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Despite their ecological and economical relevance, a lack of data is still occurring about the distribution, abundance, bioaccumulation and risks for consumption of some edible bivalves. The present study has been carried out in the Venice Lagoon taking into account three bivalve species to investigate i) the possible relationship among the biological features-distribution, abundance and bioaccumulation patterns- of razor clams (Solen marginatus Pulteney, 1799), Manila clams (Ruditapes philippinarum Adams and Reeve, 1850) and lagoon cockles (Cerastoderma glaucum Poiret, 1789) and the hydrological features, namely sediment physico-chemical characteristics and contamination; ii) their specific role as bioindicators of inorganic contamination; iii) the possible risks for human health associated with the consumption of these edible bivalves. Results showed that species distribution and abundance, especially for razor clams, was mainly influenced by environmental conditions and sediment granulometric composition, above metal(loid) contamination. The contamination patterns were different among species, as Manila clam generally showed higher bioaccumulation values for most of the metal(loid)s, whilst lagoon cockles preferentially accumulated Ni. Eventually, a serious concern exists for the human consumption, for all species and investigated sites regarding As. This study will raise attention on the effects of bioaccumulation of inorganic pollutants by edible bivalves and risks for consumers' safety, especially concerning razor clams and cockles, for which a critical lack of data on metal(loid) bioaccumulation occurs from the Northern Adriatic Sea.

Purpose: Canal dredging and shaping produce considerable amounts of sediments whose reuse on- and off-site depends on their pollution level. This study explores the potential of a calcium chloride washing to remove potentially toxic elements (PTEs) from a freshly dredged calcareous sediment and to affect the aggregate stability of the washed sediment. Materials and methods: The ability of 0.5-M CaCl2 washing to restore the sediment samples polluted with Cu and Zn dredged from a freshwater canal was assessed. The distribution of the two metals among the sediment geochemical phases, the potential availability to plants (DTPA), and the water aggregate stability of particles were evaluated on the washed samples and compared to control. Results and discussion: In the most polluted sample (similar to 200 and 500mgkg(-1) of Cu and Zn, respectively), the washing decreased the amount of Cu by 26%, mainly in the sulfide/organic fraction, and of Zn by 10%, mainly in carbonates. A decrease in the dispersivity of clay fraction was observed due to the well-known effect of Ca2+ ions on flocculation of colloidal clay particles. At large scale, the aggregates formed by the interaction between large particles and flocculated clay showed lower water stability with respect to the control, thus suggesting the need to improve the physical properties of the treated material. Noteworthy, after 1 year of dry storage from washing, the average percentage of potentially bioavailable (DTPA) Cu and Zn (23 and 13% of pseudo-total amount, respectively) dropped with respect to the control (40 and 19%) and a concomitant carbonate increase (+33%) was observed. Conclusions: The CaCl2 washing is a promising method to reduce Cu and, to a minor extent, Zn in wet calcareous sediments dredged from freshwater canals. However, there is still room for improvement. The PTE fractions remaining in carbonates and organic component of washed sediment clearly indicate the potential of combining mild acidic pH values with calcium chloride in the washing to enhance the PTE removal.

The Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) is now one of the top 5 most commercially valuable bivalve species worldwide. Originally from the Indo-Pacific region, it has been introduced in many countries for fisheries and aquaculture, including estuarine environments along Atlantic and Mediterranean European coasts. Yet despite its commercial value and widespread distribution, the precise origins of stocks remain speculative and the genetic diversity of introduced populations is poorly known. Thus, the aim of this work was to collect mtDNA COI (Cytochrome oxidase I) gene sequences from 5 European countries with Manila clam stocks and compare them with native Asian populations to evaluate their genetic diversity and identify possible routes of invasion. The COI gene sequencing supported a strong founder effect in the European populations with 3 main haplotypes occurring at high frequencies, derived from Japan. However, high haplotype diversity was also observed due to the occurrence of 10 rare haplotypes. This supports hypotheses (i) there have been additional, previous unrecorded, introductions as previously hypothesized by analysis of 16S rDNA, and (ii) there has been a limited loss of genetic diversity in introduced populations, as previously suggested by microsatellite data. This is the first genetic comparison of Manila clam populations introduced in to Europe with native clams. Genetic data herein presented are fundamentally important for the traceability of clam products and stock management programmes and will also inform discussion on the potential resilience of exploited Manila clam populations.

Cadmium (Cd) is a toxic trace element released into the environment by industrial and agricultural practices, threatening the health of plants and contaminating the food/feed chain. Biotechnology can be used to develop plant varieties with a higher capacity for Cd accumulation (for use in phytoremediation programs) or a lower capacity for Cd accumulation (to reduce Cd levels in food and feed). Here we generated transgenic tobacco plants expressing components of the Pseudomonas putida CzcCBA efflux system. Plants were transformed with combinations of the CzcC, CzcB and CzcA genes, and the impact on Cd mobilization was analysed. Plants expressing PpCzcC showed no differences in Cd accumulation, whereas those expressing PpCzcB or PpCzcA accumulated less Cd in the shoots, but more Cd in the roots. Plants expressing both PpCzcB and PpCzcA accumulated less Cd in the shoots and roots compared to controls, whereas plants expressing all three genes showed a significant reduction in Cd levels only in shoots. These results show that components of the CzcCBA system can be expressed in plants and may be useful for developing plants with a reduced capacity to accumulate Cd in the shoots, potentially reducing the toxicity of food/feed crops cultivated in Cd-contaminated soils.

