New olive-pomace fertilizer tested with a 2-tiers approach: Biomarkers on Eisenia fetida, physiochemical effects on Solanum lycopersicum and Olea europaea.

Every year, the olive oil industry generates a substantial amount of pomace, a semi-solid residue made up of skin, pulp, pit, and kernel fragments. Rather than being disposed of, the pomace can be dried and transported to an extraction facility where pomace oil can be extracted. Utilizing its high thermal capacity, the extracted pomace can be used as a supplementary fuel in the drying process, resulting in the production of ashes. In this study, the effect of pomace waste applied to the soil was investigated by testing two mixtures with different proportions of de-oiled pomace flour and kernel ash (50:50 and 70:30, respectively) in powder and pellet form. We used a dual approach, evaluating the effects of the mixtures on both soil communities and plant physiology and productivity, to assess the actual usability of the fertilizer in agriculture. The biomarker approach was valuable in assessing the sublethal effects of the two mixtures in powder form in soil. After 30 days of exposure, the bioindicator organism Eisena fetida showed lipid peroxidation, glutathione S-transferase and lactate dehydrogenase levels similar to the control, while lysozyme activity was reduced in all treatments. The powder mixture was lethal to the tomato plants, while there was no evidence of any damage to the olive trees. During 60 days of monitoring, both mixtures in pellet form showed a slight increase in physiological parameters, suggesting a benefit to the photosynthetic system. The improved carbon assimilation in tomato plants treated with the mixtures results in increased plant productivity, both in terms of number and weight of fruits, while maintaining the antioxidant content. This study paves the way for the use of the pomace mixture as a soil improver, thus increasing the value of this waste product.

Use of biomarkers to investigate toxicological effects of produced water treated with conventional and innovative methods.

The aim of this study was to develop and apply a multi-biomarker system to assess the toxicological effects of produced water (PW) from a Mediterranean off-shore oil platform. The selected bioindicator organism, mosquitofish (Gambusia affinis), was exposed in the laboratory to high concentrations of different PW: PW before treatment (BT), after conventional treatment (ACT) and after innovative treatment with zeolites in a prototype system (AIT). A set of biomarkers (benzo(alpha)pyrene monooxygenase, ethoxyresorufin-o-deethylase, vitellogenin, porphyrins, PAH bile metabolites, esterases, catalase, micronuclei) and PAH concentrations were measured in the experimental organism. The methodology proved to be appropriate and biomarker responses (CYP 1A1, PAH bile metabolites, micronuclei, esterases, porphyrins) affected by BT were less affected by ACT. PW treated with zeolites (AIT) had the lowest toxicological impact. The results obtained applying this multi-biomarker approach suggest that the system using zeolites is effective for treating produced water.

Chlamydia pneumoniae antibodies and angiographically demonstrated coronary artery disease in a sample population from Italy.

Recent reports suggest an association between Chlamydia pneumoniae and chronic coronary heart disease. This case-control study investigates the relationship between the presence of immunoglobin G (IgG) and immunoglobin A (IgA) when measured by means of microimmunofluorescence (MIF) and angiographically diagnosed coronary disease. Cases (n = 150) were angiography patients with at least one coronary artery lesion occupying at least 50% of the luminal diameter. Controls (n = 49) were angiography patients with no detectable signs of coronary artery disease and patients (n = 56) without signs or symptoms of coronary disease and with normal ECG results. No significant differences were revealed between the seroprevalence of IgG and IaA and geometric mean titers (GMT) as measured in cases and controls. When cases were compared with controls whose angiographic results were normal, after adjusting for established risk factors (cholesterol, smoking, hypertension, diabetes, age, gender and family history), the estimated risk of coronary artery disease was 0.79 (95% confidence interval (C.I.), 0.31-1.99) for the presence of IgG and was 0.94 (95 C.I., 0.37-2.39) for IgA. When cases were compared with controls with normal ECG results, the adjusted odds ratio (O.R.) for coronary artery disease was 1.17 (95%, C.I., 0.52-2.62) for the presence of IgG and 0.82 195% C.I., 0.36-1.86) for the presence of IgA. These results do not support an association between C. pneumoniae infection and coronary disease.

