Technical Note: Dissolved organic matter fluorescence a finite mixture approach to deconvolve excitation-emission matrices

The analysis of the shape of excitation-emission matrices (EEMs) is a relevant tool for exploring the origin, transport and fate of dissolved organic matter (DOM) in aquatic ecosystems. Within this context, the decomposition of EEMs is acquiring a notable relevance. A simple mathematical algorithm that automatically deconvolves individual EEMs is described, creating new possibilities for the comparison of DOM fluorescence properties and EEMs that are very different from each other. A mixture model approach is adopted to decompose complex surfaces into sub-peaks. The laplacian operator and the Nelder-Mead optimisation algorithm are implemented to individuate and automatically locate potential peaks in the EEM landscape. The EEMs of a simple artificial mixture of fluorophores and DOM samples collected in a Mediterranean river are used to describe the model application and to illustrate a strategy that optimises the search for the optimal output.

Bioavailability of sinking organic matter in the Blanes canyon and the adjacent open slope (NW Mediterranean Sea)

Submarine canyons are sites of intense energy and material exchange between the shelf and the deep adjacent basins. To test the hypothesis that active submarine canyons represent preferential conduits of available food for the deep-sea benthos, two mooring lines were deployed at 1200 m depth from November 2008 to November 2009 inside the Blanes canyon and on the adjacent open slope (Catalan Margin, NW Mediterranean Sea). We investigated the fluxes, biochemical composition and food quality of sinking organic carbon (OC). OC fluxes in the canyon and the open slope varied among sampling periods, though not onsistently in the two sites. In particular, while in the open slope the highest OC fluxes were observed in August 2009, in the canyon the highest OC fluxes occurred in April-May 2009. For almost the entire study period, the OC fluxes in the canyon were significantly higher than those in the open slope, whereas OC contents of sinking particles collected in the open slope were consistently higher than those in the canyon. This result confirms that submarine canyons are effective conveyors of OC to the deep sea. Particles transferred to the deep sea floor through the canyons are predominantly of inorganic origin, significantly higher than that reaching the open slope at a similar water depth. Using multivariate statistical tests, two major clusters of sampling periods were identified: one in the canyon that grouped trap samples collected in December 2008, oncurrently with the occurrence of a major storm at the sea surface, and associated with increased fluxes of nutritionally available particles from the upper shelf. Another cluster grouped samples from both the canyon and the open slope collected in March 2009, concurrently with the occurrence of the seasonal phytoplankton bloom at the sea surface, and associated with increased fluxes of total phytopigments. Our results confirm the key ecological role of submarine canyons for the functioning of deep-sea ecosystems, and highlight the importance of canyons in linking episodic storms and primary production occurring at the sea surface to the deep sea floor.

The effects of milk as a food matrix for polyphenols on the excretion profile of cocoa (-)-Epicatechin metabolites in healthy humans subjects

The effect of different food matrices on the metabolism and excretion of polyphenols is uncertain. The objective of the study was to evaluate the possible effect of milk on the excretion of (2)-epicatechin metabolites from cocoa powder after its ingestion with and without milk. Twenty-one volunteers received the following three test meals each in a randomised cross-over design with a 1-week interval between meals: (1) 250 ml whole milk as a control; (2) 40 g cocoa powder dissolved in 250 ml whole milk (CC-M); (3) 40 g cocoa powder dissolved in 250 ml water (CC-W). Urine was collected before consumption and during the 0-6, 6-12 and 12-24 h periods after consumption. (2)-Epicatechin metabolite excretion was measured using liquid chromatography-MS. One (2)-epicatechin glucuronide and three (2)-epicatechin sulfates were detected in urine excreted after the intake of the two cocoa beverages (CC-M and CC-W). The results show that milk does not significantly affect the total amount of metabolites excreted in urine. However, differences in metabolite excretion profiles were observed; there were changes in the glucuronide and sulfate excretion rates, and the sulfation position between the period of excretion and the matrix. The matrix in which polyphenols are consumed can affect their metabolism and excretion, and this may affect their biological activity. Thus, more studies are needed to evaluate the effect of these different metabolite profiles on the body.

