Chemical and structural aspects of fresh and fossil marine mollusc shells investigated by mid-infrared and near-infrared spectroscopy with the support of statistical and multivariate methods.

In the present study, we applied Fourier transform infrared (FTIR) and Fourier transform near infrared (FTNIR) spectroscopy to investigate some specific structural aspects of Patella caerulea, Mytilus edulis, Ostrea edulis, and Calista chione shells sampled in different sites. Moreover, for Ostrea edulis and Calista chione, the present study also included fossil samples. As far as FTIR spectroscopy is concerned, the support of statistical and multivariate methods such as the average spectrum (AV), spectral deconvolution, and two-dimensional correlation analysis (2DCOS) allowed to detect structural differences existing within the same mollusc species as a function of the sites they come. These differences can be reasonably linked to the local environmental conditions, which affect the biomineralization pattern of shell formation and growth. These structural differences are related to the calcite, aragonite, Mg-calcite contents, and interactions, as presently observed for fresh and fossil shells. The application of 2DCOS and deconvolution to FTIR spectra also showed the role of the amorphous calcium carbonate (ACC) in the structural characterization of shells, then suggesting the use of a new parameter, the calcite and aragonite to ACC (CAACC) ratio, as a new measurement for the structural characterization of shells. At last, FTNIR spectroscopy allowed detecting the presence of α-helix and ß-sheet protein structures in the shells. The results of this study show that also FTIR and FTNIR spectroscopy are able to discern differences in structural characteristics of mollusc shells, a field of environmental studies where scanning electron microscopy and X-ray diffraction are the more widely used methods.

Antarctic snow: metals bound to high molecular weight dissolved organic matter.

In this paper we studied some heavy metals (Cu, Zn, Cd, Pb, As, U) probably associated to high molecular weight organic compounds present in the Antarctic snow. Snow-pit samples were collected and analysed for high molecular weight fraction and heavy metals bound to them by means of ultrafiltration treatment. High molecular weight dissolved organic matter (HMW-DOM) recovered by ultrafiltration showed a dissolved organic carbon concentration (HMW-DOC) of about 18-83% of the total dissolved organic carbon measured in Antarctic snow. The characterisation of HMW-DOM fraction evidenced an ageing of organic compounds going from surface layers to the deepest ones with a shift from aliphatic compounds and proteins/amino sugars to more high unsaturated character and less nitrogen content. The heavy metals associated to HMW-DOM fraction follows the order: Zn > Cu > Pb >> Cd âˆ¼ As âˆ¼ U. The percentage fraction of metals bound to HMW-DOM respect to total metal content follows the order: Cu >> Pb > Zn, Cd in agreement with humic substance binding ability (Irwing-William series). Going down to depth of trench, all metals except arsenic, showed a high concentration peak corresponding to 2.0-2.5 m layer. This result was attributed to particular structural characteristic of organic matter able to form different type of complexes (1:1, 1:2, 1:n) with metals.

Chemical composition and surfactant characteristics of marine foams investigated by means of UV-vis, FTIR and FTNIR spectroscopy.

In this study, we collected the ultraviolet-visible (UV-vis), Fourier transform infrared (FTIR) and Fourier transform near-infrared (FTNIR) spectra of marine foams from different sites and foams produced by marine living organisms (i.e. algae and molluscs) to retrieve information about their molecular and structural composition. UV-vis spectra gave information concerning the lipid and pigment contents of foams. FTIR spectroscopy gave a more detailed qualitative information regarding carbohydrates, lipids and proteins in addition with information about the mineral contents of foams. FTNIR spectra confirmed the presence of carbohydrates, lipids and proteins in foams. Then, due to the higher content of structural information of FTIR spectroscopy with respect to FTNIR and UV-vis, we join the FTIR spectra of marine foams to those of humic substance from marine sediments and to the spectra of foams obtained by living organisms. We submitted this resulting FTIR spectral dataset to statistical multivariate methods to investigate specific aspects of foams such as structural similarity among foams and in addition, contributions from the organic matter of living organisms. Cluster analysis (CA) evidenced several cases (i.e. clusters) of marine foams having high structural similarity with foams from vegetal and animal samples and with humic substance extracted from sediments. These results suggested that all the living organisms of the marine environment can give contributions to the chemical composition of foams. Moreover, as CA also evidenced cases of structural differences within foam samples, we applied two-dimensional correlation analysis (2DCORR) to the FTIR spectra of marine foams to investigate the molecular characteristics which caused these structural differences. Asynchronous spectra of two-dimensional correlation analysis showed that the structural heterogeneity among foam samples depended reasonably on the presence and on the qualitative difference of electrostatic (hydrogen bonds) and nonpolar (van der Waals and π-π) interactions involving carbohydrate proteins and lipids present. The presence and relevance of these interactions agree with the supramolecular and surfactant characteristics of marine organic matter described in the scientific literature.