Manila clam Ruditapes philippinarum represents an important economic resource of Italian aquaculture. However, especially for Venice lagoon, health and environmental risks should be evaluated. A multidisciplinary approach was used. In particular, local production chain analysis and end-user requirements were investigated to assess manila clam safety and traceability in Northern Adriatic Sea. Moreover, the molecular and biochemical techniques demonstrated a good quality of the investigated farming sites, due to the absence of biotoxins. Molecular markers provided informations on the population genetics and specific genetic traits of Ruditapes philippinarum in Northern Adriatic Sea, which was investigated for the first time. The complexity of all these informations was simplified in a single useful tool: a database containing all the informations about the seafood product.

Arbuscular mycorrhizal (AM) fungi are key organisms of the soil/plant system, influencing soil fertility and plant nutrition, and contributing to soil aggregation and soil structure stability by the combined action of extraradical hyphae and of an insoluble, hydrophobic proteinaceous substance named glomalin-related soil protein (GRSP). Since the GRSP extraction procedures have recently revealed problems related to co-extracting substances, the relationship between GRSP and AM fungi still remains to be verified. In this work the hypothesis that GRSP concentration is positively correlated with the occurrence of AM fungi was tested by using Medicago sativa plants inoculated with different isolates of Glomus mosseae and Glomus intraradices in a microcosm experiment. Our results show that (i) mycorrhizal establishment produced an increase in GRSP concentration – compared to initial values – in contrast with non-mycorrhizal plants, which did not produce any change; (ii) aggregate stability, evaluated as mean weight diameter (MWD) of macroaggregates of 1–2 mm diameter, was significantly higher in mycorrhizal soils compared to non-mycorrhizal soil; (iii) GRSP concentration and soil aggregate stability were positively correlated with mycorrhizal root volume and weakly correlated with total root volume; (iv) MWD values of soil aggregates were positively correlated with values of total hyphal length and hyphal density of the AM fungi utilized. The different ability of AM fungal isolates to affect GRSP concentration and to form extensive and dense mycelial networks, which may directly affect soil aggregates stability by hyphal enmeshment of soil particles, suggests the possibility of selecting the most efficient isolates to be utilized for soil quality improvement and land restoration programs.

The concentration changes of 12 metals (As, Hg, Cd, Cr, Co, Cu, Pb, Mn, Ni, V, Zn, Fe, and Hg) in the soft tissues of Tapes philippinarum during growth were investigated. Clams were seeded in two contaminated areas of the Venice lagoon (San Giuliano and Fusina) and in an area of the Marano lagoon (Lignano Sabbiadoro) close to the clam-farm where the seed was produced. Metal trends were very different according to the considered element, the study site and the growth period. Arsenic was always higher in clam tissues than in surface sediments and suspended particles in all the three stations. Mercury, Cd and Cu were higher in the clams from Marano and Fusina but not in those from San Giuliano. Zinc and Co in clams exceeded the concentrations in the sediment and suspended particles only at Marano. The other elements (Cr, Pb, Mn, Ni, and Fe) were always higher in SPM and sediments. In general metal concentrations in clams were more highly correlated to concentrations in the suspended particles rather than in the surface sediments and in suspended clams rather than in bottom clams, nevertheless significant differences between stations and contaminants were found. Metal concentrations in clams were always lower than the European regulatory limits.

A phytotoxicity assay based on seed germination/root elongation has been optimized and used to evaluate the toxic effects of some phenolic herbicides. The method has been improved by investigating the influence of experimental conditions. Lepidium sativum was chosen as the most suitable species, showing high germinability, good repeatability of root length measurements, and low sensitivity to seed pretreatment. DMSO was the most appropriate solvent carrier for less water-soluble compounds. Three dinitrophenols and three hydroxybenzonitriles were tested: dinoterb, DNOC, 2,4-dinitrophenol, chloroxynil, bromoxynil, and ioxynil. Toxicity was also determined using the Vibrio fischeri Microtox® test, and a highly significant correlation was found between EC50 values obtained by the two assays. Dinoterb was the most toxic compound. The toxicity of hydroxybenzonitriles followed the order: ioxynil >bromoxynil >chloroxynil; L. sativum exhibited a slightly higher sensitivity than V. fischeri to these compounds. A QSAR analysis highlighted the importance of hydrophobic, electronic, and hydrogen-bonding interactions, in accordance with a mechanism of toxic action based on protonophoric uncoupling of oxidative phosphorylation. The results suggest that the seed germination/root elongation assay with L. sativum is a valid tool for the assessment of xenobiotic toxicity and can be recommended as part of a test battery.

We present in this work preliminary results on the translocation of copper, manganese and iron from soil to leaves of Sambucus nigra L. seedlings in the presence of increasing copper concentrations in the growth medium. The use of electron spin resonance spectroscopy and the comparison with atomic absorption spectroscopy is not presently common in the study of metal absorption in plants. This approach gave promising results both on the detection of some metal ions, as well as of radical species related to the presence of Cu2+ in plant tissues. Copper was found to act synergistically in manganese uptake.

Over time, anthropogenic activities have led to severe cadmium (Cd) and arsenic (As) pollution in several environments. Plants inhabiting metal(loid)-contaminated areas should be able to sequester and detoxify these toxic elements as soon as they enter roots and leaves. We postulated here that an important role in protecting plants from excessive metal(loid) accumulation and toxicity might be played by arbuscular mycorrhizal (AM) fungi. In fact, human exploitation of plant material derived from Cd- and As-polluted environments may lead to a noxious intake of these toxic elements; in particular, a possible source of Cd and As for humans is given by cigarette and cigar smoke. We investigated the role of AM fungus Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schusler in protecting Nicotiana tabacum L. (cv. Petit Havana) from the above-mentioned metal(loid) stress. Our findings proved that the AM symbiosis is effective in increasing the plant tissue content of the antioxidant glutathione (GSH), in influencing the amount of metal(loid)-induced chelators as phytochelatins, and in reducing the Cd and As content in leaves and roots of adult tobacco plants. These results might also prove useful in improving the quality of commercial tobacco, thus reducing the risks to human health due to inhalation of toxic elements contained in smoking products.