Are the mediterraneantop predators exposed to toxicological risk due to endocrine disrupters?

Man-made endocrine disrupting chemicals (EDCs) range across all continents and oceans; some geographic areas are potentially more threatened than others: one of these is the Mediterranean Sea. This basin has limited exchange of water with the Atlantic Ocean and is surrounded by some of the most heavily populated and industrialized countries in the world. Accordingly, levels of some xenobiotics are much higher here than in other seas and oceans. In this research the unexplored hypothesis that Mediterranean top predator species (such as large pelagic fish and marine mammals) are potentially at risk due to EDCs is investigated. Here we illustrate the development of sensitive biomarkers (Vitellogenin, Zona Radiata proteins) for evaluation of toxicological risk in top marine predators (Xiphias gladius, Thunnus thynnus thynnus), and nonlethal techniques, such as nondestructive biomarkers (BPMO activities in skin biopsy), for the hazard assessment of threatened species exposed to EDCs, such as marine mammals (Stenella coeruleoalba, Tursiops truncatus, Delphinus delphis, and Balaenoptera physalus).

Mixed function oxidase induction in Carcinus aestuarii. Field and experimental studies for the evaluation of toxicological risk due to Mediterranean contaminants.

The aim of this study was to test and validate the use of mixed function oxidase (MFO) induction, in the crab Carcinus aestuarii, under experimental and field studies, for the evaluation of toxicological risk due to the main contaminants in the Mediterranean. Two different experiments were performed in the laboratory in order to identify the most suitable tissues for MFO studies in this species and the most suitable and sensitive MFO responses for evaluating chemical stress due to lipophilic contaminants. In order to validate this methodology in the field, two studies were carried out in two polluted Mediterranean lagoons: a transplant experiment in Orbetello Lagoon and an in situ experiment in Venice Lagoon. The following MFO responses were investigated in hepatopancreas and gills of the crabs: ethoxyresorufin-O-deethylase (EROD) and benzo(a)pyrene hydroxylase (BPH) activities and reductase enzyme activities. The main results can be summarised as follows: midgut-gland and gills were confirmed to be useful for MFO tests; BPH activity in hepatopancreas was the most suitable and sensitive MFO response for evaluating chemical stress due to Mediterranean contaminants in laboratory and field studies; in the Orbetello Lagoon experiment, a statistically significant difference was found between sites subject to different human impact.

Nondestructive biomarkers of exposure to endocrine disrupting chemicals in endangered species of wildlife.

This paper explores the problem of endocrine disrupting chemicals (EDCs) from the ecotoxicological point of view, focusing on nondestructive biomarkers of exposure to EDCs for risk assessment of endangered species of wildlife. Several EDCs, such as polyhalogenated aromatic hydrocarbons and toxic metals, tend to be biomagnified in the terrestrial and particularly the marine food chains. Top predators tend to accumulate high concentrations of these contaminants which places them in a situation of high toxicological risk. Hence, there is a need to develop nondestructive techniques, such as nondestructive biomarkers, for hazard assessment, protection, and conservation of endangered species exposed to EDCs. The biological materials proposed for this approach (for example blood, faeces, fur, skin biopsy specimens) are easily obtained with minimal stress for individuals and populations. Some validation data are reported on porphyrins in sea bird excreta (Larus dominicanus, Phalacrocorax olivaceus, Pelecanus occidentalis thagus), as nondestructive biomarkers of exposure to organochlorines, and on benzopyrene monooxygenase activities in marine mammal skin biopsy specimens (Stenella coeruleoalba, Balaenoptera physalus), as early indicators of exposure to p,p'-DDE and other endocrine disrupting organochlorines.

Fingerprint of polycyclic aromatic hydrocarbons in two populations of southern sea lions (Otaria flavescens).

The fingerprint of 14 polycyclic aromatic hydrocarbons (PAHs) was investigated in biopsy, fur, blood, liver and faeces of live and dead specimens of two Argentinian population of southern sea lion (Otaria flavescens). One colony lives in Mar del Plata harbour which is particularly polluted with petroleum, the second (control) colony lives at Punta Bermeja (Patagonia). The highest concentrations of the five carcinogenic PAHs were found in the Mar del Plata sea lions.

