whales (Balaenoptera physalus) from the Gulf of California.

The present study reports unique data on concentrations of several classes of organochlorine pesticides and polychlorinated biphenyls in blubber biopsies from healthy living fin whales (Balaenoptera physalus) from the Gulf of California, Mexico, one of the most isolated and unstudied population in the world. OC levels in this population were generally lower than levels reported in fin whales from other regions. The rank order of OCs were SigmaDDTs (range from 300 to 2400 ng g(-1) lw) > SigmaPCBs (range from 40 to 290 ng g(-1) lw) > SigmaHCHs (range from or = SigmaCHLORs (from < LOQ to 100 ng g(-1) lw). The most abundant OC pesticide measured was the DDT metabolite, p,p'-DDE. The PCBs 138, 153, and 180 were the most abundant PCBs congeners found in the fin whales samples. Males had significant higher concentrations of SigmaOC, SigmaDDTs and SigmaPCBs than females (P < 0.05), although the p,p'-DDE/SigmaDDTs ratios were similar between the sexes. Although the OC concentrations found in this population were generally below the levels that would be expected to cause deleterious health effects, the maximum values observed (2700 ng g(-1) lw) in some animals were higher than those associated with reproductive effects in whales. Given the small population size and highly isolated characteristics of Gulf of California fin whales, health effects in individuals could readily translate into population-level effects. Future research on this topic will be necessary to better understand the role that these compounds may have on the health of this population.