Estuarine hydrodynamic processes driving the molecular changes of terrestrial dissolved organic nitrogen: From mixing to biological modification.

Estuaries receive substantial amounts of terrestrial dissolved organic nitrogen (tDON), which will be transported from the freshwater to the oceanic terminus through vigorous exchange processes. However, the intricate migration and transformation dynamics of tDON during this transportation, particularly at a molecular level, remain constrained. To address this knowledge gap, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was used for the analysis of DON molecular composition in the Pearl River Estuary (PRE), a river-dominated estuarine system influenced by intensified anthropogenic activities in southern China. The results showed a pronounced spatial-temporal variation in DON concentration in the study area. At the molecular level, tDON exhibited reduced unsaturation and aromaticity, coupled with an elevated abundance of DON compounds containing one­nitrogen atom (1 N-DON, 53.17 %) and compounds containing carbon, hydrogen, oxygen, nitrogen, and sulfur (CHONS) (27.46 %). It was evident that lignin was depleted while more oxygenated tannin compounds were generated in the freshwater-seawater mixing zone. This transformation is attributed to heightened biological activities, likely influenced by the priming effect of terrestrial nutrient inputs. In summer, the prevailing plume combined with biological activities in the strong mixing area and outer estuary increased the abundance of 3 N-DON molecules and a concurrent rise in the abundance of DON compounds containing only carbon, hydrogen, oxygen, and nitrogen (CHON), DON compounds containing carbon, hydrogen, oxygen, nitrogen, sulfur, and phosphorus (CHONSP), and CHONS. This trend also underscores the expanding role of marine plankton and microbes in the utilization of DON compounds containing carbon, hydrogen, oxygen, nitrogen, and phosphorus (CHONP). These findings provide details of tDON transformation processes at the molecular level in a river-dominated estuary and underline the estuarine hydrodynamics involved in transporting and altering DON within the estuary.

Novel Cytotoxic Sesquiterpene Coumarin Ethers and Sulfur-Containing Compounds from the Roots of Ferula turcica.

Six new sesquiterpene coumarin ethers, namely turcicanol A (1), turcicanol A acetate (2), turcicanol B (3), turcica ketone (4), 11'-dehydrokaratavicinol (5), and galbanaldehyde (6), and one new sulfur-containing compound, namely turcicasulphide (7), along with thirty-two known secondary metabolites were isolated from the root of the endemic species Ferula turcica Akalin, Miski, & Tuncay through a bioassay-guided isolation approach. The structures of the new compounds were elucidated by spectroscopic analysis and comparison with the literature. Cell growth inhibition of colon cancer cell lines (COLO205 and HCT116) and kidney cancer cell lines (UO31 and A498) was used to guide isolation. Seventeen of the compounds showed significant activity against the cell lines.

Metabolomics analysis of peony root using NMR spectroscopy and impact of the preprocessing method for NMR data in multivariate analysis.

Peony root is an important herbal drug used as an antispasmodic analgesic. To evaluate peony roots with different botanical origins, producing areas, and post-harvest processing, 1H NMR-based metabolomics analysis was employed. Five types of monoterpenoids, including albiflorin (4), paeoniflorin (6), and sulfonated paeoniflorin (25), and six other compounds, including 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (18), benzoic acid (21), gallic acid (22), and sucrose (26) were detected in the extracts of peony root samples. Among them, compounds 4, 6, 18, and total monoterpenoids including 21 were quantified by quantitative 1H NMR (qHNMR). Compound 25 was detected in 1H NMR spectra of sulfur-fumigated white peony root (WPR) extracts indicating that 1H NMR was a fast and effective method for identifying sulfur-fumigated WPR. The content of 26, the main factor affecting extract yield, increased significantly in peony root after low-temperature storage for one month, whereas that in WPR did not increase due to the boiling treatment after harvesting. We investigated the impact of preprocessing methods to such analysis for NMR data from commercial samples, resulting that the data matrix transformed from qHNMR spectra and normalized to internal standard were optimum for multivariate analysis. The multivariate analysis demonstrated that among commercial samples derived from P. lactiflora, peony root samples in Japanese market (PR) had high contents of 18 and 22, and red peony root (RPR) samples had high content of monoterpenoids represented by 6; and among RPR samples, those derived from P. veitchii showed higher contents of 18 and 22 than those from P. lactiflora. The 1H NMR-based metabolomics method coupled with qHNMR was useful for evaluation of peony root and would be applicable for other crude drugs.

Effect of DAP and glutamine supplementation on sulfur-containing volatiles and sensory properties of Chardonnay wine fermented with Saccharomyces cerevisiae yeast.