FTIR spectroscopy supported by statistical techniques for the structural characterization of plastic debris in the marine environment: Application to monitoring studies.

We inserted 190 FTIR spectra of plastic samples in a digital database and submitted it to Independent Component Analysis (ICA) to extract the "pure" plastic polymers present. These identified plastics were polypropylene (PP), high density polyethylene (HDPE), low density polyethylene (LDPE), high density polyethylene terephthalate (HDPET), low density polyethylene terephthalate (LDPET), polystyrene (PS), Nylon (NL), polyethylene oxide (OPE), and Teflon (TEF) and they were used to establish the similarity with unknown plastics using the correlation coefficient (r), and the crosscorrelation function (CC). For samples with r<0.8 we determined the Mahalanobis Distance (MD) as additional tool of identification. For instance, for the four plastic fragments found in the Carretta carretta, one plastic sample was assigned to OPE due to its r=0.87; for all the other three plastic samples, due to the r values ranging between 0.83 and0.70, the support of MD suggested LDPET and OPE as co-polymer constituents.

Monitoring of marine mucilage formation in Italian seas investigated by infrared spectroscopy and independent component analysis.

The aim of this study is to present and to discuss some characteristics of recalcitrant organic matter mechanism and formation. These aggregates called mucilages that are produced by the degradation reactions of several algae, have been investigated by infrared (FTIR) spectroscopy. FTIR spectra of macroaggregates produced by different algal samples have been daily collected in order to investigate the steps of aggregation. Afterwards, they have been elaborated by means of Independent Component Analysis (ICA). ICA investigation of FTIR spectra showed that the global aggregation process of marine mucilage always consisted of two different phases or independent components (ICs). One IC is related to the first degradation step of algal cells leading to the production of mono and oligosaccharides with aminoacids and oligopeptides. The second IC is related to the polymerization of oligosaccharides with aminoacids and oligopeptides and to their interaction with less polar compounds such as lipids thus producing supramolecular structures. The emerging mechanisms of anomalous size aggregates of organic matter match those of natural organic matter aggregation. The approach we suggest is to use synthetic mucilages which allows to monitor the macroaggregates formation because it can hardly be performed by means of natural marine macroaggregates.

Molecular and structural characteristics in toxic algae cultures of Ostreopsis ovata and Ostreopsis spp. evidenced by FTIR and FTNIR spectroscopy.

In this article we investigated the compositional and structural characteristics of the principal biomolecules such as carbohydrates, proteins, lipids, nucleic acids and chlorophyll pigments present in biofilm cultures of Ostreopsis spp. and in batch cultures of Ostreopsis ovata. Our approach based on the use of infrared (FTIR) and near infrared (FTNIR) spectroscopy showed the marked differences existing between biofilm cultures and batch cultures. FTIR spectroscopy showed the higher contents of polysaccharides and chlorophyll pigments in O. ovata from batch cultures with respect to Ostreopsis spp. Second derivative FTIR spectroscopy showed different features concerning the secondary structure of proteins because in O. ovata samples the beta sheet and beta turn structures were observed whereas in Ostreopsis spp. samples the alpha helix structure was the most evident. FTNIR spectroscopy showed other structural differences observed existing between O. ovata and Ostreopsis spp. mainly related to hydrogen bond interactions determining more packed structures in the nucleus of O. ovata. In addition, the interpretation of FTIR and FTNIR spectral information was also supported by the application of two statistical methods, the independent component analysis (ICA) and the spectral cross correlation analysis (SCCA). ICA was used as spectral deconvolution technique to separate the effects of the interference bicarbonate ion from algal FTIR spectra so to verify the high similar qualitative composition of the three biofilm samples of Ostreopsis spp. At last, SCCA applied to FTIR and FTNIR spectra was useful to evidence some structural differences involving -CH and CH(2) groups of aliphatic chains in O. ovata and Ostreopsis spp. samples. Though preliminary, these results agree with some previous studies suggesting that the presence of different ecophysiological characteristics in O. ovata and Ostreopsis spp. depending on the parameters related to the condition growth.