Skin biopsy as a nondestructive tool for the toxicological assessment of endangered populations of pinnipeds: preliminary results on mixed function oxidase in Otaria flavescens.

The aim of this study was to develop and validate a method for assessing the toxicological risk of endangered populations of pinnipeds based on a nondestructive biological tool, the skin biopsy specimen. Skin biopsies can be obtained from pinnipeds by anaesthetising the animals and taking a small amount of skin in the anterior flipper area, or by shooting a biopsy dart with a crossbow. Skin biopsy material is suitable for a wide range of chemical and biomarker analysis. Organochlorines and polycyclic aromatic hydrocarbons can be analysed in subcutaneous fat and MFO activity (BPMO), Cyt.P450 isoforms, and DNA damage can be detected in epidermis.

Biomarker responses at different levels of biological organisation in crabs (Carcinus aestuarii) experimentally exposed to benzo(alpha)pyrene.

The aim of this study was to validate a multi-trial biomarker approach for the evaluation of toxicological risk due to benzo(alpha)pyrene. Carcinus aestuarii, exposed to increasing concentrations of B(alpha)P in the water, was used as the bioindicator organism. A set of biomarkers were tested in order to: identify biological materials for biomarker and residue analysis; determine a group of sensitive techniques for the assessment of PAH contamination; investigate correlation between responses at different levels of biological organisation. The results underlined that BPMO activities in hepatopancreas and gills were a good biomarker of exposure to PAH-type compounds. B esterases activities in hemolymph and porphyrin patterns in excreta could be proposed as a non-destructive approach for evaluating chemical exposure in this species.

Do endocrine disrupting chemicals threaten Mediterranean swordfish? Preliminary results of vitellogenin and Zona radiata proteins in Xiphias gladius.

Endocrine Disrupting Chemicals (EDCs) have the potential to alter hormone pathways that regulate reproductive processes in wildlife and fishes. In this research the hypothesis that Mediterranean top predator species (such as large pelagic fish) are potentially at risk due to EDCs is investigated. These marine organisms tend to accumulate high concentrations of EDCs such as polyhalogenated aromatic hydrocarbons (PHAHs). The potential effects of EDCs on a fish species of commercial interest, the top predator Xiphias gladius (swordfish), were investigated using vitellogenin (Vtg) and Zona radiata proteins (Zrp) as diagnostic and prognostic biomarkers. Dramatic induction of typically female proteins (Vtg and Zrp) was detected by ELISA and Western Blot in adult males of the species. These results are the first warning of the potential risk for reproductive function of Mediterranean top predators, and suggest the need for continuous monitoring of this fragile marine environment.

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.

Skin biopsies for cell cultures from Mediterranean free-ranging cetaceans.

The aim of this study was to develop a useful method for obtaining viable tissue samples for establishing cell cultures from skin biopsies of free-ranging cetaceans. The skin biopsies were performed by two methods: dart from an air gun and dart from a crossbow. The dart tip was modified to collect tissue. The tissue was kept in tissue culture medium at ambient temperature, then processed within 24 h. Many modifications in culture technique, with respect to conventional culture methods for human fibroblasts, were made. The cultures thus obtained can be used for many purposes, including genetic and toxicological studies. In toxicology they are an alternative in vitro system for studying threatened animals such as marine mammals. In particular, fibroblasts can be used to test the vulnerability of cetaceans and pinnipeds to different environmental contaminants such as organochlorine compounds, heavy metals and polycyclic aromatic hydrocarbons.

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.

Multi-trial biomarker approach in Meganyctiphanes norvegica: a potential early indicator of health status of the Mediterranean "whale sanctuary.".

