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.

Molecular and structural changes induced by essential oil treatments in Vicia faba roots detected by FTIR and FTNIR spectroscopy.

Essential oils have recognized antimicrobial and antifungal properties which allow their utilization in agriculture like an alternative to pesticides, but their utilization requires the knowledge of all the potential structural changes and damages produced by the interaction with the vegetal organisms. In this paper, we investigated the effects of two essential oils, the tea tree oil (TTO) and the mixture of clove and rosemary oils (C + R), on the molecular structure of Vicia faba roots by Fourier transform infrared (FTIR) and Fourier near infrared transform (FTNIR) spectroscopy. FTIR spectroscopy showed structural modifications of the absorption bands related to DNA (1100 and 1050 cm-1, carbohydrate backbones, and nucleotide bands within 900 and 850 cm-1), proteins (1700 and 1600 cm-1 amide I band, 1580 and 1520 cm-1 amide II band), and lipids (methylene group of aliphatic chains between 2950 and 2800 cm-1). The changes in the secondary structures of proteins consisted of a denaturation depending on increased presence of random coil structures. In addition, in the samples treated with TTO oils, we observed the presence of protein oxidation, an effect negligible instead for the C + R-treated samples. The modified shapes of the infrared methyl bands of aliphatic chains suggested an increased lipid disorder which could also determine lipid peroxidation. The changes observed for the DNA structures at the highest concentration of the above essential oils can be related to the genotoxic effect of eugenol, an important constituent of both TTO and C + R mixture oils. FTNIR spectroscopy showed the modified shape of the second overtone bands belonging to methyl and methylene groups, between 8500 and 8000 cm-1. This confirmed the increased lipid disorders already observed by FTIR spectroscopy. The results obtained on the probe organism V. faba show that FTIR and FTNIR spectroscopy can become a useful support to the conventional cytogenetic tests used in the evaluation of the allelopathic uses of essential oils in agriculture.

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.

Molecular and structural changes induced by essential oils treatments in Vicia faba roots detected by genotoxicity testing.

Over the last few years, there has been an increased interest in exploiting allelopathy in organic agriculture. The aim of this investigation was to examine the effects of essential oil mixtures in order to establish their allelopathic use in agriculture. Two mixtures of essential oils consisting respectively of tea tree oil (TTO) and clove plus rosemary (C + R) oils were tested. Phytotoxicity and genotoxicity tests on the root meristems of Vicia faba minor were performed. A phytotoxic influence was particularly relevant for C + R mixture, while genotoxicity tests revealed significant results with both C + R oil mixture and TTO. Phenotypic analysis on Vicia faba minor primary roots following C + R oil mixture treatment resulted in callose production, an early symptom attributed to lipid peroxidation. The approach described in this study, based on genotoxicity bioassays, might identify specific DNA damage induced by essential oil treatments. These tests may represent a powerful method to evaluate potential adverse effects of different mixtures of essential oils that might be useful in alternative agriculture. Future studies are focusing on the positive synergism of more complex mixtures of essential oils in order to reduce concentrations of potentially toxic components while at the same time maintaining efficacy in antimicrobial and antifungal management.

Effects of essential oil treatments on the secondary protein structure of Vicia faba: a mid-infrared spectroscopic study supported by two-dimensional correlation analysis.

In this study we investigated the effects of essential oil treatments on the secondary protein structure of the Vicia faba roots, a bioindicator plant, in order to obtain information for the potential allelopathic uses of these oils as alternative to the use of pesticides in agriculture. We tested two mixtures of essential oils consisting of Tween 20-emulsions of tea tree oil (TTO) and Tween 20-emulsion of Clove and Rosemary (GARROM) essential oils respectively at three different oil concentrations each. The molecular modifications caused in Vicia faba by exposure to oil emulsions were investigated by FTIR spectroscopy in diffuse reflectance (DRIFT) mode. We considered the specific Amide I, Amide II and Amide VI bands by ordinary and second derivative spectroscopy and the results showed that both Tween 20-emulsion of GARROM and Tween 20-emulsion of TTO oils cause transitions among the secondary (α-helix, ß-sheet and ß-turn) structures with in addition the appearance of random coil structures in exposed samples. The Amide VI bands, placed between 500 and 600 cm(-1), confirmed the structural transitions observed for the Amide I bands. In addition we observed the presence of a protein oxidation effect for TTO treated samples, oxidation which resulted negligible instead for the GARROM oil samples. At last, FTIR spectra were also submitted to two-dimensional correlation analysis (2DCORR) and double two-dimensional correlation analysis (D2DCORR); the results confirmed the different effects caused by the two typologies of essential oils on the secondary protein structures of Vicia faba roots.

Determination of trace metal baseline values in Posidonia oceanica, Cystoseira sp., and other marine environmental biomonitors: a quality control method for a study in South Tyrrhenian coastal areas.

