Cannabis sativa L. and Brassica juncea L. grown on arsenic-contaminated industrial soil: potentiality and limitation for phytoremediation.

Phytoremediation represents a natural method to remove contaminants from soil. The goal of this study was to investigate the potential of phosphate-assisted phytoremediation by two energy crops, Cannabis sativa L. and Brassica juncea L., for the sustainable remediation of heavily arsenic-contaminated industrial soil. The two species were investigated for uptake, translocation, and physiological effects of arsenic and phosphate in a microcosm test. Although C. sativa and B. juncea were symptomless when grown in arsenic-contaminated soil, an important reduction of biomass (50 and 25%, respectively) was observed as a stress marker. Phytotoxicity and cytotoxicity effects promoted by contaminated soils were investigated in both the species and a model plant for ecotoxicity studies, Vicia faba L., which is the most developed model to test genotoxicity effects in terms of chromosomal aberration and micronuclei presence. The higher amount of arsenic was found in C. sativa and B. juncea roots (on average 1473 and 778 mg kg-1, respectively), but both species were able to uptake and translocate arsenic in leaves and stems, up to 47.0 and 189 mg kg-1, respectively. Phosphate treatment had no effect on arsenic uptake in none of the crop, but significantly improved the plant performance. Biomass production resulted similar to that of B. juncea control plants. Antioxidant enzymatic activities and photosynthetic performance responded differently in the two crops. The present investigation provides new insight for a proficient selection of the most suitable crop species for sustainable phytomanagement of a highly polluted As-contaminated site by coupled phytoremediation-bioenergy approach.

Interplay between extracellular polymeric substances (EPS) from a marine diatom and model nanoplastic through eco-corona formation.

The occurrence of nanoplastics in oceans' surface waters is no more a hypothesis and it could severely affect marine organisms from different trophic levels. Nanoscale particles interaction with dissolved natural organic matter (NOM) significantly influence their behaviour and consequently bioavailability and toxicity to marine species. Extracellular polymeric substances (EPS) are among the main components of the NOM pool in seawater yet have been so far little investigated for their effect in altering the physical-chemical properties of nanosized objects. Here we employed EPS from marine diatom Phaeodactylum tricornutum to study the evolution of an eco-corona formation upon incubation with 60 nm carboxylated polystyrene nanoparticles (PS-COOH NPs), as proxy for nanoplastics in seawater. EPS significantly reduced PS-COOH NPs aggregation rate compared to biomolecule free natural seawater (NSW) and caused the formation of complexes constituted by both carbohydrate and protein components. Size Exclusion Chromatography (SEC) revealed four main distinct groups of peaks, spanning from high (>100 kDa) to low molecular weight (20 kDa) molecules, characterized by a high chemical heterogeneity. The lowering of the chromatographic signals detected after EPS incubation with PS-COOH NPs, mainly in the eluates at high molecular weight, suggests that an important fraction of EPS remained adsorbed on PS-COOH NPs. In agreement, SDS-PAGE analysis of proteins adsorbed on PS-COOH showed the occurrence of an eco-corona formed by proteins in the range of molecular weight 30-100 kDa. No toxicity to diatoms was observed upon PS-COOH exposure (72 h, 1-100 mg L-1) even by adding a further source of exogenous EPS during exposure. Moreover, the addition of EPS reduced ROS production, even when cells were incubated with PS-COOH NPs at 10 and 50 mg L-1, suggesting an antioxidant scavenging activity of EPS.

Chemical composition of essential oil from plants of abandoned mining site of Elba island.

The essential oil composition of three spontaneous species growing in an abandoned mining of Elba island was analyzed by GC-MS. A total of 194 compounds were identified representing 73.7-100% of the whole oil composition. The essential oils of Cistus salvifolius and Dittrichia viscosa from this site showed different profiles in comparison with those from not polluted area, where oxygenated sesquiterpenes were the main class. Volatiles from D. viscosa growing in ex-mining area presented 10-epi-γ-eudesmol and α-eudesmol as main compounds while ß-caryophyllene and limonene were the main ones in not polluted area. Ambroxide and ambrial were the most important compounds in the essential oil from C. salvifolius harvested in polluted area while nonanal and tridecanal were the main compounds in control samples. Oxygenated monoterpenes were the most abundant class from both Lavandula stoechas samples, with fenchone and camphor as main compounds.

