S. Maltese

Pacific Ocean–Wide Profile of CYP1A1 Expression, Stable Carbon and Nitrogen Isotope Ratios, and Organic Contaminant Burden in Sperm Whale Skin Biopsies

Background Ocean pollution affects marine organisms and ecosystems as well as humans. The International Oceanographic Commission recommends ocean health monitoring programs to investigate the presence of marine contaminants and the health of threatened species and the use of multiple and early-warning biomarker approaches. Objective We explored the hypothesis that biomarker and contaminant analyses in skin biopsies of the threatened sperm whale (Physeter macrocephalus) could reveal geographical trends in exposure on an oceanwide scale. Methods We analyzed cytochrome P450 1A1 (CYP1A1) expression (by immunohistochemistry), stable nitrogen and carbon isotope ratios (as general indicators of trophic position and latitude, respectively), and contaminant burdens in skin biopsies to explore regional trends in the Pacific Ocean. Results Biomarker analyses revealed significant regional differences within the Pacific Ocean. CYP1A1 expression was highest in whales from the Galapagos, a United Nations Educational, Scientific, and Cultural Organization World Heritage marine reserve, and was lowest in the sampling sites farthest away from continents. We examined the possible influence of the whales’ sex, diet, or range and other parameters on regional variation in CYP1A1 expression, but data were inconclusive. In general, CYP1A1 expression was not significantly correlated with contaminant burdens in blubber. However, small sample sizes precluded detailed chemical analyses, and power to detect significant associations was limited. Conclusions Our large-scale monitoring study was successful at identifying regional differences in CYP1A1 expression, providing a baseline for this known biomarker of exposure to aryl hydrocarbon receptor agonists. However, we could not identify factors that explained this variation. Future oceanwide CYP1A1 expression profiles in cetacean skin biopsies are warranted and could reveal whether globally distributed chemicals occur at biochemically relevant concentrations on a global basis, which may provide a measure of ocean integrity.

The Pelagos Sanctuary for Mediterranean marine mammals: Marine Protected Area (MPA) or marine polluted area? The case study of the striped dolphin (Stenella coeruleoalba)

The concurrence of man-made pressures on cetaceans in the Mediterranean Sea is potentially affecting population stability and marine biodiversity. This needs to be proven for the only pelagic marine protected area in the Mediterranean Sea: the Pelagos Sanctuary for Mediterranean Marine Mammals. Here we applied a multidisciplinary tool, using diagnostic markers elaborated in a statistical model to rank toxicological stress in Mediterranean cetaceans. As a case study we analyzed persistent, bioaccumulative and toxic chemicals combined with a wide range of diagnostic markers of exposure to anthropogenic contaminants and genetic variation as marker of genetic erosion in striped dolphin (Stenella coeruleoalba) skin biopsies. Finally, a statistical model was applied to obtain a complete toxicological profile of the striped dolphin in the Pelagos Sanctuary and other Mediterranean areas (Ionian Sea and Strait of Gibraltar). Here we provide the first complete evidence of the toxicological stress in cetaceans living in Pelagos Sanctuary.

An "ex vivo" model to evaluate toxicological responses to mixtures of contaminants in cetaceans: integumentum biopsy slices.

The need for powerful new tools to detect the effects of chemical pollution, in particular of persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons (PAHs) on Mediterranean cetaceans led us to develop and apply a suite of sensitive biomarkers for integument biopsies of stranded and free‐ranging animals. This multi‐response ex vivo method has the aim to detect toxicological effects of contaminant mixtures. In the present study, we applied an ex vivo assay using skin biopsy and liver slices, combining molecular biomarkers [Western blot of Cytochrome P450 1A1 (CYP1A1) and Cytochrome P450 2B (CYP2B)] and gene expression biomarkers (Quantitative real‐time PCR of CYP1A1, heat shock protein 70, estrogen receptor alpha and E2F transcription factor) in response to chemical exposure [organochlorines compounds (OCs), polybrominated diphenyl ethers (PBDEs), and PAHs] for stranded Mediterranean Stenella coeruleoalba. The main goal of this experiment was to identify the biomarker and/or a suite of biomarkers that could best detect the presence of a specific class of pollutants (OCs, PBDEs, and PAHs) or a mixture of them. This multi‐response biomarker methodology revealed an high sensitivity and selectivity of responses (such as CYP1A and ER α mRNA variations after OCs and PAHs exposure) and could represent a valid future approach for the study of inter‐ and intra‐species sensitivities to various classes of environmental contaminants.