Preliminary evaluation of hydrocarbon removal power of Caulerpa racemosa in seawater by means of infrared and visible spectroscopic measurements.

In this paper we tested the power of Caulerpa racemosa for removal hydrocarbons from seawater. C. racemosa was implanted in two aquariums filled with natural seawater having a hydrocarbon content lower than 0.05mg/L which is the detection limit of the FTIR spectrophotometric method used for the determination. One aquarium was submitted to sequential additions of hydrocarbons (n-esadecane 10, 20 and 40mg/L, n-docosane 15mg/L) and diesel fuels (20mg/L) while the second one remained uncontaminated and used as control. After any addition, hydrocarbon content in seawater was determined at regular time intervals (one or two days) and when comparable hydrocarbon contents (i.e. lower than 0.05mg/L) were again observed, the real removal power of hydrocarbons was verified by several spectroscopic measurements performed on algae from both aquariums. Total hydrocarbon contents in algae determined by infrared (FTIR) spectroscopy, always resulted higher in the polluted aquarium for all the concentrations of added pollutants. Further FTIR studies performed on algae showed the presence of marked quantitative and structural molecular modifications involving carbohydrates, proteins, lipids, nucleic acids and chlorophyll pigments in C. racemosa from the aquarium test. In addition, visible (VIS) spectroscopic examination of C. racemosa showed a reduction of chlorophyll pigments in the polluted aquarium with respect to the control one. At last, FTIR spectra all the algal samples submitted to hydrocarbon pollution were re-examined by means of two-dimensional correlation analysis, a statistical tool helpful for studying the dynamic evolution of any molecular and biological system submitted to an external perturbation producing compositional and structural changes. This approach showed differences among the molecular modifications caused by any type of hydrocarbon used, modifications related reasonably to the molecular dimensions and concentration of the added pollutants. All these spectroscopic evidences suggested that the removal power of C. racemosa depends on its metabolic activities and not only on a simple adsorption process.

The roles of carbohydrates, proteins and lipids in the process of aggregation of natural marine organic matter investigated by means of 2D correlation spectroscopy applied to infrared spectra.

In this paper the marine organic matter soluble in an alkaline medium called extractable humic substance (EHS), was extracted from three sediment samples of Tyrrhenian Sea and separated by precipitation at pH 2 in the two fractions of fulvic acids (FAs) and humic acids (HAs). FAs were further fractionated in seven sub-samples of different molecular weight (mw) by means of seven different ultrafiltration membranes operating in the range between mw<1 kDa and mw>100 kDa. Then the qualitative composition of each sample of fractionated FAs and HAs was studied by means of one-dimensional Fourier transform infrared spectroscopy in reflectance mode (FTIR-DRIFT) and by two-dimensional (2D) correlation spectroscopy both in wavelength-wavelength (WW) and in sample-sample (SS) mode. The application of 2D correlation WW spectroscopy allows to elucidate the different roles played by carbohydrates and proteins with respect to some lipid compounds such as fatty acids and ester fatty acids during the process of aggregate formations from mw approximately 1 kDa to higher size aggregates. In addition, 2D correlation WW spectroscopy allows to observe some peculiar interactions between carbohydrates and proteins in the formation of EHS aggregates, interactions which vary from a sample to another sample. The results of 2D correlation SS spectroscopy confirm the general evidences obtained by 2D WW spectroscopy and moreover, they also describe the formation of EHS aggregates as a complex process where evolutionary links and connectivity between aggregates of neighbour molecular size ranges are not evident. Two-dimensional correlation spectroscopy applied to FTIR spectroscopy shows to be a powerful tool for the investigation of the mechanisms involved in EHS aggregation because it supports the acquisition of structural information which sometimes can be hardly obtained by one-dimensional FTIR spectroscopy.

