Letizia Marsili

Are baleen whales exposed to the threat of microplastics? A case study of the Mediterranean fin whale (Balaenoptera physalus)

Baleen whales are potentially exposed to micro-litter ingestion as a result of their filter-feeding activity. However, the impacts of microplastics on baleen whales are largely unknown. In this case study of the Mediterranean fin whale (Balaenoptera physalus), we explore the toxicological effects of microplastics on mysticetes. The study included the following three steps: (1) the collection/count of microplastics in the Pelagos Sanctuary (Mediterranean Sea), (2) the detection of phthalates in surface neustonic/planktonic samples, and (3) the detection of phthalates in stranded fin whales. A total of 56% of the surface neustonic/planktonic samples contained microplastic particles. The highest abundance of microplastics (9.63 items/m(3)) was found in the Portofino MPA (Ligurian Sea). High concentrations of phthalates (DEHP and MEHP) were detected in the neustonic/planktonic samples. The concentrations of MEHP found in the blubber of stranded fin whales suggested that phthalates could serve as a tracer of the intake of microplastics. The results of this study represent the first warning of this emerging threat to baleen whales.

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies

BackgroundOdontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers.ResultsTen commonly used housekeeping genes (HKGs) were partially sequenced in the striped dolphin (Stenella coeruleoalba) and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper) which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine 3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase 1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and β-2-microglobin (B2M) show variable expression among the studied samples and appear as less suitable reference genes for data normalization.ConclusionIn this work, we have provided essential background information for the selection of control genes in qRT-PCR studies of cetacean skin biopsies, as a molecular technique to investigate ecotoxicological hazard in marine mammals. Of 10 HKGs tested, those encoding for YWHAZ and GAPDH appear as the most reliable control genes for the normalization of qRT-PCR data in the analysis of striped dolphin skin biopsies. Potentially useful reference genes are also those encoding for ribosomal proteins L4 and S18.

The use of non-destructive biomarker in Mediterranean cetaceans: Preliminary data on MFO activity in skin biopsy

Abstract Cetaceans have been subjected to heavy anthropogenic pressure in the last century. In addition to hunting there is now the subtle threat of pollution which may be responsible for metabolic impairment and involved in stranding episodes and population decline. The need to study pollutants and their effect on cetacean populations is often in contrast with the need to protect these animals. In this paper a new method of collecting skin and hypodermic biopsies in the Fin whale (Balaenoptera physalus) and Striped dolphin (Stenella coeruleoalba) in order to analyse Mixed Function Oxidase (MFO) activity and organochlorine contents, is described. The results show marked differences between the two species. MFO activity in skin and organochlorines in blubber are higher in Striped dolphin than the Fin whale. The new method enables the main effects of exposure to lipophilic contaminants to be assessed in a nondestructive way in biomarker studies.

Trace elements in striped dolphins (Stenella coeruleoalba) from the western Mediterranean

Tissues obtained from Stenella coeruleoalba stranded along the Spanish and Italian Mediterranean coasts from 1987 to 1994 were analysed for cadmium (Cd), copper (Cu), mercury (Hg), selenium (Se) and zinc (Zn). The age, length and weight of the dolphins were recorded. Hg levels were also assayed in skin biopsies from dolphins of the same species in the waters off northeastern Spain and in the Tyrrhenian and Ligurian Seas. Levels of all elements differed in muscle of stranded dolphins from the two areas. Hg was higher in tissues from animals stranded on the Italian coasts and in skin biopsies obtained in the Tyrrhenian and Ligurian Seas, than in the respective Spanish samples. This is probably related to Hg pollution from the natural weathering of cinnabar ores in central Italy. Se and Cd levels had similar accumulation patterns to those of Hg. Accumulation of Hg and Se is explained by the existence of a detoxification pathway involving both elements, however the reason for the similar Cd trend is unclear. Geographical differences in the accumulation pattern of these elements may reflect the existence of two different populations of Stenella coeruleoalba in the western Mediterranean.