The activity of bovine Cu,Zn superoxide dismutase (SOD) was determined by steady-state voltammetry using mercury microelectrodes having close hemisphere shapes. The measurements were carried out in aerated aqueous solutions saturated with the surfactant triphenylphosphine oxide (TPO). The influence of pH on oxygen reduction process, either in the presence or in the absence of SOD, was studied over the range 7.5–12.3. Well-defined steady-state voltammetric waves, whose heights were reproducible within 2%, were obtained. A theoretical equation was derived for the calculation of the kinetic constant involved in the SOD-catalysed dismutation of the superoxide ion. The results obtained with the approach employed here agreed satisfactorily with those obtained by a dropping mercury electrode with a short dropping time.

Sphere-cap mercury microelectrodes fabricated on a platinum disk substrate were tested in aqueous solutions over the temperature range 295–353 K. The performance of these electrodes was assessed in solutions containing Ru(NH3)6Cl3 and LiClO4 as supporting electrolyte. From the steady-state limiting current obtained at three different mercury microelectrodes, the diffusion coefficient of the electroactive species at different temperatures was determined. It was found that the diffusion coefficient values were consistent with the Stokes–Einstein and activation energy models. The results obtained also allowed us to conclude that sphere-cap mercury microelectrodes can be useful for elevated temperature electroanalysis in aqueous solutions. Keywords: Sphere-cap microelectrodes, Mercury, Voltammetry, Steady state, Temperature

Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) – is one of the most successful marine invaders, being introduced worldwide for aquaculture and fisheries. Genetic diversity and structure of its populations are largely unknown, especially in the invaded environments. Herein we present molecular genetic data on 12 introduced Manila clam populations in Italy, Spain, and Portugal from both Mediterranean (Adriatic) and Atlantic sampling sites. The phylogenetic information was investigated by the direct sequencing of 16S mitochondrial DNA. Results of mtDNA analyses showed the occurrence of 11 haplotypes for European introduced populations, as a consequence of multiple founder effects from different source populations. Rp hap1 was the most frequent, shared among all populations. The other 10 haplotypes were rare and distributed at local scale, in agreement with what was observed in other invasive bivalves. Biogeographic and phylogenetic analyses based on 16S rDNA of introduced Manila clam populations showed a complex scenario, dominated by multiple pools of individuals coming from different sources, as a consequence of multiple introductions.

The space-time distribution of some pollutants (Cu, Pb, Zn, Cd, Fe, Mn, V, Ni, Cr) in the sludge of the canals of Venice was studied. The contamination levels were comparable to, or higher, than those measured in the most polluted sediments of the Lagoon of Venice. Sediments were collected by two different sampling techniques: 1) collection of sediment cores (upper 5 cm) by a syringe-type corer; 2) collection by traps, placed on the bottom of the canal. Traps permitted the sampling of sediments essentially resuspended by overlying water turbulence. This sediment fraction is subjected to variations of its physicochemical parameters (principally change of redox conditions) and therefore to pollutant exchange at the water/sediment interface.

The effects on mitochondrial respiratory parameters of heavy metals, such as Cu, Ni, Pb, Cd, Zn, Ag, Hg, were recorded by using thein vitro response of submitochondrial particles (SMP) from beef heart mitochondria.

Human activities such as metals mining and milling operations provide one of the most important sources of contamination in the environment. Abandoned mines can be an important source of toxic elements. The threat of heavy metal pollution posed by mine soils generally concerns more than one metal. The aim of this study was to assess total concentration of six potentially toxic metals (Cd, Cr, Cu, Pb, Zn and Fe) in the soil and plant samples of three dominant willow species ( Salix purpurea L., Salix caprea L. and Salix eleagnos Scop.) collected from abandoned mixed sulphide mine dumps (Imperina Valley, North-east Italy). Results demonstrate that metal concentrations in soils are in general above the Italian average limits and they are also significantly (except Cr), as compared with controls (p ≤ 0.05), with averages of 2.12 mg Cd kg − 1 , 2267 mg Cu kg − 1 , 9552 mg Pb kg − 1 , 1243 mg Zn kg − 1 and 299,973 mg Fe kg − 1 . The phytoremediation ability of selected Salix species for heavy metals was estimated. The results have revealed significant differences among willow species (p ≤ 0.05) regardless of the species selected. The transfer factor and bioaccumulation coefficient of selected metals varied among plant species and from different sites. Some of the investigated species have potential for soil stabilization and extraction of heavy metals. The results indicate that there is an increasing need for further research projects mainly focused on the mechanisms whereby such willows are able to survive in contaminated soils.

Pteris vittata can tolerate very high soil arsenic concentration and rapidly accumulates the metalloid in its fronds. However, its tolerance to arsenic has not been completely explored. Arbuscular mycorrhizal (AM) fungi colonize the root of most terrestrial plants, including ferns. Mycorrhizae are known to affect plant responses in many ways: improving plant nutrition, promoting plant tolerance or resistance to pathogens, drought, salinity and heavy metal stresses. It has been observed that plants growing on arsenic polluted soils are usually mycorrhizal and that AM fungi enhance arsenic tolerance in a number of plant species. The aim of the present work was to study the effects of the AM fungus Glomus mosseae on P. vittata plants treated with arsenic using a proteomic approach. Image analysis showed that 37 spots were differently affected (21 identified). Arsenic treatment affected the expression of 14 spots (12 up-regulated and 2 down-regulated), while in presence of G. mosseae modulated 3 spots (1 up-regulated and 2 down-regulated). G. mosseae, in absence of arsenic, modulated 17 spots (13 up-regulated and 4 down-regulated). Arsenic stress was observed even in an arsenic tolerant plant as P. vittata and a protective effect of AM symbiosis toward arsenic stress was observed.