The aim of this study was to propose a suite of biomarkers (BPMO activity, NADPH-cytocrome c reductase, NADH-ferricyanide reductase. esterases, porphyrins, vitellogenin and zona radiata proteins) and residue levels (organochlorines, PAHs and heavy metals) in the zooplanktonic euphausiid Meganyctiphanes norvegica as a potential multi-disciplinary diagnostic tool for assessment of the health status of the Mediterranean "whale sanctuary". Very little difference in BPMO was detected between sites, with values ranging from 0.75 to 2.68 U.A.F./mg prot/h. On the other hand larger differences between sites were found for reductase activities. Esterases (AChE), porphyrins (Copro-, Uro-, Proto-porphyrins) vitellogenin and zona radiata proteins were also detectable in this zooplanctonic species. Hg showed mean levels of 0.141 ppm d.w., Cd 0.119 ppm d.w. and Pb 0.496 ppm d.w. Total PAHs ranged from 860.7 to 5,037.9 ng/g d.w., carcinogenic PAHs from 40.3 to 141.7 ng/g d.w., HCB from 3.5 to 11.6 ng/g d.w., DDTs from 45.3 to 163.2 ng/g d.w. and the PCBs from 84.6 to 210.2 ng/g d.w.

Plastic litter in the sea.

On June 2013 a workshop at the University of Siena (Italy) was organized to review current knowledge and to clarify what is known, and what remains to be investigated, concerning plastic litter in the sea. The content of the workshop was designed to contribute further to the European Marine Strategy Framework Directive (MSFD) following an inaugural workshop in 2012. Here we report a number of statements relevant to policymakers and scientists that was overwhelming agreement from the participants. Many might view this as already providing sufficient grounds for policy action. At the very least, this early warning of the problems that lie ahead should be taken seriously, and serve as a stimulus for further research.

Repolarization reserve determines drug responses in human pluripotent stem cell derived cardiomyocytes.

Unexpected induction of arrhythmias in the heart is still one of the major risks of new drugs despite recent improvements in cardiac safety assays. Here we address this in a novel emerging assay system. Eleven reference compounds were administrated to spontaneously beating clusters of cardiomyocytes from human pluripotent stem cells (hPSC-CM) and the responses determined using multi-electrode arrays. Nine showed clear dose-dependence effects on field potential (FP) duration. Of these, the Ca(2+) channel blockers caused profound shortening of action potentials, whereas the classical hERG blockers, like dofetilide and d,l-sotalol, induced prolongation, as expected. Unexpectedly, two potent blockers of the slow component of the delayed rectifier potassium current (I(Ks)), HMR1556 and JNJ303, had only minor effects on the extracellular FP of wild-type hPSC-CM despite evidence of functional I(Ks) channels. These compounds were therefore re-evaluated under conditions that mimicked reduced "repolarization reserve," a parameter reflecting the capacity of cardiomyocytes to repolarize and a strong risk factor for the development of ventricular arrhythmias. Strikingly, in both pharmacological and genetic models of diminished repolarization reserve, HMR1556 and JNJ03 strongly increased the FP duration. These profound effects indicate that I(Ks) plays an important role in limiting action potential prolongation when repolarization reserve is attenuated. The findings have important clinical implications and indicate that enhanced sensitization to repolarization-prolonging compounds through pharmacotherapy or genetic predisposition should be taken into account when assessing drug safety.

The Italian wall lizard (Podarcis sicula) as a bioindicator of oil field activity.

The aim of this study was to develop and to validate a methodology based on biomarker responses and residue analysis on the terrestrial lizard Podarcis sicula to assess the ecotoxicological effects associated with on-shore oil extraction. The oil treatment plant investigated is located in Val d'Agri (southern Italy). Italian wall lizards were sampled on four stations along a transect determined on the basis of prevailing winds downwind of the oil plant. Cytochrome P450 1A1 activities (EROD and BPMO), AChE activity, PAH bile metabolites and contaminant levels (PAHs and trace elements) were measured. Major results in the evaluation of toxicological impact of oil field activity in the Italian wall lizards were obtained for Cd, Hg, total and carcinogenic PAH levels, and PAH metabolites in bile. Results obtained validate, for the first time, P. sicula as a terrestrial bioindicator for the assessment of the toxicological impact of on-shore extraction activity.

Potential toxicological hazard due to endocrine-disrupting chemicals on Mediterranean top predators: state of art, gender differences and methodological tools.