Anthropogenic contaminants in Indo-Pacific humpback and Australian snubfin dolphins from the central and southern Great Barrier Reef

We present the first evidence of accumulation of organochlorine compounds (DDTs, PCBs, HCB) and polycyclic aromatic hydrocarbons (PAHs) in Indo-Pacific humpback and Australian snubfin dolphins from the central and southern Great Barrier Reef. These dolphins are considered by the Great Barrier Marine Park Authority to be high priority species for management. Analyses of biopsy samples, collected from free ranging individuals, showed PAHs levels comparable to those reported from highly industrialized countries. DDTs and HCB were found at low levels, while in some individuals, PCBs were above thresholds over which immunosuppression and reproductive anomalies occur. These results highlight the need for ongoing monitoring of these and other contaminants, and their potential adverse effects on dolphins and other marine fauna. This is particularly important given the current strategic assessment of the Great Barrier Reef World Heritage Area being undertaken by the Australian Government and the Queensland Government.

Release of Polycyclic Aromatic Hydrocarbons and Heavy Metals from Rubber Crumb in Synthetic Turf Fields: Preliminary Hazard Assessment forAthletes

Synthetic turf, made with an infill of rubber crumb from used tyres or virgin rubber, is now common in many sporting facilities. It is known that it contains compounds such as polycyclic aromatic hydrocarbons (PAHs) and heavy metals. We evaluated in nine samples of rubber crumb the total content of some heavy metals (Zn, Cd, Pb, Cu, Cr, Ni, Fe) normally found in tyres by microwave mineralization and the levels of the 14 US EPA priority PAHs by Soxhlet extraction and HPLC analysis. The results showed high levels of PAHs and zinc in all rubber crumb samples compared to rubber granulate limits set by Italian National Amateur League (LND). Following the precautionary principle, a risk assessment at 25°C was done, using the Average Daily Dose (ADD) assumed by athletes, expressed in terms of mass of contaminant per unit of body weight per day (mg/kg day), and the Lifetime Average Daily Dose (LADD) and then evaluating the Hazard Index (HI) and the Cumulative Excess Cancer Risk (ΣECR). In the different rubber granulates samples the HI ranges from a minimum of 8.94×10-7 to a maximum of 1.16×10-6, while the ΣECR ranges from a minimum of 4.91×10-9 to a maximum of 1.10×10-8. Finally, the aim of this study was to estimate the “hazard” for athletes inhaling PAHs released at the high temperatures this synthetic turf may reach. Then a sequence of proofs was carried out at 60°C, a temperature that this rubber crumb can easily reach in sporting installations, to see whether PAH release occurs. The toxicity equivalent (TEQ) of evaporates from rubber crumb is not negligible and represents a major contribution to the total daily intake of PAHs by different routes.