The coastal lagoons of the Northern Adriatic Sea are among the most worldwide productive locations of Manila clam Ruditapes philippinarum. Although introduced in Italy in 1983 from the Indo-Pacific, fishing and exploitation of Manila clam improved during the years as Italy became the leading country in Europe for production of this shellfish. Despite its commercial importance, genetic structure of R. philippinarum in Northern Adriatic Sea has not been previously investigated. Here we present the first genetic study on Manila clam populations inhabiting a Mediterranean area, assessed by both mitochondrial (16S rDNA) and nuclear DNA (microsatellite loci). Our study showed that this species has a limited genetic differentiation at the mitochondrial level, but a higher rate of genetic diversity can be identified by polymorphic markers as microsatellites. In particular, out of 28 alleles, 7 private ones were recorded for the Venice Lagoon populations, 2 for those of Scardovari and one for the Po River Delta populations. These molecular markers suggest the occurrence of at least two different introduction events from different recruitment stocks, representing a powerful tool not only to assess genetic diversity of an introduced species, but also helpful information to manage aquaculture and fishery stocks, and to warrant food quality, safety and for the authentication of shellfish products, and traceabilty path.

Ab initio in silico design of proteins and enzymes has emerged as a powerful tool to design application-tailored proteins and catalysts for a wide range of applications. Several enzymes exploit the unique features of metal cofactors to achieve catalytic activity otherwise unattainable through the use of only natural amino acid residues. One of the major bottlenecks in ab initio design of novel proteins relies on long-range and epistatic effects that severely limit the possibility of a rational design. Within this framework there is an ongoing effort to reduce protein length and complexity to unlock the full potential of in silico protein design. In this work we specifically address this problem designing and investigating the dynamic features of 10 in silico designed minimal metallo-proteins. In particular, in this paper we investigate whether and to what extent it is possible to design a minimal metallo-enzyme made of only residues involved in metal binding. In this research we address these questions by investigating the ability of 10 different “mini-proteins” with a length shorter than 15 residues. Molecular dynamics studies clearly show that it is possible to design a minimal protein able to bind a metal atom with the correct geometry. It is noteworthy that designed mini-proteins cannot achieve the formation of a canonical hydrophobic core, rather the metal ion provides a “metal core” around which the entire protein is organized. This opens the possibility of designing synthetic enzymes composed of only functional residues organized around a “metal core” which acts as both structural and functional determinat.

In this work the arbuscular mycorrhizal (AM) fungal communities colonizing a polluted ash dump island, downtown Venice, were studied by using a multimodal approach. The island, Sacca San Biagio, was covered with a thick layer of municipal solid waste residues produced by an incinerator operating from 1973, to 1984. Such residues contained high levels of heavy metals (Cu, Pb and Zn). We characterized the AMF communities present in soils on Sacca San Biagio island by using molecular methods. Nine AM fungal sequence types were detected in the roots of three plant species, representative of the dominant flora, by using partial SSU ribosomal RNA genes. The most abundant sequence types corresponded to Glomus intraradices / Glomus fasciculatum , and to Glo18, a sequence detected so far only in planta . Two sequences were new to science. Glomalin-related soil protein (GRSP), extracted from rhizosphere soil of dominant plant species, ranged from 1.6 to 2.3 mg g −1 . The occurrence of an active AM fungal community able to live in such harsh environment was evinced by the correlation between mycorrhizal colonization and GRSP content.

Bottom ash from a municipal solid waste incinerator on a former contaminated site, the island of Sacca San Biagio (Lagoon of Venice), was examined in order to evaluate levels of pollutants and their potential mobility and availability. Heavy metal concentrations were determined and the actual contamination of the site was compared with national legislation on polluted sites. The site was mainly contaminated by zinc, copper and lead. Physico-chemical characterization of bottom ash was carried out by SEM (Scanning Electron Microscopy) with micro-analysis by EDS (Energy Dispersive X-ray Spectroscopy) and XRD (X-ray Diffractometry), for information on newly formed minerals. SEM-EDS analysis revealed the presence of particles, compounds and clusters containing heavy metals and, in particular, the presence of barium sulfate, which was assumed to be a site-specific compound. Similarities between bottom ash and atmospheric PM10 collected on the adjacent island of Sacca Fisola were studied and a risk of aerodispersion of the fine fraction of ash was assumed. Lastly, in order to evaluate the potentially available fraction of metals (non-residual fraction) and the directly exchangeable fraction, two single extraction procedures with HCl and citric acid were carried out, respectively. Results showed a relatively low concentration of readily phyto-available metals, as well as the high concentrations found for some heavy metals (Cu, Pb, Zn) in the potentially mobilizable fraction.

Acid extractions represent a simple and effective tool for the assessment of metal contamination, origin and bioavailability in the aquatic environment. Optimized microwave-assisted procedures utilizing two extractions (8 M HNO 3 and 1 M HCl) were developed. Particular attention was given to the evaluation of efficiency and selectivity of the developed procedures. This involved an examination of the sediment and the extraction residues by Mossbauer spectroscopy and X-ray diffractometry. These instrumental approaches were used to identify the geochemical phases associated with acid decomposition. Microwave digestion with 8 M HNO 3 produced an efficient solubilization of the most important metal-bearing phases and left unaffected only the minerals (quartz, feldspars, muscovite) with a negligible metal content, thus providing a good estimate of the total metal concentrations. The 1 M HCl extraction, despite the limitation due to the incomplete solubilization of pyrite and to the partial dissolution of sheet silicates, represents a simple and effective technique for providing an estimate of the bioavailable metal fraction in sediments. The combined use of the two microwave-assisted acid extractions can provide a rapid and simple approach for the risk assessment of metal-polluted sediments.