Effects of inorganic nitrogen (NH4Cl) and biodegradable organic carbon (CH3COONa) additions on a pilot-scale seawater biofilter

Biofilters degrade only a small fraction of the natural organic matter (NOM) contained in seawater which is the leading cause of biofouling in downstream processes. This work studies the effects of chemical additions on NOM biodegradation by biofilters. In this work, biofiltration of seawater with an empty bed contact time (EBCT) of 6 min and a hydraulic loading rate of 10 m h-1 reduces the biological oxygen demand (BOD7) by 8%, the dissolved organic carbon (DOC) by 6% and the UV absorbance at 254 nm (A254) by 7%. Different amounts of ammonium chloride are added to the seawater (up to twice the total dissolved nitrogen in untreated seawater) to study its possible effect on the removal of NOM by a pilot-scale biofilter. Seawater is amended with different amounts of easily biodegradable dissolved organic carbon (BDOC) supplied as sodium acetate (up to twice the DOC) for the same purpose. The results of this work reveal that the ammonium chloride additions do not significantly affect NOM removal and the sodium acetate is completely consumed by the biofiltration process. For both types of chemical additions, the BOD7, DOC and A254 in the outlet stream of the biofilter are similar to the values for the untreated control. These results indicate that this biofilter easily removes the BDOC from the seawater when the EBCT is not above 6 min. Furthermore, nitrogen does not limit the NOM biodegradation in seawater under these experimental conditions.

Residues of b-lactams and quinolones in tissues and milk samples. Confirmatory analysis by liquid chromatography-mass spectrometry

The aim of this work is to optimize and validate methods for the multiresidue determination of series of families of antibiotics as quinolones, penicillins and cephalosporins included in European regulation in food samples using LC-MS/MS. Different extraction techniques and clean-up applied to antibiotics in meat were compared. The quality parameters were established according with EU guideline. The developed method was applied to 49 positive raw milk samples from animal medicated with different antibiotics; the 63% of the analyzed samples were found to be compliant. ___________________________________________________________________________________________

Self-Organising Maps and Correlation Analysis as a Tool to Explore Patterns in Excitation-Emission Matrix Data Sets and to Discriminate Dissolved Organic Matter Fluorescence Components

Dissolved organic matter (DOM) is a complex mixture of organic compounds, ubiquitous in marine and freshwater systems. Fluorescence spectroscopy, by means of Excitation-Emission Matrices (EEM), has become an indispensable tool to study DOM sources, transport and fate in aquatic ecosystems. However the statistical treatment of large and heterogeneous EEM data sets still represents an important challenge for biogeochemists. Recently, Self-Organising Maps (SOM) has been proposed as a tool to explore patterns in large EEM data sets. SOM is a pattern recognition method which clusterizes and reduces the dimensionality of input EEMs without relying on any assumption about the data structure. In this paper, we show how SOM, coupled with a correlation analysis of the component planes, can be used both to explore patterns among samples, as well as to identify individual fluorescence components. We analysed a large and heterogeneous EEM data set, including samples from a river catchment collected under a range of hydrological conditions, along a 60-km downstream gradient, and under the influence of different degrees of anthropogenic impact. According to our results, chemical industry effluents appeared to have unique and distinctive spectral characteristics. On the other hand, river samples collected under flash flood conditions showed homogeneous EEM shapes. The correlation analysis of the component planes suggested the presence of four fluorescence components, consistent with DOM components previously described in the literature. A remarkable strength of this methodology was that outlier samples appeared naturally integrated in the analysis. We conclude that SOM coupled with a correlation analysis procedure is a promising tool for studying large and heterogeneous EEM data sets.

Fertilizing Methods and Nutrient Balance at the End of Traditional Organic Agriculture in the Mediterranean Bioregion: Catalonia (Spain) in the 1860s

By reconstructing the nutrient balance of a Catalan v illage circa 1861-65 we examine the sustainability of organic agricultural sy stems in the northwest Mediterranean bioregion prior to the green rev olution and the question of whether the nutrients extracted f rom the soil were replenished. With a population density of 59 inhabitants per square km, similar to other northern European rural areas at that time, and a lower liv estock density per cropland unit, this v illage experienced a manure shortage. The gap was f illed by other labour-intensiv e way s of transf erring nutrients f rom uncultiv ated areas into the cropland. Key elements in this agricultural sy stem were v iney ards because they hav e f ew nutrient requirements, and woodland and scrublands as sources of relev ant amounts of nutrients collected in sev eral ways.

Headspace needle-trap analysis of priority volatile organic compounds from aqueous samples: application to the analysis of natural and waste waters

Combining headspace (HS) sampling with a needle-trap device (NTD) to determine priority volatile organic compounds (VOCs) in water samples results in improved sensitivity and efficiency when compared to conventional static HS sampling. A 22 gauge stainless steel, 51-mm needle packed with Tenax TA and Carboxen 1000 particles is used as the NTD. Three different HS-NTD sampling methodologies are evaluated and all give limits of detection for the target VOCs in the ng L−1 range. Active (purge-and-trap) HS-NTD sampling is found to give the best sensitivity but requires exhaustive control of the sampling conditions. The use of the NTD to collect the headspace gas sample results in a combined adsorption/desorption mechanism. The testing of different temperatures for the HS thermostating reveals a greater desorption effect when the sample is allowed to diffuse, whether passively or actively, through the sorbent particles. The limits of detection obtained in the simplest sampling methodology, static HS-NTD (5 mL aqueous sample in 20 mL HS vials, thermostating at 50 °C for 30 min with agitation), are sufficiently low as to permit its application to the analysis of 18 priority VOCs in natural and waste waters. In all cases compounds were detected below regulated levels