Overcoming limitation of "recalcitrant areas" to phytoextraction process: The synergistic effects of exogenous cytokinins and nitrogen treatments.

The aim of the present work was to test the efficiency of the phytoextraction process involving the use of exogenous phytohormone (cytokinins, CKs) and fertilizer (nitrogen, N) treatments in phytotechnologies to address risk management in "recalcitrant areas". The CKs and N treatments, alone or combined (CKs + N) in a Modulated Application (MA), were tested on the crop plant Helianthus annuus, common to Mediterranean area, fast growing and with high biomass production. Plants were grown on boron (B) contaminated sediments (collected from a geothermal area located in Tuscany (Italy). Plant growth, B uptake, together with plant stress parameters were investigated. Boron is easily taken up and translocated by some crop plants, but the high phytotoxicity can dramatically impact the plant growth and consequently the applicability and efficiency of the phytoextraction process. As indicators of plant stress, oxidative balance and photosynthetic parameters were investigated to give a deeper insight of phytotoxic mechanisms. Results showed that while each treatment (CKs and N alone) had significantly positive effects on plant health, the MA treatment provided a synergistic effect on morphological parameters and biomass production as a whole. After MA treatment, plants showed antioxidant activity comparable to that of the control (unpolluted sediments) and showed an increase of net photosynthesis. Moreover, our data showed very high values of B uptake and translocation (about 800 mg kg-1 in shoots), without any alteration triggered by the treatments (CKs and N alone or combined in MA). B phytoextraction resulted increased about fivefold with the MA treatments, while each treatment alone increased only two or three folds when treated with either CKs or N. The MA treatment is not "contaminant specific", so it could be applied in other "recalcitrant areas" where different types of contaminations occur, in order to overcome limitations of plant growth.

TiO2 nanoparticles in seawater: Aggregation and interactions with the green alga Dunaliella tertiolecta.

Titanium dioxide nanoparticles (TiO2 NPs) have been widely employed in industrial applications, thus rising concern about their impact in the aquatic environment. In this study we investigated the chemical behaviour of TiO2 NPs in the culture medium and its effect on the green alga Dunaliella tertiolecta, in terms of growth inhibition, oxidative stress, ROS (Reactive Oxygen Species) accumulation and chlorophyll content. In addition, the influence of exopolymeric substances (EPS) excreted by the microalgae on the stability of NPs has been evaluated. The physicochemical characterization showed a high propensity of TiO2 NPs to form micrometric-sized aggregates within 30min, large enough to partially settle to the bottom of the test vessel. Indeed, an increasing amount of TiO2 particles settled out with time, but the presence of EPS seemed to mitigate this behaviour in the first 6h of exposure where the main effects in D. tertiolecta were observed. TiO2 NPs did not inhibit the 72-h growth rate of D. tertiolecta, nor affected the cellular chlorophyll concentration in the range 0.01-10mgL-1. The time-course of ROS production showed an initial transient increase of ROS in TiO2 NP-exposed algae compared to the control, concomitant with an enhancement of catalase activity. Interestingly, intracellular ROS was a small fraction of total ROS, the highest amount being extracellular. The occurrence of cell-mediated chemical transformations of TiO2 NPs in the external medium, related to the presence of EPS, has been evaluated. Our results showed that carbohydrates were the major component of EPS, whereas proteins of medium molecular weight (20-80kDa) were preferentially bound to TiO2 NPs, likely influencing their biological fate.

Response of spontaneous plants from an ex-mining site of Elba island (Tuscany, Italy) to metal(loid) contamination.