The maintenance of soil health and productivity is a central aim of sustainable agriculture. Arbuscular mycorrhizal fungi (AMF) are soil biota fundamental for soil fertility and plant nutrition, which may be used in the evaluation of the impact of agronomic practices on soil quality. In the present study we evaluated the influence of three different land uses on AMF populations and correlated glomalin-related soil protein (GRSP) content with AMF biomass parameters, such as spore density and biovolume. Among the differently managed sites – maize monoculture, grassland and poplar grove – maize soil showed the lowest AMF spore number and GRSP content. The same morphological taxa were found in the three sites, except for one additional morphotype in poplar grove. A good correlation between GRSP and spore biovolume was found, suggesting that GRSP may represent a useful biochemical parameter for the assessment of biological soil fertility in sustainable agriculture.

Samples of Mytilus galloprovincialis collected in different sites of the Venice lagoon (Italy) were investigated for total arsenic concentrations by ICP-AES and for single arsenic species by HPLC-ICP-MS. For this purpose, an analytical procedure for the sensitive and efficient speciation of the arsenic species As(III), As(V), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AB), arsenocholine (AC), and four arsenosugars was optimised. The total arsenic and the single arsenic species were determined in both the hepatopancreas (digestive gland) and the remaining soft tissues in order to verify the different arsenic accumulation in the body parts of mussels. Arsenic compounds were extracted from the mussels with a methanol/water mixture; the extracts were evaporated to dryness, redissolved in water, and chromatographed in an anion-exchange column, a Hamilton PRP-X100. Only small quantities or traces of inorganic arsenic were detected in the mussels. The majority of arsenic compounds detected in the extracts were organic species, with a predominance of arsenobetaine and of an arsenosugar. In addition, a greater arsenic accumulation in the digestive glands of mussels was observed.

A simple and rapid in vitro toxicological assay, utilizing submitochondrial particles (SMP), has been used to evaluate the toxic effects of fifteen herbicides belonging to the phenol and phenoxyalkanoic acid chemical classes. The SMP assay allows the quantitative evaluation of the toxicity of compounds with different mechanisms of action: uncouplers, inhibitors of the enzyme complexes involved in reverse electron transfer and in oxidative phosphorylation and chemicals that alter the membrane structure. The two groups of herbicides showed different levels of toxicity. For phenol derivatives, EC 50 values ranged from 0.16 μM (ioxynil) to 6.7 μM (2,4-dinitrophenol), whereas for phenoxy herbicides EC 50 values ranged from 21 μM (2,4,5-trichlorophenoxyacetic acid, 2,4,5-T) to 110 μM (4-chloro-2-methylphenoxyacetic acid, MCPA). On the average, the toxicity of phenolic compounds is greater than that of phenoxyalkanoic acids by two orders of magnitude. Quantitative structure–activity relationships (QSAR) were developed between EC 50 values and various molecular descriptors. The results suggest the existence of different mechanisms of action for the two classes of compounds. The findings obtained for phenolic herbicides are consistent with a protonophoric uncoupling mechanism, whereas for phenoxy herbicides a non-specific mode of action at membrane level can be hypothesized.

In this study a procedure of sequential extraction of metals from sediments was applied to samples from the lagoon of Venice. Compared to the measurement of total concentrations, the knowledge of metal partitioning among sediment phases can help in estimating the risk posed by metal polluted sediments and give information on contamination sources and on the factors that influence metal enrichment in sediments. The results permitted to estimate for each metal the percentage of the total content that could be potentially remobilized from sediments, owing to changes in environmental conditions. The immediately available fraction represented in general a low proportion of the total content. An important proportion of most metals (except for Zn, Cd and Cu) was associated to residual phases and therefore was not expected to be released under the normal environmental conditions. The large amounts of metals bound to Fe-Mn oxides/hydroxides confirmed the strong scavenging efficiency of this phase. The carbonate phase was the preferential associative form for Pb, Cd and Zn, whereas Cu was bound in prevalence to the organic matter/sulphide phase.

In this study a procedure of sequential extraction of metals from sediments was applied to samples from the lagoon of Venice. Compared to the measurement of total concentrations, the knowledge of metal partitioning among sediment phases can help in estimating the risk posed by metal polluted sediments and give information on contamination sources and on the factors that influence metal enrichment in sediments. The results permitted to estimate for each metal the percentage of the total content that could be potentially remobilized from sediments, owing to changes in environmental conditions. The immediately available fraction represented in general a low proportion of the total content. An important proportion of most metals (except for Zn, Cd and Cu) was associated to residual phases and therefore was not expected to be released under the normal environmental conditions. The large amounts of metals bound to Fe-Mn oxides/hydroxides confirmed the strong scavenging efficiency of this phase. The carbonate phase was the preferential associative form for Pb, Cd and Zn, whereas Cu was bound in prevalence to the organic matter/sulphide phase.

The concentrations of heavy metals (Fe, Zn, Cu, Cd, Ni, Pb, Cr, As) were determined in seven seaweeds of environmental and commercial relevance (Ulva rigida C. Ag., Gracilaria gracilis (Stackhouse) Steentoft, L. Irvine and Farnham, Porphyra leucosticta Thuret, Grateloupia doryphora (Montagne) Howe., Undaria pinnatifida (Harv.) Suringar, Fucus virsoides J. Agardh, Cystoseira barbata (Good. et Wood.) Ag.) collected in four sampling sites in the lagoon of Venice, in spring and autumn 1999. Metals were extracted using hot concentrated acids in a Microwave Digestion Rotor and analysed by absorption spectrophotometry using a flame mode for Fe and Zn and a graphite furnace for Pb, Cr, Cd, Cu, Ni and As. High contamination levels, especially for Pb, were detected in Ulva and to a lesser extent in Gracilaria. Brown seaweeds, especially Cystoseira was highly contaminated by As. The least contaminated genera with all metals except As were Porphyra and Undaria. A concentration decrease for Zn and Cd was observed from the inner parts of the central lagoon, close to the industrial district, towards the lagoon openings to the sea.