Morphological and physiological responses of barley plants grown on soils characterised by metal toxicity and metal deficiency.

Aim of this work was to investigate which are the effects on barley crops grown on two different soils: a soil lacking in Cu, an essential micronutrient (A) and a naturally polluted soil rich in lead, zinc, copper (B). In particular we investigated the relationship between some ecophysiological parameters such as biomass, chlorophyll concentration and guaiacolo peroxidase activity and the chemical-physical properties of the soils like pH, organic matter and heavy metal content. Because metals uptake by plants is strongly correlated with the bioavailable fraction rather then their total amount in a soil, we have measured also metal exchangeable forms, using a single extraction method (MgCl2 as extractant). Plants grown on soil B showed a metal content higher than background limits, whereas plants grown on soil A were characterised by a background Fe and Zn concentrations and by a tolerant Pb concentration. Conversely, Cu content in tissues of plants grown in soil A is found to be under the background limits. Copper-deficiency plants present chlorotic leaves followed by a reduced clorophyll content, while plants grown on metals contaminated soil showed an increase of peroxidase activity.

Metal bioavailability: how does its significance change in the time?

The significance of terms "metal bioavailability" and "bioavailable metal fraction" is evolved in the time, passing from a very simple concept to a complex concept bound to abiotic and biotic aspects. At the beginning metal toxicity was related to metal fraction present in water phase, than only free metal ion activity was considered and the free ion activity model (FIAM) was proposed. Successively, due to the exceptions observed and to the consciousness that metal bioavailability could be considered as dynamic characteristic the concept of metal bioavailability became very complex, depending on physical, chemical and biological factors.

The increase of iron phytoavailability in soils ammended with paper mill sludge.

In soils characterized by low organic matter and high pH values (7.5-8.59) iron availability to plants is limited even if the content of total Fe(III) is high. We have studied by Hordeum distichum plants the capability of paper mill sludge to increase the iron phytoavailable fraction in an alkaline soil. The effect of paper mill sludges adding to an iron-deficient soil was evaluated both considering the phyto-available fraction of iron and the distribution of organic carbon (hydrophobic, fulvic and humic carbon) in the soil before and after sludge adding. Iron concentration was determined in the different portions of Hordeum distichum plants grown on soils with and without paper mill sludges. Application of paper mill sludge induces an increase in the concentration of available Fe. This effect is probably due to the production of Fe chelators by soil microorganisms acting on sludge organic matter. The Fe chelators produced result in the solubilization of not soluble Fe-complexes present in the soil.

Transport and modification of humic substances present in Antarctic snow and ancient ice.

We performed a study of fulvic acids extracted from fresh and aged snow, and from recent and ancient ice in Antarctica. The fresh snow samples were collected in coastal and inland sites to evaluate the influence of the distance from the sea on organic matter transport. Moreover, in a site (Melbourne Mountain) samples were collected at different heights to study the influence of altitude on transport. The obtained results showed that dissolved fulvic acid concentrations are influenced neither by distance nor by height while particulate fulvic acid concentrations are influenced by both parameters. Moreover, the results showed that fulvic acids transported for a long distance can undergo chemical modifications. Chemical modifications are better evidenced by the analysis of samples taken in trenches at different depth, which showed structural changes attributable to the loss of nitrogen-containing compounds and to an increase in aromatic character of the structures due to reduction and/or condensation processes. With ageing, the humification process proceeds with heavy carbon losses as demonstrated by results obtained from fulvic acids isolated from ice aged between twenty-five thousand and seventy thousand years.

Laboratory studies on waste water sludge application for acid soil remediation.

In this work the effects of sludge disposal on pH values, buffer capacity and adsorption capacity of an acid soil were studied. A stabilized waste water sludge and a paper mill sludge were employed; the pH values of both sludges were higher than 8. The observed differences between soil-urban sludge and soil-paper mill sludge systems can be ascribed to the nature of the compounds present in the sludges and adsorbed on the soil. Both kinds of sludge are able to modify the natural pH of the soil, the buffer capacity and its capacity to retain metal. These modifications increase with increasing soil-sludge contact time and are higher for paper mill sludges. Temperature affects only the modifications due to urban sludge treatment.