Volatile compounds in wine have a critical impact on the consumers' senses. In this study, the effect of diammonium phosphate (DAP) and glutamine on sulfur-containing volatiles and sensory properties of Chardonnay wine fermented with Saccharomyces cerevisiae yeast were evaluated. Fermentation kinetics was determined by monitoring reducing sugar consumption rates during fermentation. The volatile profile of wines was analyzed by headspace solid phase microextraction (HS-SPME) coupled with gas-chromatography-mass spectrometry (GC-MS). The volatile sulfur compounds (VSCs) were analyzed by HS-SPME-GC-MS/MS. Flavor attributes of wines were assessed by a sensory panel with quantitative descriptive analysis. A total of 53 volatiles, including 6 VSCs, were identified and quantified in the Chardonnay wine. The results suggested that glutamine supplementation at the beginning of fermentation could help to initiate fermentation earlier and promote the formation of isoamyl acetate, phenethyl acetate, ethyl nonanoate, methyl decanoate, diethyl succinate and phenethyl alcohol, isobutanol, while DAP supplementation had no obvious effect on the volatile composition of the resulting wine and fermentation kinetics. PRACTICAL APPLICATION: Suitable nitrogen source is helpful to a healthy fermentation, and can also prevent the off-flavor and regulate aroma profile of wine. This study provides insights on the volatile and sensory characteristics of Chardonnay wines affected by different nitrogen source addition.

Sulfur dioxide-free Verdejo wines through the use of a pure stilbene extract: exploring possible synergistic effect with glutathione.

BACKGROUND: The search for alternatives to sulfur dioxide (SO2 ) in white wine production is a tough challenge for the oenological industry. The current article investigates the effectiveness of several preservatives in protecting Verdejo white wines. The following wines were elaborated: without preservative (CT wines), with SO2 (SO2 wines), with stilbene-enriched extract (ST99 wines), with glutathione (GSH wines) and finally with the combination stilbene extract plus glutathione (ST99-GSH wines). RESULTS: ST99 extract preserved the wines from malolactic fermentation (MLF), maintained the quality parameters of the wines, but affected their color from the beginning of the process. Synergistic effects between ST99 and GSH were found for oxygen consumption rates, related to oxidation processes. The content of amino acids and biogenic amines was slightly affected by the treatments, but the quality or safety of the wines was not compromised. However, after 12 months of ageing in bottle, the SO2 wines showed the highest score in the sensory analysis. The reasons for the evolution of treated wines and the implications of this study for maintaining the quality of free SO2 white wines are discussed. CONCLUSION: ST99 may be proposed as an alternative to SO2 in wines to be consumed in the short term. Its combination with GSH does not prolong its shelf-life. © 2022 Society of Chemical Industry.

Phytochemistry Meets Geochemistry­Blumenol C Sulfate: A New Megastigmane Sulfate from Palicourea luxurians (Rubiaceae: Palicoureeae)

There is a previously neglected influence of geochemical conditions on plant phytochemistry. In particular, high concentrations of dissolved salts can affect their biosynthesis of natural products. Detoxification is most likely an important aspect for the plant, but additional natural products can also give it an expanded range of bioactivities. During the phytochemical analysis a Palicourea luxurians plant collected in a sulfate-rich environment (near the Río Sucio, Costa Rica) showed an interesting natural product in this regard. The structure of this compound was determined using spectroscopic and computational methods (NMR, MS, UV, IR, CD, optical rotation, quantum chemical calculations) and resulted in a megastigmane sulfate ester possessing a ß-ionone core structure, namely blumenol C sulfate (1, C13H22O5S). The levels of sulfur and sulfate ions in the leaves of the plant were determined using elemental analysis and compared to the corresponding levels in comparable plant leaves from a less sulfate-rich environments. The analyses show the leaves from which we isolated blumenol C sulfate (1) to contain 35% more sulfur and 80% more sulfate than the other samples. Antimicrobial and antioxidant activities of compound 1 were tested against Escherichia coli, E. coli ampR and Bacillus subtilis as well as measured using complementary in vitro FRAP and ATBS assays, respectively. These bioactivities are comparable to those determined for structurally related megastigmanes. The sulfur and sulfate content of the plant leaves from the sulfate-rich environment was significantly higher than that of the other plants. Against this background of salt stress, we discuss a possible biosynthesis of blumenol C sulfate (1). Furthermore, there appears to be no benefit for the plant in terms of extended bioactivities. Hence, the formation of blumenol C sulfate (1) probably primarily serves the plant detoxification process.

Identification of Photocatalytic Alkaloids from Coptidis Rhizome by an Offline HPLC/CC/SCD Approach.