Man-made endocrine-disrupting chemicals (EDCs) range across all continents and oceans. Some geographic areas are potentially more threatened than others: one of these is the Mediterranean Sea. Levels of some xenobiotics are much higher here than in other seas and oceans. In this paper we review the final results of a project supported by the Italian Ministry of the Environment, in which the hypothesis that Mediterranean top predator species (such as large pelagic fish and marine mammals) are potentially at risk due to EDCs was investigated. We illustrate the need to develop and apply sensitive methodological tools, such as biomarkers (Vitellogenin, Zona Radiata proteins and CYP1A activities) for evaluation of toxicological risk in large pelagic fish top predators (Swordfish, (Xiphias gladius), Bluefin Tuna (Thunnus thynnus thynnus)) and nondestructive biomarkers (CYP1A activities and fibroblast cell culture in skin biopsy), for the hazard assessment of threatened marine mammals species (Striped Dolphin, (Stenella coeruleoalba), Bottlenose Dolphin (Tursiops truncatus), Common Dolphin (Delphinus delphis) and Fin Whale (Balaenoptera physalus))exposed to EDCs. Differential gender susceptibility to EDCs is also explored both in large pelagic fish and in cetaceans. In cetaceans, male specimens showed higher cytochrome P450 induction (BPMO in skyn biopsies, CYP2B in fibroblasts cell cultures) by xenobiotics with respect to females.

Interspecies variation of "B" esterases in birds: the influence of size and feeding habits.

Interspecies differences in "B" esterases were evaluated in seven species of wild birds in order to investigate their different susceptibilities to organophosphorus (OP) insecticides. The relationship between "B" esterases and body size and the influence of feeding habits in the evolution of esterase activities were explored. An in vitro inhibition test was carried out with paraoxon and azinphos methyl oxon in order to investigate the role of equilibrium binding parameters (acetylcholinesterase/ organophosphorus compounds affinity) in determining susceptibility of the different species to parathion and azinphos methyl. Esterase activities were different in all species. The smallest bird studied (the sparrow) had the highest brain acetylcholinesterase (AChE) and plasma carboxylesterase (CbE) activities. The largest bird (the cormorant) had the lowest level of both activities. For microsomal CbE, the coot had the highest activity. An inverse correlation was found between brain AChE and plasma CbE activity in relation to body size. Omnivorous species showed the highest brain AChE and plasma CbE activities which were low in species with a specialised diet. The in vitro inhibition test demonstrated the active involvement of equilibrium binding parameters in determining different susceptibilities to these contaminants in birds.

Porphyrins as biomarkers of methylmercury and PCB exposure in experimental quail.

Chemicals such as heavy metals and polyhalogenated hydrocarbons have a high capacity to interfere with the enzymatic processes responsible for haem biosynthesis. These compounds can produce accumulation in tissues and organs and increased elimination of porphyrins in excreta (Andrew et al, 1990). The development of fast and easy analytical methods and the wide variety of biological media in which porphyrins can be detected have suggested their use as biomarkers of environmental pollution (Akins et al, 1993; De Matteis and Lim 1994). The analysis of porphyrins in the excreta is of special interest because it enables nondestructive monitoring of wild animals in the assessment of threatened or endangered species (Fossi et al, 1994). Methylmercury and PCBs are ubiquitous global pollutants and there is evidence they accumulate in terminal consumers, particularly those belonging to marine trophic chain (Renzoni et al, 1986; Yamashita et al, 1993). There have been some reports on methylmercury-induced (e.g. Woods et al, 1991; Bowers et al, 1992; Miller and Woods 1993) and PCB-induced porphyria (e.g. Vos and Pennings, 1971; Miranda et al, 1987; Elliot et al, 1990; Miranda et al, 1992) but little data on their combined effect. In order to investigate the quality of porphyrins as biomarkers we performed an experiment in which Japanese quail were fed a diet containing methylmercury and polychlorobyphenyls (PCBs as Arochlor 1260) individually or combined in different ratios. The present study aims to provide preliminary data on liver and fecal levels of porphyrins in response to methylmercury and PCB administration, and on whether the indicator is sensitive to synergism or antagonism between the two compounds, administered simultaneously.