In this study, we investigated Cd, Cr, Cu, Pb, and Zn in the seagrass Posidonia oceanica (L.) Delile leaves and in the brown algae Cystoseira sp. sampled along a 280-km transect in the Tyrrhenian Sea, from the Ustica to Linosa Islands (Sicily, Italy) with the aim to determine their control charts (baseline levels). By applying the Johnson's (Biometrika 36:149-175, 1949) probabilistic method, we determined the metal concentration overlap ranges in a group of five biomonitors. Here, we propose the use of the indexes of bioaccumulation with respect to the lowest (L'i) and the highest (L i) extreme values of the overlap metal concentration ranges. These indexes allow the identification of the most opportune organism (or a suite of them) to better managing particular environmental conditions. Posidonia leaves have generally high L i indexes for Cd, Cu, Pb, and Zn, and this suggests its use as biomonitor for baseline marine areas. Our results confirm the high aptitude of Patella as a good biomonitor for Cd levels in seawater. From this study, Ustica resulted with higher levels of Cd, Cu, Pb, and Zn than the other Sicilian Islands.

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.

Baseline trace metals in seagrass, algae, and mollusks in a southern Tyrrhenian ecosystem (Linosa Island, Sicily).

Trace elements were analyzed in organisms collected at five sampling stations along coastal areas of Linosa Island, Sicily (southern Tyrrhenian Sea, Italy). Concentrations of Cd, Cr, Cu, Pb, and Zn were measured in Posidonia oceanica L. Delile tissues, the two brown algae Padina pavonica (L.) Thivy and Cystoseira sp., and the two gastropod mollusks Monodonta turbinata Born and Patella caerulea L. Seawater samples were also collected at each site to assess soluble metal concentrations and to gain relevant information on their bioaccumulation ability. Data were processed by multivariate techniques, such as principal component analysis (PCA) and linear discriminant analysis on PCA factors. The scoreplots obtained showed that the pollutant distribution is more significantly correlated with species than with sites. For seaweeds, P. oceanica was associated with higher Cd, Cu, and Zn levels; Padina species had higher Cr concentrations, and Cystoseira had higher Pb levels. For mollusks, Monodonta species had high concentrations of Cu and Cr and Patella species were associated with Cd. Some general metal bioaccumulation patterns are described but no one sampling site was more contaminated than the others. The hypothesis of Linosa island serving as a reference ecosystem for baseline trace metal levels in southern Tyrrhenian areas is indeed supported by the statistical comparison among other southern Tyrrhenian ecosystems performed with Kruskall Wallis and Mann-Whitney tests. For P. oceanica leaves, P. pavonica, M. turbinata, and P. caerulea, this study confirms their usefulness as possible cosmopolitan biomonitors of trace metals in marine Mediterranean areas.

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.

Application of curve fitting in thin-layer chromatography-flame ionization detection analysis of the carbohydrate fraction in marine mucilage and marine snow samples from Italian seas.

This paper presents a thin-layer chromatographic-flame ionization detection (TLC-FID) procedure to characterize the carbohydrate fraction of marine mucilage and marine snow samples from the Italian Seas. The identification of the different carbohydrate subfractions is supported by the application of a deconvolution procedure based on a new mathematical function for describing chromatographic peaks and enhancing their resolution. The joint-approach TLC-FID analysis and deconvolution procedure allows for the characterization of the carbohydrate fraction of the marine samples in a single step without using the different derivatization procedures requested by the most common gas chromatography and high-performance liquid chromatographic methods for carbohydrate analysis. In fact, the results obtained by the TLC-FID procedure show that different neutral, uronic acid, and aminosugar subfractions can be present simultaneously in these samples. Moreover, the results support some hypotheses about the causes of the presence of mucilages in the Italian Seas.

Effects of two diluents in the Microtox toxicity bioassay with marine sediments.

This paper compares the use of two different diluents, EPA synthetic seawater (salinity 31 per thousand ) and NaCl standard diluent (salinity 35 per thousand ), in the Microtox toxicity bioassay performed on elutriate and solid phase derived from marine sediments. The study was performed comparing three series of data obtained by the use of the two diluents. In the first series the intensity of the natural light output of Vibrio fischeri was considered; in the second series pH value, sulphite and ammonia present in the control and in the diluent after the treatment with the sediment samples; in the last series, the measured toxicity in marine sediments was considered. The light output intensity measured with respect to time, gives information about the bacterial activity due to the different osmotic conditions. pH values joint with ammonia and sulphite content, give information about the effect of the bacterial metabolic activities and of the different interaction between each diluent with the sediment sample. At last, the comparison of the two diluents on real samples show how the different osmotic and hydrogenionic conditions determine different toxicity responses. The results show that the EPA diluent allows more suitable environment for the metabolic activities of bacteria depending on lower stressing conditions than those present when the standard diluent is used. Moreover, the use of EPA diluent reduces the risk of false positive response in the execution of the toxicity bioassay.

Improvement of an ultrasound assisted method for the analysis of total carbohydrate in environmental and food samples.

The determination of the carbohydrate content of environmental and food samples, performed by spectroscopic and chromatographic methods, needs a preliminary hydrolysis which converts polysaccharides into monosaccharides prior to the analytical measurement. In this paper, a modified procedure of the ultrasound-assisted method for the determination of total carbohydrates, published by the same authors in a previous paper, is presented and discussed. The modified procedure represents an improvement with respect to the previous one because it shortens the hydrolysis time and the colorimetric determination times through the use of two acid media (formic and acetic acid) that also allows quantitative recoveries of total carbohydrates from food and environmental samples.