Natural products continue to be a valuable source of active metabolites; however, researchers of natural products are mostly focused on the biological effects, and their chemical utility has been less explored. Furthermore, low throughput is a bottleneck for classical natural product research. In this work, a new offline HPLC/CC/SCD (high performance liquid chromatography followed by co-crystallization and single crystal diffraction) workflow was developed that greatly expedites the discovery of active compounds from crude natural product extracts. The photoactive total alkaloids of the herbal medicine Coptidis rhizome were firstly separated by HPLC, and the individual peaks were collected. A suitable coformer was screened by adding it to the individual peak solution and observing the precipitation, which was then redissolved and used for co-crystallization. Seven new co-crystals were obtained, and all the single crystals were subjected to X-ray diffraction analysis. The molecular structures of seven alkaloids from milligrams of crude extract were resolved within three days. NDS greatly decreases the required crystallization amounts of alkaloids to the nanoscale and enables rapid stoichiometric inclusion of all the major alkaloids with full occupancy, typically without disorder, affording well-refined structures. It is noteworthy that anomalous scattering by the coformer sulfur atoms enables reliable assignment of absolute configuration of stereogenic centers. Moreover, the identified alkaloids were firstly found to be photocatalysts for the green synthesis of benzimidazoles. This study demonstrates a new and green phytochemical workflow that can greatly accelerate natural product discovery from complex samples.

Difference in the nutritional, in vitro, and functional characteristics of protein and fat isolates of two Indian chia (Salvia hispanica L) seed genotypes with variation in seed coat color.

This study aimed to determine the association between the seed coat color of two chia seed genotypes for their composition, protein content, amino acid, and fatty acid profiles. The optimal pH for protein isolation for both genotypes (BCPI and WCPI) was 10, based on protein purity and solubility. Fatty acid profiling indicated, overall, 18 different fatty acids higher in BCPI10 with linolenic acid domination (∼66%) followed by linoleic acid (∼19%) and oleic acid (∼6%), contributing PUFAs (∼86%). Optimized protein isolates, black (BCPI10) and white (WCPI10) chia, had shown purity, L*-value, solubility, and yields of 90.65%, 75.86%, 77.75%, 11.30%, and 90.00%, 77.83%, 76.07%, 10.69%, respectively. BCPI10 depicted higher EAA (33.19 g/100 g N) and EEA indices (57.676%) compared to WCPI10 (32.14 g/100 g N) and 56.360%, respectively. Amino acid profiling indicated higher, PER, TAA, TEAA, TNEAA, TAAA, TBA, acidic AA values for BCPI10, and higher leucine/isoleucine ratio for WCPI10 having leucine and sulfur amino acids as limiting amino acids. BCPI10 had higher sulfur-containing amino acid contents, as the main contributor to the albumin a water-soluble fraction, leading to its higher in vitro digestibility (71.97%) than WCPI10 (67.70%). Both isolates exhibited good WHC and OHC of 3.18, 2.39 and 3.00, 2.20, respectively. Both protein isolates had similar ∆Td (°C) values with some variation in FTIR spectrum from 1000 cm-1 to 1651 cm-1 having more peak intensity for BCPI10. SDS-PAGE indicated bands at 150 kDa, representing globulin and mild bands at 25-33 kDa for glutelin and albumin. A significant (p < 0.05) variation reported in this study for protein and lipid profiles of both genotype attributes to genetic differences between the seeds. PRACTICAL APPLICATION: Based on the nutritional profile, both chia seed isolates (black and white) are suitable for consumption with an edge for black seed when supplemented with their limiting amino acids. The high values of the functional properties and structural characteristics combined with high nutritional values make the chia protein isolate an excellent source of raw material for various food formulations. Fatty acid profile of the oils from the genotypes showed the presence of high amounts of unsaturated fatty acids, especially the PUFAs with more number of fatty acids in black chia seed. The excellent lipid profile of chia seed oil indicates the benefit of using chia seed oil as a source of essential fatty acids in the human diet for optimal health.

Sulfur- and phosphorus-standardized metal quantification of biological specimens using inductively coupled plasma mass spectrometry.

This protocol describes how inductively coupled plasma mass spectrometry (ICP-MS) can quantify metals, sulfur, and phosphorus present in biological specimens. The high sensitivity of ICP-MS enables detection of these elements at very low concentrations, and absolute quantification is achieved with standard curves. Sulfur or phosphorus standardization reduces variability that arises because of slight differences in sample composition. This protocol bypasses challenges because of limited sample amounts and facilitates studies examining the biological roles of metals in health and disease. For complete details on the use and execution of this protocol, please refer to Hartwig et al. (2020).

Proximate analysis, HPTLC finger print analysis and multi spectrometric analysis of Strychnos nux-vomica nuts.