Could feeding habit and migratory behaviour be the causes of different toxicological hazard to cetaceans of Gulf of California (Mexico)

In this work, a suite of diagnostic biomarkers was applied to seven cetacean species to evaluate the role of the feeding habits and migratory behavior in the toxicological status of these species from the Gulf of California, Mexico. We investigate the interspecific differences in cytochrome P450 1A1 and 2B (CYP1A1 and CYP2B, respectively), aryl hydrocarbon receptor and E2F transcription factor 1 and the contaminants levels [organochlorine compounds, polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs)] in four odontocete species (common bottlenose dolphin, long-beaked common dolphin, sperm whale and killer whale) and three mysticete species (blue whale, fin whale, and Bryde’s whale) using skin biopsy. Differences in contaminant levels and molecular biomarker responses between the odontocete and mysticete species have been pointed out. The canonical discriminant analysis on principal component analysis factors, performed to reveal clustering variables, shows that odontocete are characterised by the highest levels of lipophilic contaminants compared to the mysticete, with the highest levels of polychlorinated biphenyls, dichlorodiphenyltrichloroethanes and PBDEs detected in killer whale and the lowest levels in Bryde’s whale. The biomarker data show interspecific differences amongst the seven species, revealing highest CYP1A and CYP2B protein levels in the mysticete fish-eating species (Bryde’s whale). In conclusion, three main factors seem to regulate the biomarker responses in these species: (a) the inductive ability of persistent organic pollutants and PAHs; (b) the different evolutionary process of the two CYPs related to the different feeding habits of the species; (c) the migratory/resident behaviour of the mysticete species in this area.

Biological threats and environmental pollutants, a lethal mixture for mediterranean cetaceans?

Department of Environmental Sciences, University of Siena,Via P.A. Mattioli 4, 53100, Siena, ItalyThe possible existence of any cause–effect relationships between the concentrations of organochlorines (OCs) and the presenceof Morbillivirus and Toxoplasma gondii infections was investigated in both free-living and stranded specimens of Stenellacoeruleoalba, Tursiops truncatus, Globicephala melas, Balaenoptera physalus and Physeter macrocephalus from theMediterranean Sea. High blubber concentrations of polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane(DDT) were recorded in free-ranging G. melas. Tissue concentrations of PCBs and DDT in stranded T. truncatus(367 lipid weight (l.w.) and 143.7 mg/kg l.w., respectively) and S. coeruleoalba (139.9 l.w.; 92.9 mg/kg l.w.) were beyondthe PCB threshold value for the appearance of adverse effects in marine mammals. Evidence of T. gondii infection was mole-cularly detected in three S. coeruleoalba and six T. truncatus.Keywords: environmental toxicology, pollution monitoring, organochlorines, Morbillivirus, Toxoplasma gondii, disease, cetaceans,Mediterranean Sea

First ecotoxicological assessment of Caretta caretta (Linnaeus, 1758) in the Mediterranean Sea using an integrated nondestructive protocol

The loggerhead sea turtle (Caretta caretta) is a long-lived carnivorous reptile included in the IUCN Red List. The IUCN assessment for this species underlines the lack of information regarding pollution and pathogens and indicates as a priority effort to investigate and reduce the impacts of these threats. In this contest, the aim of our study was to conduct the first ecotoxicological assessment of this species in the Mediterranean Sea using a nondestructive integrated methodology. We set up and applied a monitoring protocol which includes endpoints, such as CYP1A, LPO, ENA assay, B esterases, never investigated before in this species. Seventy-five loggerhead turtles were sampled in a nondestructive way in Italian Sea Turtle Rescue Centers or free-ranging along the Spanish coasts. Blood, skin and carapace samples were used to test biomarker responses (CYP1A, VTG, LPO, Comet and ENA assay, BChE, GGT) and contaminant (OCs, PAHs, Pb, Cd, Hg) levels. Elaboration of experimental results was carried out taking also into consideration different age classes of the specimens. Among the main results obtained we should underline the statistically significant correlation between carcinogenic PAHs in blood and DNA fragmentation as well as between Cd in carapace and GGT in plasma. We also evidenced as youngest animals showed significantly higher DNA fragmentations, BChE inhibition and increase of GGT. Older specimens showed the highest levels of erythrocyte nuclear abnormalities which may indicate a long term toxicological stress. This study contributed to expand the knowledge about the ecotoxicology of C. caretta in the Mediterranean. The nondestructive protocol could also be applied to other marine ecosystems and other sea turtle species.