Organochlorine levels in subcutaneous blubber biopsies of fin whales (Balaenoptera physalus) and striped dolphins (Stenella coeruleoalba) from the Mediterranean Sea.

Polychlorinated biphenyls (PCBs) and DDT compounds were determined in subcutaneous blubber of fin whales (Balaenoptera physalus) and striped dolphins (Stenella coeruleoalba) from the Mediterranean Sea. From 1990 to 1993, 68 fin whale and 89 dolphin blubber biopsies were analysed. The whales were sampled while passing through the Ligurian Sea, whereas the dolphins were collected in different areas of the Mediterranean: the Ligurian, Tyrrhenian and Ionian Seas. Total PCBs and DDTs were 5.5-7.1 ppm and 4.2-9.5 ppm, respectively, in the whales and 15.5-86.0 ppm and 15.6-63.5 ppm, respectively, in the dolphins. Thirty PCB congeners were identified for each sample, IUPAC numbers 153, 138, 187, 180 and 170 being the most abundant, totalling an average of 55% of total PCBs in the whale and 60% in the dolphin. The large differences in accumulation are related to position in the food chain. In striped dolphins significant differences were found in relation to sampling site, and in fin whales in relation to sex.

CHLORINATED HYDROCARBON (HCB, DDTs AND PCBs LEVELS IN CETACEANS STRANDED ALONG THE ITALIAN COASTS: AN OVERVIEW

Concentrations of HCB, DDTs and PCBs in the tissues andorgans of cetaceans ( Stenella coeruleoalba, Tursiops truncatus, Balaenoptera physalus, Steno bredanensis}, Grampus griseus} and Globicephala melaena) strandedalong the Italian coasts in the period 1987–1993 are reported. The values are compared between speciesand between specimens of the samespecies. Chlorinated hydrocarbon (CH) levels were found toincrease in relation to the quantity andtype of lipids in each tissue and organ. Differences inaccumulation encountered in the differentspecies are principally due to different feeding habitats.Remarkable differences found between malesand females of each species confirm that during gestation andlactation, females undergo disintoxication by passing much of their total burden of CHs to their young.

Biomarkers for endocrine disruptors in three species of Mediterranean large pelagic fish

The hypothesis that Mediterranean top predator species, such as large pelagic fish, are potentially at risk due to endocrine disrupting chemicals (EDCs), is investigated. The potential estrogenic effects of PHAHs in three fish species of commercial interest, the top predators bluefin tuna (Thunnus thynnus thynnus), swordfish (Xiphias gladius), and Mediterranean spearfish (Tetrapturus belone), were investigated using vitellogenin (Vtg), zona radiata proteins (Zrp) and mixed function oxidases (EROD, BPMO) as diagnostic tools. High induction of Vtg and Zrp was detected by western blot and ELISA techniques in adult males of X. gladius and T. thynnus thynnus, suggesting that these species are at high toxicological risk in the Mediterranean sea. Comparison of BPMO and EROD activities in the three species indicated, both in male and female, much higher MFO activity in bluefin tuna. This data suggests high exposure of this species to lipophilic xenobiotic contaminants in the Mediterranean environment.

Use of nondestructive biomarkers and residue analysis to assess the health status of endangered species of pinnipeds in the south-west Atlantic

Total porphyrins, EROD, PROD, BROD, organochlorines, PAHs and total Hg were determined in specific organs and tissues of pinnipeds collected from the south-west Atlantic. The results confirm the existence of metabolic imbalance between high toxifying and low detoxifying potential in pinnipeds, as reported in other marine mammals. The non destructive biomarker approach based on the analysis of metabolic responses (porphyrins) and residues (DDTs, PCBs, PAHs and Hg) in non-invasive samples (excreta and fur) appear to be a useful tool for the hazard assessment of endangered species.

The use of non destructive biomarkers in the study of marine mammals.