Sperm cell and embryo toxicity tests using the Mediterranean sea urchin Paracentrotus lividus were performed to assess the toxicity of tributyltin chloride, bis(tributyltin)oxide, triphenyltin acetate, and triphenyltin hydroxide. Toxicity values (mean effective concentration [EC50]) ranged from 2.97 to 18.5 microg/L for sperm cells and from 1.11 to 2.62 microg/L for embryos. For sperm cells, the toxicity of the two tributyl compounds was significantly greater than that of two triphenyl compounds; for embryos, the triphenyl compounds appeared to be more toxic. Study of embryotoxic effects highlighted closely concentration-dependent damages, the most sensitive stages corresponding to the crucial phases of differentiation (gastrula and prisma). Both EC50 and no-observed-effect concentration values for the four organotin compounds are similar to those reported in the literature for early life stages of other marine organisms.

Sperm cell and embryo toxicity tests using the Mediterranean sea urchin Paracentrotus lividus were performed to assess the toxicity of tributyltin chloride, bis(tributyltin)oxide, triphenyltin acetate, and triphenyltin hydroxide. Toxicity values (mean effective concentration [EC50]) ranged from 2.97 to 18.5 μg/L for sperm cells and from 1.11 to 2.62 μg/L for embryos. For sperm cells, the toxicity of the two tributyl compounds was significantly greater than that of two triphenyl compounds; for embryos, the triphenyl compounds appeared to be more toxic. Study of embryotoxic effects highlighted closely concentration-dependent damages, the most sensitive stages corresponding to the crucial phases of differentiation (gastrula and prisma). Both EC50 and no-observed-effect concentration values for the four organotin compounds are similar to those reported in the literature for early life stages of other marine organisms.

The effects on mitochondrial respiration of 15 chloroanilines were recorded by using the in vitro response of submitochondrial particles (SMP) from beef heart mitochondria. The bioassay procedure for SMP is based on the process of reverse electron transfer, which can be negatively affected by inhibitors of electron transport, by uncouplers, and by chemicals that impair membrane integrity. The EC50 values, determined for the tested chloroanilines, indicate a general tendency of increasing toxicity with increasing chlorine substitution. In order to validate the results obtained and to evaluate the capability of the SMP assay to reproduce the toxic effects of the examined compounds on different freshwater species, the EC50 values were compared with literature data from other biological assays regarding both in vitro systems and whole organisms. A good correlation was found in particular with two widely used testing systems, the Microtox and the Tetrahymena assays. In addition, quantitative structure-activity relationships (QSARs) were established between the EC50 values and various molecular descriptors for hydrophobic, steric, and electronic interactions. The results obtained were utilized to elucidate the mechanism of toxic action of chloroanilines, which are commonly reported to act by the polar narcosis mode of action. Moreover, they confirmed that the SMP assay can be a useful tool for studying the toxicity of chemicals that act nonspecifically by impairing membrane structure and functions.

The toxicity of a series of chlorophenols, determined by a short-term in vitro assay utilizing mammalian submitochondrial particles, was related to the physicochemical and structural properties of these compounds. Quantitative Structure-Activity Relationships were defined by correlating EC50 values with six molecular descriptors, chosen to represent lipophilic, electronic and steric effects: the n-octanol/water partition coefficient (log Kow), the constant of Hammett (sigma sigma), the acid dissociation constant (pKa), the first order valence molecular connectivity index (1 chi v), the perimeter of the efficacious section (sigma D) and the melting point (m.p.). The results of regression analysis showed that log Kow is the most successful descriptor, indicating that the ability of chlorophenols to partition into the lipid bilayer of the mitochondrial membrane has an important role in determining their toxic effects. These results are consistent with a molecular mechanism of uncoupling action based on the chemiosmotic theory and on the protonophoric properties of chlorophenols. The quality of the QSAR models confirms the suitability of the SMP assay as a short-term prediction tool for aquatic toxicity of environmental pollutants acting on respiratory functions.

The toxicity of 14 organotin compounds was investigated by means of two short-term in vitro bioassays, the submitochondrial particle (SMP) test and the Microtox® test. The first bioassay makes use of SMPs and is based on the effects of toxicants on reverse electron transport, which is induced by adenosine triphosphate and succinate at the first site level of the respiratory chain. Microtox is a well known test system that uses marine luminescent bacteria and quantifies toxicity by measuring the reduction of luminescence caused by toxic chemicals. Very good agreement was observed between the median effective concentration (EC50) values determined for organotin compounds by means of the two bioassays. Toxicity depended on both the number and kind of organic substituents bound to the tin atom. It followed the order triorganotins > diorganotins ≈ tetraorganotins > monoorganotins. Within each series, butyltin and phenyltin compounds exhibited the highest toxicity. Microtox and SMP EC50 values were successfully correlated with toxicity data for aquatic organisms, demonstrating the usefulness of these bioassays as prescreening or complementary tools for monitoring aquatic toxicity. Moreover, to investigate the suitability of the two assays in providing information on the mechanism of toxic action of organotins, EC50 values were correlated with various physicochemical and structural parameters of the tested compounds. The results obtained showed that these parameters are poor descriptors of organotin toxicity; in particular, the poor correlations found between EC50 values and log Kow could be ascribed to the fact that different modes of action govern the biological activity of mono-, di-, tri-, and tetraorganotin compounds, respectively.