Identification of metabolites and thermal transformation products of quinolones in raw cow milk by liquid chromatography coupled to high resolution mass spectrometry

The presence of residues of antibiotics, metabolites, and thermal transformation products (TPs), produced during thermal treatment to eliminate pathogenic microorganisms in milk, could represent a risk for people. Cow"s milk samples spiked with enrofloxacin (ENR), ciprofloxacin (CIP), difloxacin (DIF), and sarafloxacin (SAR) and milk samples from cows medicated with ENR were submitted to several thermal treatments. The milk samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) to find and identify TPs and metabolites. In this work, 27 TPs of 4 quinolones and 24 metabolites of ENR were found. Some of these compounds had been reported previously, but others were characterized for the first time, including lactose-conjugated CIP, the formamidation reaction for CIP and SAR, and hydroxylation or ketone formation to produce three different isomers for all quinolones studied.

Identification of metabolites and thermal transformation products of quinolones in raw cow milk by liquid chromatography coupled to high resolution mass spectrometry.

The presence of residues of antibiotics, metabolites, and thermal transformation products (TPs), produced during thermal treatment to eliminate pathogenic microorganisms in milk, could represent a risk for people. Cow"s milk samples spiked with enrofloxacin (ENR), ciprofloxacin (CIP), difloxacin (DIF), and sarafloxacin (SAR) and milk samples from cows medicated with ENR were submitted to several thermal treatments. The milk samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) to find and identify TPs and metabolites. In this work, 27 TPs of 4 quinolones and 24 metabolites of ENR were found. Some of these compounds had been reported previously, but others were characterized for the first time, including lactose-conjugated CIP, the formamidation reaction for CIP and SAR, and hydroxylation or ketone formation to produce three different isomers for all quinolones studied.

A Headspace needle-trap method for the analysis of volatile organic compounds in whole blood

Needle trap devices (NTDs) are a relatively new and promising tool for headspace (HS) analysis. In this study, a dynamic HS sampling procedure is evaluated for the determination of volatile organic compounds (VOCs) in whole blood samples. A full factorial design was used to evaluate the influence of the number of cycles and incubation time and it is demonstrated that the controlling factor in the process is the number of cycles. A mathematical model can be used to determine the most appropriate number of cycles required to adsorb a prefixed amount of VOCs present in the HS phase whenever quantitative adsorption is reached in each cycle. Matrix effect is of great importance when complex biological samples, such as blood, are analyzed. The evaluation of the salting out effect showed a significant improvement in the volatilization of VOCs to the HS in this type of matrices. Moreover, a 1:4 (blood:water) dilution is required to obtain quantitative recoveries of the target analytes when external calibration is used. The method developed gives detection limits in the 0.020–0.080 μg L−1 range (0.1–0.4 μg L−1 range for undiluted blood samples) with appropriate repeatability values (RSD < 15% at high level and <23% at LOQ level). Figure of merits of the method can be improved by using a smaller phase ratio (i.e., an increase in the blood volume and a decrease in the HS volume), which lead to lower detection limits, better repeatability values and greater sensibility. Twenty-eight blood samples have been evaluated with the proposed method and the results agree with those indicated in other studies. Benzene was the only target compound that gave significant differences between blood levels detected in volunteer non-smokers and smokers

New methods for the concentration of viruses from urban sewage using quantitative PCR.

Viruses are among the most important pathogens present in water contaminated with feces or urine and represent a serious risk to human health. Four procedures for concentrating viruses from sewage have been compared in this work, three of which were developed in the present study. Viruses were quantified using PCR techniques. According to statistical analysis and the sensitivity to detect human adenoviruses (HAdV), JC polyomaviruses (JCPyV) and noroviruses genogroup II (NoV GGII): (i) a new procedure (elution and skimmed-milk flocculation procedure (ESMP)) based on the elution of the viruses with glycine-alkaline buffer followed by organic flocculation with skimmed-milk was found to be the most efficient method when compared to (ii) ultrafiltration and glycine-alkaline elution, (iii) a lyophilization-based method and (iv) ultracentrifugation and glycine-alkaline elution. Through the analysis of replicate sewage samples, ESMP showed reproducible results with a coefficient of variation (CV) of 16% for HAdV, 12% for JCPyV and 17% for NoV GGII. Using spiked samples, the viral recoveries were estimated at 30-95% for HAdV, 55-90% for JCPyV and 45-50% for NoV GGII. ESMP was validated in a field study using twelve 24-h composite sewage samples collected in an urban sewage treatment plant in the North of Spain that reported 100% positive samples with mean values of HAdV, JCPyV and NoV GGII similar to those observed in other studies. Although all of the methods compared in this work yield consistently high values of virus detection and recovery in urban sewage, some require expensive laboratory equipment. ESMP is an effective low-cost procedure which allows a large number of samples to be processed simultaneously and is easily standardizable for its performance in a routine laboratory working in water monitoring. Moreover, in the present study, a CV was applied and proposed as a parameter to evaluate and compare the methods for detecting viruses in sewage samples.