The release of large amounts of toxic metals in the neighboring sites of abandoned mine areas represents an important environmental risk for the ecosystem, because it adversely affects soil, water, and plant growth. The aim of the present study was to investigate the metal(loid) (As, Cr, Cu, Ni, Pb, and Zn) contents of native Mediterranean plants grown on the ex-mining area of Elba island (Italy), with the prospective of its recovery by further phytoremediation technology. Soil samples were collected and characterized for metal(loid) content in total and potentially available (EDTA-extractable) fractions. Arsenic was particularly high, being 338 and 2.1 mg kg-1 as total and available fractions, respectively. Predominant native species, namely Dittrichia viscosa L. Greuter, Cistus salviifolius L., Lavandula stoechas L., and Bituminaria bituminosa L., were analyzed for metal content in the different plant organs. D. viscosa exhibited the highest metal(loid) content in the leaves and the singular behavior of translocating arsenic to the leaves (transfer factor about 2.06 and mean bioconcentration factor about 12.48). To assess the healthy status of D. viscosa plants, the leaves were investigated further. The activities of the main antioxidant enzymes and the levels of secondary metabolites linked to oxidative stress in plants from the ex-mining area were not significantly different from those of control plants, except for a lower content of carotenoids, indicating that native plants were adapted to grow in these polluted soils. These results indicate that D. viscosa can be suitable for the revegetation of highly metal-contaminated areas.

Toxicological effects of CdSe/ZnS quantum dots on marine planktonic organisms.

Quantum dot nanoparticles (QDs) are proposed as novel materials for photovoltaic technologies, light emitting devices, and biomedical applications. In this study we investigated the effect of CdSe/ZnS QDs on the growth rate of four microalgae: the diatom Phaeodactylum tricornutum, the cryptophyte Rhinomonas reticulata, the prymnesiophyte Isochrysis galbana and the green alga Dunaliella tertiolecta. In addition we analyzed the effect of QDs on the copepod Acartia tonsa. A classical acute test (48-h) with embryos was carried out to evaluate naupliar survival. Moreover, a 4-day chronic test with adult copepods was conducted to evaluate their fecundity (embryos f(-1)day(-1)) and egg hatching success. QDs in the range from 1 to 4nM gradually inhibited the growth rate of P. tricornutum, I. galbana, R. reticulata and D. tertiolecta with an EC50 of 1.5, 2.4, 2.5 and 4.2nM, respectively. Acute tests with A. tonsa (QD concentration tested from 0.15 to 1.5nM) showed an increased naupliar mortality in response to QD treatment, exhibiting an EC50 of 0.7nM. Chronic test showed no negative effect on egg production, except on the last two days at the highest QD concentration (2.5nM). No significant reduction of the percentage of egg hatching success was recorded during the exposure. Toxicity assessment of QDs was also investigated at the molecular level, studying heat shock protein 70 gene expression (hsp 70). Our results indicate that hsp70 was upregulated in adults exposed 3 days to 0.5nM QDs. Overall, these results suggest that species unable to swim along the water column, like P. tricornutum and early hatched copepods, could be more exposed to toxic effects of QDs which tend to aggregate and settle in seawater.

A SELDI-TOF approach to ecotoxicology: comparative profiling of low molecular weight proteins from a marine diatom exposed to CdSe/ZnS quantum dots.

Quantum dots (QDs), namely semiconductor nanocrystals, due to their particular optical and electronic properties, have growing applications in device technology, biotechnology and biomedical fields. Nevertheless, the possible threat to human health and the environment have attracted increasing attention as the production and applications of QDs increases rapidly while standard evaluation of safety lags. In the present study we performed proteomic analyses, by means of 2D gel electrophoresis and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry (SELDI-TOF-MS). We aimed to identify potential biomarkers of exposure to CdSe/ZnS quantum dots. The marine diatom Phaeodactylum tricornutum exposed to 2.5nM QDs was used as a model system. Both 2DE and SELDI showed the presence of differentially expressed proteins. By Principal Component Analysis (PCA) we were able to show that the differentially expressed proteins can discriminate between exposed and not exposed cells. Furthermore, a protein profile specific for exposed cells was obtained by SELDI analysis. To our knowledge, this is the first example of the application of SELDI technology to the analysis of microorganisms used as biological sentinel model of marine environmental pollution.