An enzymatically active monomeric analog of human copper,zinc superoxide dismutase (SOD) was produced by replacing four hydrophobic residues at the dimer interface of wild-type SOD (WT) with hydrophilic residues in a manner which has maintained the overall protein charge (i.e., Phe50Glu, Gly51Glu, Val148Lys, Ile151Lys). This analog has been characterized by (1) molecular weight determination, (2) several spectroscopic techniques probing catalytic site geometry and (3) enzymatic activity measurements at various ionic strengths. At physiological ionic strength the present monomer has sizable activity being five times that of a previously reported monomeric analog carrying only two of these substitutions with an overall charge two units more negative than WT (i.e., Phe50Glu, Gly51Glu). Unlike the catalytic activity of the latter analog, this one reveals an ionic strength dependency like that of WT. Enzymatic behavior is discussed with regard to factors affecting substrate diffusion towards the catalytic site.

New 3-dimensional molecular indices (WHIM descriptors) contain information about the whole molecular structure in terms of size, shape, symmetry and atom distribution. These indices are calculated from (x,y,z)-coordinates of a molecule within different weighting schemes in a straightforward manner and represent a very general approach to describe molecules in a unitary conceptual framework. The obtained models confirm the high modeling power of the WHIM descriptors. In this work WHIM descriptors are used for modeling chlorophenols toxicity measured by other not previously considered biosensors. In particular, the attention is stressed on submitochondral particles (SMP) as toxicity biosensors recently proposed and their relationships with other biosensors are further investigated.

A mineralogical and micro‐chemical study was performed by X‐ray diffraction and SEM/EDS techniques in a reduced sediment sample from the Venice Lagoon to investigate selectivity and significance of two widely used acid extractions (cold IN HCl and hot 8N HNO3) for the evaluation of anthropogenic metal concentrations. Hydrochloric acid determines an incomplete and variable solubilization of authigenic iron sulphides, yielding a partial extraction of the associated metals (Hg, Mn, Zn and less Ni and Cu). Nitric acid is effective in solubilizing all sulphides, however, it leads to an overestimate of the anthropogenic metal content since it partially dissolves the lattice of some clay‐minerals, particularly chlorites. When these acid extractions are used to asses metal pollution in aquatic ecosystems, the possible over or under‐estimate of the anthropogenic concentration must be taken into account, considering the mineralogical composition of the sediment.

The dependence of the activity of iron-containing superoxide dismutase from Escherichia coli on pH and ionic strength was investigated extensively in the ranges 7.7–11.0 and 0.02–0.25 M respectively. The results show that the enzymatic activity diminishes with both increasing pH and increasing ionic strength, reaching a minimum value at about pH 11. The activity decrease with increasing pH was ascribed to a gradual titration, by OH− ion, of the acid group Fe3+-H2O. This gives rise to a deprotonated enzymatic form with a residual activity which is about 10% that of the protonated form. The dependence of the activity on ionic strength was interpreted as a Debye-Huckel effect.

An in vitro bioassay procedure was used to investigate the toxic action of chlorophenols on mitochondrial respiratory parameters. The toxicity of these compounds was evaluated by determining their effects on the energy-coupled reverse electron transfer (RET) in submitochondrial particles (SMPs) from beef heart mitochondria. The bioassay procedure is based on the spectrophotometric recording of the effects of toxicants on the rate of NAD{sup +} reduction, induced by ATP and succinate at the first site level of the respiratory chain. The toxicity end point was expressed as the toxicant concentrations that causes 50% inhibition of NAD{sup +} reduction rate (EC50). The EC50 values determined for the 14 tested chlorophenols ranged from 17 mg/L for 2-chlorophenol to 0.081 mg/L for pentachlorophenol, indicating a general trend of increasing toxicity with increasing chlorine substitution. Among chlorophenol isomers, which have the same number of chlorine atoms, a lesser toxicity was associated with ortho-substituted chlorophenols, whereas meta-substituted chlorophenols were much more toxic. The EC50 values were compared with the toxicity data for a variety of bioassays, by means of linear regression analysis. High degrees of correlation obtained with toxicity tests involving different freshwater species demonstrate the ability of SMPs to reproduce the toxic effects of themore » tested compounds upon aquatic organisms. This supports the assessment that the respiratory chain is the main target of this class of toxicants. Results obtained with chlorophenols and, in previous studies, with other environmental contaminants confirm the suitability of the SMP bioassay as a prescreening or complementary short-term test for monitoring aquatic toxicity.« less

Grain-size and redox potential distributions in sediments were used as tracers to investigate environmental conditions in a shallow water area of the Venice Lagoon subjected to summer anoxic events. Data are presented showing different environmental characteristics within the study area. The results illustrate the reliability of these tracers to acquire a preliminary knowledge of the aquatic ecosystem behavior. Even small differences in morphology and hydrodynamics are observable because of the marked influence they exert on sediment parameters.

The effects on mitochondrial respiratory parameters of linear alkylbenzene sulfonates (LAS), nonylphenol polyeth-oxylates (NPEO), and some of their biotransformation products, namely sulfophenyl carboxylates (SPCs), nonylphenol (NP), and nonylphenoxy acetic acid (NP1EC), were recorded by using the in vitro response of submitochondrial particles (SMP) from beef heart. The toxicity of these compounds was estimated by determining their effects on the energy-coupled reverse electron transfer (RET), which is induced by ATP and succinate at the first site level of the respiratory chain and reduces exogenous NAD+ to NADH. The toxicity of the substances, expressed as the toxicant concentration decreasing the reduction rate of NAD+ to an extent of 50% (EC50), ranged from 0.61 mg/L for a commercial LAS mixture to 18,000 mg/L for individual SPCs; from 1.3 mg/L for NPEO, with an average of 10 ethoxy units, to 8.2 and 1.8 mg/L for NP1EC and NP, respectively. These results were related to the molecular structure of each compound class and compared with the toxicity values obtained by a variety of biological systems currently used for toxicity testing. The acute toxicity data have demonstrated that (a) the SMP bioassay is suitable for reproducing the toxicological response of whole organisms, such as fishes and invertebrates, to the tested chemicals; and (b) the hydrophobic moiety of these compounds plays a significant role in eliciting their toxic effects. From a toxicological standpoint, attention must be paid to the occurrence in natural waters of residual LAS, whereas in the case of NPEO both unaltered surfactant and all biotransformation products need to be identified and quantified.