New methods for the concentration of viruses from urban sewage using quantitative PCR.

Viruses are among the most important pathogens present in water contaminated with feces or urine and represent a serious risk to human health. Four procedures for concentrating viruses from sewage have been compared in this work, three of which were developed in the present study. Viruses were quantified using PCR techniques. According to statistical analysis and the sensitivity to detect human adenoviruses (HAdV), JC polyomaviruses (JCPyV) and noroviruses genogroup II (NoV GGII): (i) a new procedure (elution and skimmed-milk flocculation procedure (ESMP)) based on the elution of the viruses with glycine-alkaline buffer followed by organic flocculation with skimmed-milk was found to be the most efficient method when compared to (ii) ultrafiltration and glycine-alkaline elution, (iii) a lyophilization-based method and (iv) ultracentrifugation and glycine-alkaline elution. Through the analysis of replicate sewage samples, ESMP showed reproducible results with a coefficient of variation (CV) of 16% for HAdV, 12% for JCPyV and 17% for NoV GGII. Using spiked samples, the viral recoveries were estimated at 30-95% for HAdV, 55-90% for JCPyV and 45-50% for NoV GGII. ESMP was validated in a field study using twelve 24-h composite sewage samples collected in an urban sewage treatment plant in the North of Spain that reported 100% positive samples with mean values of HAdV, JCPyV and NoV GGII similar to those observed in other studies. Although all of the methods compared in this work yield consistently high values of virus detection and recovery in urban sewage, some require expensive laboratory equipment. ESMP is an effective low-cost procedure which allows a large number of samples to be processed simultaneously and is easily standardizable for its performance in a routine laboratory working in water monitoring. Moreover, in the present study, a CV was applied and proposed as a parameter to evaluate and compare the methods for detecting viruses in sewage samples.

Organic chicken product authentication: state-of-the-art and future perspectives

Organic food products are highly susceptible to fraud. Currently, administrative controls are conducted to detect fraud, but having an analytical tool able to verify the organic identity of food would be very supportive. The state-of-the-art in food authentication relies on fingerprinting approaches that find characteristic analytical patterns to unequivocally identify authentic products. While wide research on authentication has been conducted for other commodities, the authentication of organic chicken products is still in its infancy. Challenges include finding fingerprints to discriminate organic from conventional products, and recruiting sample sets that cover natural variability. Future research might be oriented towards developing new authentication models for organic feed, eggs and chicken meat, keeping models updated and implementing them into regulations. Meanwhile, these models might be very supportive to the administrative controls directing inspections towards suspicious fraudulent samples.

The influence of substratum type and nutrient supply on biofilm organic matter utilization in streams

We investigated the effect of benthic substratum type (sand and rocks) and nutrient supply (N and P) on biofilm structure and heterotrophic metabolism in a field experiment in a forested Mediterranean stream (Fuirosos). Rock and sand colonization and biofilm formation was intensively studied for 44 d at two stream reaches: control and experimental (continuous addition of phosphate, ammonia, and nitrate). Structural (C, N, and polysaccharide content and bacterial and chlorophyll density) and metabolic biofilm parameters (b-glucosidase, peptidase, and phosphatase enzyme activities) were analyzed throughout the colonization process. The epilithic biofilm (grown on rocks) had a higher peptidase activity at the impacted reach, together with a higher algal and bacterial biomass. The positive relationship between the peptidase activity per cell and the N content of the epilithic biofilm suggested that heterotrophic utilization of proteinaceous compounds from within the biofilm was occurring. In contrast, nutrient addition caused the epipsammic biofilm (grown on sand) to exhibit lower b-glucosidase and phosphatase activities, without a significant increase in bacterial and algal biomass. The differential response to nutrient addition was related to different structural characteristics within each biofilm. The epipsammic biofilm had a constant and high C:N ratio (22.7) throughout the colonization. The epilithic biofilm had a higher C:N ratio at the beginning of the colonization (43.2) and evolved toward a more complex structure (high polysaccharide content and low C:N ratio) during later stages. The epipsammic biofilm was a site for the accumulation and degradation of organic matter: polysaccharides and organic phosphorus compounds had higher degradation activities