The response of Phaeodactylum tricornutum to quantum dot exposure: Acclimation and changes in protein expression.

Nanotechnology has a great potential to improve life and environmental quality, however the fate of nanomaterials in the ecosystems, their bioavailability and potential toxicity on living organisms are still largely unknown, mainly in the marine environment. Genomics and proteomics are powerful tools for understanding molecular mechanisms triggered by nanoparticle exposure. In this work we investigated the effect of exposure to CdSe/ZnS quantum dots (QDs) in the marine diatom Phaeodactylum tricornutum, using different physiological, biochemical and molecular approaches. The results show that acclimation to QDs reduced the growth inhibition induced by nanoparticles in P. tricornutum cultures. The increase of glutathione observed at the end of the lag phase pointed to cellular stress. Transcriptional expression of selected stress responsive genes showed up-regulation in the QD-exposed algae. A comparison of the proteomes of exposed and unexposed cells highlighted a large number of differentially expressed proteins. To our knowledge, this is the first report on proteome analysis of a marine microalga exposed to nanoparticles.

Temperature and pressure dependence of azurin stability as monitored by tryptophan fluorescence and phosphorescence. The case of F29A mutant.

The effects of a single-point, F29A, cavity-forming mutation on the unfolding thermodynamic parameters of azurin from Pseudomonas aeruginosa and on the internal dynamics of the protein fold under pressure were probed by the fluorescence and phosphorescence emission of Trp48, deeply buried in the compact hydrophobic core of the macromolecule. Pressure-induced unfolding, monitored by the shift in the fluorescence spectrum, led to a volume change of 70-90mlmol(-1). The difference in the unfolding volume between F29A and wild type azurin was smaller than the volume of the space theoretically created in the mutant, indicating that the cavity is, at least partially, filled with water molecules. The complex temperature dependence of the unfolding volume, for temperatures up to 20°C, suggests the formation of an expanded form of the protein and highlights how the packing efficiency of azurin appears to contribute to the magnitude of internal void volume at any given temperature. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime. At 40°C the application of pressure in the predenaturation range initially decreases the internal flexibility of azurin, the trend eventually reverting on approaching unfolding. The main difference between wild type and the cavity mutant is the inversion point which happens at 300MPa for wild type and at 150MPa for F29A. This suggests that, for the cavity mutant, pressure-induced internal hydration is more dominant than any compaction of the globular fold at relatively low pressures.

Interaction of CdSe/ZnS quantum dots with the marine diatom Phaeodactylum tricornutum and the green alga Dunaliella tertiolecta: a biophysical approach.

In this study, we investigated the interaction of nanoparticles, such as CdSe/ZnS quantum dots (QDs), with the marine diatom Phaeodactylum tricornutum and the green alga Dunaliella tertiolecta, as biological models in the marine environment. Fluorescence kinetics measurements indicated that 30min after dispersion in seawater QDs lost the 60% of the initial emission intensity, possibly due to the occurrence of aggregation processes. However, the presence of algae seemed to mitigate this effect. By using confocal microscopy, we highlighted the presence of QDs adsorbed on the surface of both algae, but not inside the cells. The toxicity of QDs was evaluated in terms of inhibition of growth rate, oxidative stress, and lipid peroxidation. QDs in the range of 1-2.5nM gradually inhibited the growth rate of P. tricornutum and increased the oxidative stress, as evinced by the increase in lipid peroxidation, reactive oxygen species (ROS) production and activity of two main antioxidant enzymes (superoxide dismutase and catalase). On the contrary, QDs did not inhibit the growth rate of D. tertiolecta, at most a modest stimulation was observed in the range of 0.5-2nM, suggesting a hormetic response. No effect in the parameters indicating oxidative stress was observed in the green alga. In conclusion our results showed that the biological effects were species-specific.