Marine mammals have been subject to heavy anthropogenic pressure by direct killing and chemical pollution all over the world. Most studies of contamination and biomarker responses in marine mammals have been conducted using animals killed by hunting out of a total of 12 cetacean species studied, 45 of the specimens were obtained by sacrificing the animal; out of a total of eight pinniped species studied, 40 of the specimens were obtained by killing. The development of a series of non destructive techniques to evaluate biomarker responses and residue levels is recommended for the hazard assessment and conservation of endangered species of marine mammals. Here we review the current status of the non destructive biomarker approach in marine mammals, describing the biological materials available for non destructive tests in stranded brain, liver, blood, skin, subcutaneous blubber, muscle and fur and free ranging animals blood, skin biopsy, fur and faeces and the respective biomarker techniques mixed function oxidase activity and DNA damage in skin biopsy samples; porphyrins in faeces and fur; esterases, porphyrins, clinical biochemical parameter, vitamin A and micronuclei in blood samples. Residue analysis can be carried out in the various biological materials. We also report the results of applying this methodological approach to cetaceans minke whale Balaenoptera acutorostrata, fin whale-- Balaenoptera physalus, beluga whale-- Delphinapterus leucas, short finned pilot whale-- Globicephala macrorhynchus, harbour porpoise -- Phocoena phocoena, Rissos dolphin-- Risso s Grampus griseus, Dall s porpoise-- Phocoenoides dalli dalli, melon headed whale-- Peponocephala electra, bottlenose dolphin -- Tursiops truncatus, striped dolphin-- Stenella coeruleoalba, spinner dolphin-- Stenella longirostris, killer whale-- Orcinus orca and pinnipeds northern fur seal- Callorhinus ursinus, hooded seal-- Cystophora cristata, grey seal-- Halichoerus grypus, harbour seal-- Phoca vitulina, ringed seal-- Phoca hispida, harp seal-- Phoca groenlandica, ribbon seal-- Phoca fasciata, largha seal- Phoca largha, southern sea lion-- Otaria flavescens in field studies for prognostic and diagnostic purposes.Marine mammals have been subject to heavy anthropogenic pressure by direct killing and chemical pollution all over the world. Most studies of contamination and biomarker responses in marine mammals have been conducted using animals killed by hunting out of a total of 12 cetacean species studied, 45 of the specimens were obtained by sacrificing the animal; out of a total of eight pinniped species studied, 40 of the specimens were obtained by killing. The development of a series of non destructive techniques to evaluate biomarker responses and residue levels is recommended for the hazard assessment and conservation of endangered species of marine mammals. Here we review the current status of the non destructive biomarker approach in marine mammals, describing the biological materials available for non destructive tests in stranded brain, liver, blood, skin, subcutaneous blubber, muscle and fur and free ranging animals blood, skin biopsy, fur and faeces and the respective biomarker techniques mixed function ...

Skin biopsy of Mediterranean cetaceans for the investigation of interspecies susceptibility to xenobiotic contaminants

Various studies on Mediterranean cetaceans have revealed bioaccumulation of contaminants such as organochlorines (OCs) and heavy metals. The susceptibility of these animals to organic pollutants and the relationship between bioaccumulation and population decline (as in the case of Delphinus delphis) are unexplored fields. In this study, we used a non-destructive approach (skin biopsy) to explore OC bioaccumulation processes and mixed-function oxidase activity (BPMO) in four species of cetaceans: striped dolphin (Stenella coeruleoalba), bottlenose dolphin (Tursiops truncatus), common dolphin (D. delphis) and fin whale (Balaenoptera physalus). Significant differences in BPMO induction and OC levels were found between odontocetes and mysticetes, the former having mixed-function oxidase activities four times higher than the latter, binding with levels of OCs one order of magnitude higher in odontocetes. A significant correlation (P < 0.05) between BPMO activities and OC levels was found in B. physalus. In an ongoing project, fibroblast cultures have been used as an alternative in vitro method of evaluating interspecies susceptibility to contaminants such as OCs and polycyclic aromatic hydrocarbons (PAHs). These results suggest that cetacean skin biopsies are a powerful non-invasive tool for assessing ecotoxicological risk to Mediterranean marine mammals species.