Anthropogenic heavy metals and grain size were determined in 15 surface sediment samples from the Cona Marsh, an estuarine area of the Venice Lagoon (Italy). The investigation is based on separate analyses of both metal concentrations obtained with two acid extractions and particle-size percentages by laser light scattering. The grain size was also measured in sample aliquots previously submitted to the organic matter removal to disperse mineral-organic aggregates, visualized by scanning electron microscopy. These aggregates apparently shift the particle-size spectrum toward larger diameters, and the intimate association between metals and sediment particles is more evident after their dispersion. The comparison of the two distributions showed a strong correlation between heavy metals and finer particles content in the sediment (d < 7.8 μm). The investigation finally illustrated a spatial zoning of the marsh into three sectors with different degrees of pollution, in relation to the water circulation determined by river discharge and tidal forcing.

The kinetic behaviour of native bovine erythrocyte Cu,Zn superoxide dismutase (N-SOD) and of its derivatives by reaction with polyethylene glycol, acetic and succinic anhydrides has been investigated here in detail. Their responses to changes of pH and ionic strength (I) have been used as a probe for quantitatively displaying the relevance to kinetic rate constant of superficial positive charges driving the superoxide ion (O2-) toward the enzyme's active site. Overall kinetic trends indicate that this long-range O2- electrostatic guidance is essentially due to the positive charges of the amino-acid residues Lys-120 and Lys-134 which are strategically located around the active site. The comparison between the kinetic data obtained from N-SOD and those from polyethylene-glycolated SOD (PEG-SOD) enabled us to state that in PEG-SOD an O2(-)-steering positive electrostatic force, halved in comparison with N-SOD, is still operating, and that only Lys-120 is linked in the reaction of N-SOD with PEG. Elimination of the electrostatic driving force, carried out either by deprotonation of lysine amino groups at high pH, or by their neutralization with succinic anhydride and acetic anhydride, or by ionic screening at high ionic strength, always lowered the kinetic rate constant to a value of approx. 3 x 10(8) M-1.s-1. This value is about 15 times smaller than that measured in the presence of the reactant-steering mechanism and represents the k value of the reaction limited by pure diffusion. Finally, the kinetic behaviour of acetylated SOD and succinylated SOD demonstrated the inhibitor effect of OH- at strongly alkaline pH.

The purpose of this study was to evaluate the sensitivity to tributyltin (TBT) of two aquatic organisms belonging to two different levels of the feeding chain: Selenastrum capricornutum alga and Daphnia magna crustacean. The EC50 value at 96 h for Selenastrum capricornutum and the ones at 24 h and 45 h for Daphnia magna with relative confidence intervals and NOEC values were calculated. A partial stimulus effect was observed both for alga and crustacean at very low TBT concentrations. The data obtained were compared both with toxicity TBT values, obtained from bibliography, concerning invertebrates, fishes and algae and with the results obtained by a new in vitro test utilizing submitochondrial particles (SMP). The response of SMP to TBT confirmed that the inhibition of the respiratory functions is one of the mechanisms by which the TBT causes toxicity to intact cells as well as to more complex organisms.

An extensive investigation based on the redox potential and grain size distributions was made on the sediment of a Venice Lagoon mud flat subjected to excessive growth of macroalgae. Redox potential and grain size measurements are proved useful “tracers” for, respectively, oxygen bearing and consuming processes in the water-sediment column and hydrodynamical behavior inside the mud flat. Depth measurements and a considerable number of sites with respect to the size of the area studied are needed to obtain an outline of the behavior of the water body in response to stress conditions caused by human activities. With respect to the top 15-cm-thick sediment layer where EH variations occur, the mud flat is subdivisible into sectors with different characteristics. Positive or near-zero EH values were recorded in zones characterized by sparse macroalgae growth and a high content of coarse sediments (diameter ⩾44 µm). On the contrary, very negative EH values were found in zones affected by overabundant macroalgae bloom and with a higher presence of fine-grained sediment (diameter ⩽44 µm). The clear relationships between algae presence in the mud flat and both the redox potential and grain size characteristics of the sediment emphasize the hydrodynamics as a “critical factor” determining the variations of the environmental conditions in the ecosystem.

The dependence of the magnetic relaxation rates of 1H and 19F- on temperature, frequency, pH and N-3 concentration, were measured in solutions of Manganese-containing superoxide dismutase of Bacillus stearothermophilus, and were compared to activity measurements, in order to obtain some information on the structure and dynamics at Mn(III) present in the active site of the enzyme. The experimental data lead us to hypothesize the presence of two binding sites in the coordination sphere of the enzyme bound Mn(III), which are accessible to water and anions and have different chemical and spectroscopic properties. NMR measurements carried out in the presence of competitive inhibitors and the pH dependence of both NMR relaxation rates suggest that F-, N-3 and OH- ions bind to one site, while a water molecule binds to the other one. The stability constant values of the complexes between these anions and the enzyme are reported. The influence of the anions on activity and the pH dependence of NMR parameters are discussed.