[Susceptibility rate to tigecycline and antibiotic-resistance among Klebsiella pneumoniae strains isolated in Intensive Care Unit]. / Suscettibilità alla tigeciclina e antibiotico-resistenza in ceppi di Klebsiella pneumoniae isolati in Unità di Terapia Intensiva.

Antibiotic resistance in Klebsiella pneumoniae strains is an increasing problem in a lot of hospitals. It is a public health emergency because it relates with high mortality rate among patients in Intensive Care Unit (ICU). From 1/1/2009 to 31/08/2010, in ICU of SS Annunziata Hospital of Taranto, 140 isolated Klebsiella pneumoniae strains were detected. The strain identification and antimicrobial susceptibility testing were performed using a Vitek2 automated system. These isolate showed a low level of susceptibility to levofloxacin (3.4%), ciprofloxacin (6.2%), ceftazidime (2.8%) and piperacillin/tazobactam (8%). We reported also that the 10% and 13.9% of them were susceptible to meropenem and imipenem. An anti-Klebsiella pneumoniae activity in vitro to tigecycline was present in 64.6% of isolates while almost all strains (56/58) tested to colistin were susceptible. In order to our data of worryng high multiclass drug resistance including tygecicline, it needs to apply appropriate measures of surveillance and antibiotic prescription to avoid rapid spread of these mutiresistant strains in other areas.

Chemical stability of CdSe quantum dots in seawater and their effects on a marine microalga.

With the increasing use of nanotechnologies, it is expected that nanomaterials end up in natural aquatic systems, from freshwater to the sea. In this work we studied the chemical behaviour of water-soluble CdSe QDs in seawater and their effects on the marine diatom Phaeodactylum tricornutum, as a model of a biological receptor in the marine environment. We evaluated QD toxicity in terms of growth rate inhibition, oxidative stress and ROS accumulation. In addition, we used the synthesis of phytochelatins (PCs) as a biomarker of the presence of free Cd(2+) ions released from QDs. The optical and chemical characterization demonstrated the propensity of QDs to aggregate after dispersion in raw seawater. In addition, bare CdSe QDs, lacking the ZnS shell, underwent a salinity-dependent degradation process. Short-term exposure experiments showed that the ease of degradation of QDs in seawater correlated with the synthesis of PCs in P. tricornutum cells. Long-term exposure experiments, carried out with the most stable CdSe/ZnS QDs, showed that algae accumulated Cd, but synthesized negligible amounts of PCs. Since the production of PCs is a specific signal of the presence of bioavailable metal ions, our findings suggest that QDs, associated to P. tricornutum cells, did not release PC-inducing metal species. Our data also showed a gradual decrease in algal growth rate at concentrations of QDs higher than 0.5nM. Measurements of the activity of the antioxidant enzymes showed that superoxide dismutase (SOD) and catalase (CAT) activities were increased by exposure to [QDs]≥0.5 nM, whereas ascorbate peroxidase (APX) and glutathione reductase (GR) activities were not significantly affected. The increase in SOD and CAT activity can be considered a symptom of oxidative stress induced by an enhanced production of ROS. This hypothesis was confirmed by the concomitant increase in the intracellular ROS concentration.

Nitrogen fertilizer improves boron phytoextraction by Brassica juncea grown in contaminated sediments and alleviates plant stress.

In this study we evaluated the effect of different fertilizer treatments on Brassica plants grown on boron-contaminated sediments. Experiments were conducted in the laboratory and on the lysimeter scale. At laboratory scale (microcosm), five different fertilizers were tested for a 35-d period. On the lysimeter scale, nitrogen fertilization was tested at three different doses and plants were allowed to grow until the end of the vegetative phase (70 d). Results showed that nitrogen application had effectively increased plant biomass production, while B uptake was not affected. Total B phytoextracted increased three-fold when the highest nitrogen dose was applied. Phytotoxicity on Brassica was evaluated by biochemical parameters. In plants grown in unfertilized B-contaminated sediments, the activity of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and pyrogallol peroxidase (PPX) increased, whereas catalase (CAT) decreased with respect to control plants. Addition of N progressively mitigated the alteration of enzymatic activity, thus suggesting that N can aid in alleviating B-induced oxidative stress. SOD activity was restored to control levels just at the lowest N treatment, whereas the CAT inhibition was partially restored only at the highest one. N application also lowered the B-induced increase in APX and PPX activities. Increased glutathione reductase activity indicated the need to restore the oxidative balance of glutathione. Data also suggest a role of glutathione and phytochelatins in B defense mechanisms. Results suggest that the nitrogen fertilizer was effective in improving B phytoextraction by increasing Brassica biomass and by alleviating B-induced oxidative stress.

A multidisciplinary approach for assessing the toxicity of marine sediments: analysis of metal content and elutriate bioassays using metal bioavailability and genotoxicity biomarkers.

The goal of this article is to verify the applicability of two different biological assays for studying a coastal area that is subject to anthropogenic inputs. Phytochelatins in the marine diatom Thalassiosira weissflogii were used as a biomarker of metal bioavailability. The frequency of genetic damage in the sensitive D7 strain of the yeast Saccharomyces cerevisiae was used to estimate the mutagenic potential. Biological assays were carried out using sediment elutriates. Sediments were collected at three selected sites located in the Gulf of Follonica (Tuscany, Italy), during a 2-year sampling period: Cala Violina (reference site) and the mouths of the rivers Pecora and Cornia, named sites V, P and C, respectively. The chemical characterization of each site was determined in terms of metal concentrations (As, Cd, Cr, Cu, Ni, Pb), measured in 11 sediment samples for each site. The results showed that metal concentrations in sediments from sites C and P were 2-10 times higher than the reference values (site V, year 2004). In addition, we found generally higher metal concentrations in the 2007 sediments than in the 2008 ones, including those of site V, due to the occurrence of an unexpected pollution event. This enabled us to obtain a pollution gradient to validate the proposed bioassays. In fact, the bioassays showed a potential biological hazard in the 2007 elutriates. Significant mutagenic effects were found in samples exhibiting higher concentrations of Cd and Cr. The induction of phytochelatins in T. weissflogii correlated positively with the Cd concentration in the elutriates.

Changes in the non-protein thiol pool and production of dissolved gaseous mercury in the marine diatom Thalassiosira weissflogii under mercury exposure.

Two detoxification mechanisms working in the marine diatom Thalassiosira weissflogii to cope with mercury toxicity were investigated. Initially, the effect of mercury on the intracellular pool of non-protein thiols was studied in exponentially growing cultures exposed to sub-toxic HgCl(2) concentrations. T. weissflogii cells responded by synthesizing metal-binding peptides, named phytochelatins (PCs), besides increasing the intracellular pool of glutathione and gamma-glutamylcysteine (gamma-EC). Intracellular Hg and PC concentrations increased with the Hg concentration in the culture medium, exhibiting a distinct dose-response relationship. However, considerations of the PCs-SH:Hg molar ratio suggest that glutathione could also be involved in the intracellular mercury sequestration. The time course of the non-protein thiol pool and Hg intracellular concentration shows that PCs, glutathione and gamma-EC represent a rapid cellular response to mercury, although their role in Hg detoxification seems to lose importance at longer incubation times. The occurrence of a process of reduction of Hg(II) to Hg degrees and subsequent production of dissolved gaseous mercury (DGM) was also investigated at lower Hg concentrations, at which the PC synthesis doesn't seem to be involved. The significant (P<0.01) correlation between the cellular density in solution and the production of DGM suggests that this diatom is capable of directly producing DGM, both in light and dark conditions. This finding has been confirmed by the absence of DGM production in the culture media containing formaldehyde-killed cells. Finally, the relationship between these two different pathways of Hg detoxification is discussed.

A phytochelatin-based bioassay in marine diatoms useful for the assessment of bioavailability of heavy metals released by polluted sediments.

The aim of the present work was to develop a new bioassay involving the presence of phytochelatins (PCs), detoxifying intracellular metal-binding peptides, in microalgae as response to metal bioavailability in re-suspensions of metal-polluted marine sediments. For this purpose, the synthesis of PCs has been studied in laboratory cultures of three marine diatoms, namely Phaeodactylum tricornutum, Thalassiosira weissflogii and Skeletonema costatum, exposed to elutriates of sediments collected in a polluted coastal area in the province of Pisa (Tuscany, Italy). Short- and long-term incubations in the elutriates of two marine sediments (named A and B) exhibited an increase of PCs synthesis in all the phytoplanktonic species examined, when the elutriate concentration increased from 0% to 100%. Elutriate B, which was mainly contaminated by Cd, was shown to be more effective to induce PCs than elutriate A, which was richer in Cu and TOC. The results show that the PCs response, in the microalgae examined, was species-specific. Our data also show that the PCs synthesis occurred before the growth rate was affected, thereby indicating that PCs can be considered as an early warning response of metal exposure. The PCs response in exponentially growing cultures of T. weissflogii, which was found to be the most sensitive alga, increased when the initial cellular density decreased. Finally, the positive relationship obtained between the cellular PCs concentration in T. weissflogii and the degree of metal contamination of the elutriates from twelve sediments collected in a metal-polluted coastal area, confirmed that the PCs-induction test can be applied in field studies. In conclusion, the response of these marine microalgae suggests that these cellular peptides could represent an excellent biomarker of metal exposure, which is useful for the assessment of sediment toxicity, by carrying out PCs-induction tests on sediment elutriates.

Phytochelatins in the diatom Phaeodactylum tricornutum Bohlin: an evaluation of their use as biomarkers of metal exposure in marine waters.

The feasibility of a bioassay based on the synthesis of phytochelatins to assess metal pollution in aquatic environments was evaluated by using the marine diatom Phaeodactylum tricornutum. Short-term incubation experiments carried out in EDTA-buffered artificial seawater showed increasing cellular phytochelatin concentration with increasing free cadmium, lead or copper in the medium, indicating that phytochelatins behave as a biomarker of exposure to the bioavailable metal fraction. A linear dose-response relationship between metal exposure and phytochelatin synthesis was found in natural seawater samples enriched with known amounts of heavy metals. Phytochelatin induction tests carried out on polluted seawater samples showed an enhanced response compared to that obtained in unpolluted seawater. This finding was found to be consistent mainly with a copper contamination.

Determination and characterization of phytochelatins by liquid chromatography coupled with on line chemical vapour generation and atomic fluorescence spectrometric detection.

Liquid chromatography (LC) coupled on line with UV/visible diode array detector (DAD) and cold vapour generation atomic fluorescence spectrometry (CVGAFS) has been developed for the speciation, determination and characterization of phytochelatins (PCs). The method is based on a bidimensional approach, e.g. on the analysis of synthetic PC solutions (apo-PCs and Cd(2+)-complexed PCs) (i) by size exclusion chromatography coupled to UV diode array detector (SEC-DAD); (ii) by the derivatization of PC -SH groups in SEC fractions by p-hydroxymercurybenzoate (PHMB) and the indirect detection of PC-PHMB complexes by reversed phase liquid chromatography coupled to atomic fluorescence detector (RPLC-CVGAFS). MALDI-TOF/MS (matrix assisted laser desorption ionization time of flight mass spectrometry) analysis of underivatized synthetic PC samples was performed in order have a qualitative information of their composition. Quantitative analysis of synthetic PC solutions has been performed on the basis of peak area of PC-PHMB complexes of the mercury specific chromatogram and calibration curve of standard solution of glutathione (GSH) complexed to PHMB (GS-PHMB). The limit of quantitation (LOQ) in terms of GS-PHMB complex was 90 nM (CV 5%) with an injection volume of 35 microL, corresponding to 3.2 pmol (0.97 ng) of GSH. The method has been applied to analysis of extracts of cell cultures from Phaeodactylum tricornutum grown in Cd-containing nutrient solutions, analysed by SEC-DAD-CVGAFS and RPLC-DAD-CVGAFS.