OBJECTIVES: In alternative medicine, plants pay a major role. Some plants are known for their poisonous nature but still have some importance in the herbal drug industry for their medicinal value. Strychnos nux-vomica is one such plant. Its nuts are called as poison nut due to the presence of alkaloids. Both the nut and its minerals are having medicinal properties and hence the present study was indented to understand the nature of primary metabolites and multi elemental composition. METHODS: The nuts of S. nux-vomica were procured, authenticated, powdered and subjected to proximate analysis parameters, visualization of thin layer chromatographic separation (TLC) and finger print profiling through high performance thin layer chromatographic (HPTLC); surface morphology by scanning electron microscopy, energy dispersive X-ray analysis, X-ray fluorescence spectrometry, X-ray photoelectron spectrometry, powder X-ray diffractometry and inductively coupled plasma optical emission spectrometry. RESULTS: In HPTLC, 7 spots each under 254 nm, 366 nm, derivatization with vanillin sulphuric acid (VSR) reagent appeared and 2 spots with Dragendorff's reagent. In HPTLC, 12 peaks at 254 nm, 9 peaks at 366 nm, 7 peaks at 520 nm after derivatization with VSR reagent detected. Elements such as potassium, calcium, magnesium, chlorine, aluminium, iron, manganese, sodium, nickel, phosphorus, copper, zinc, sulphur and silicon were identified. PXRD revealed that the presence of potassium chloride, calcite and dolomite as major elemental composition. CONCLUSIONS: The presence of all the above elements has vital roles on human physiology. Potassium, calcium, chlorine, aluminium, nickel, phosphorus, sulphur and silicon are reported for the first time in this study.

Ecological memory of recurrent drought modifies soil processes via changes in soil microbial community.

Climate change is altering the frequency and severity of drought events. Recent evidence indicates that drought may produce legacy effects on soil microbial communities. However, it is unclear whether precedent drought events lead to ecological memory formation, i.e., the capacity of past events to influence current ecosystem response trajectories. Here, we utilize a long-term field experiment in a mountain grassland in central Austria with an experimental layout comparing 10 years of recurrent drought events to a single drought event and ambient conditions. We show that recurrent droughts increase the dissimilarity of microbial communities compared to control and single drought events, and enhance soil multifunctionality during drought (calculated via measurements of potential enzymatic activities, soil nutrients, microbial biomass stoichiometry and belowground net primary productivity). Our results indicate that soil microbial community composition changes in concert with its functioning, with consequences for soil processes. The formation of ecological memory in soil under recurrent drought may enhance the resilience of ecosystem functioning against future drought events.

Unique chemical parameters and microbial activity lead to increased archaeological preservation at the Roman frontier site of Vindolanda, UK.

Waterlogged burial conditions impact upon artefact preservation. One major determinant of preservation is presence and behaviour of microorganisms, however, unravelling the mechanisms, especially in waterlogged conditions is challenging. In this study, we analysed elemental composition, bacterial diversity and community structure from excavation trenches at the Roman Site of Vindolanda, Northumberland, UK, using pXRF and 16S rRNA gene amplicon sequencing. Excavation trenches provide information of different occupation periods. The results indicated that microbial communities were dominated by Firmicutes, Bacteroidetes and Proteobacteria at a phylum level. Samples which also had visible vivianite presence showed that there were marked increases in Methylophilus. Methylophilus might be associated with favourable preservation in these anaerobic conditions. More research is needed to clearly link the presence of Methylophilus with vivianite production. The study emphasises the need for further integration of chemical and microbiome approaches, especially in good preservation areas, to explore microbial and chemical degradation mechanisms.

Metabolite Profile and In Vitro Beneficial Effects of Black Garlic (Allium sativum L.) Polar Extract.

Over the centuries, humans have traditionally used garlic (Allium sativum L.) as a food ingredient (spice) and remedy for many diseases. To confirm this, many extensive studies recognized the therapeutic effects of garlic bulbs. More recently, black garlic (BG), made by heat-ageing white garlic bulbs, has increased its popularity in cuisine and traditional medicine around the world, but there is still limited information on its composition and potential beneficial effects. In this study, the metabolite profile of methanol extract of BG (BGE) was determined by high-performance liquid chromatography coupled to tandem mass spectrometry in high-resolution mode. Results allowed to establish that BGE major components were sulfur derivatives, saccharides, peptides, organic acids, a phenylpropanoid derivative, saponins, and compounds typical of glycerophospholipid metabolism. Characterization of the BGE action in cancer cells revealed that antioxidant, metabolic, and hepatoprotective effects occur upon treatment as well as induction of maturation of acute myeloid leukemia cells. These results are interesting from the impact point of view of BG consumption as a functional food for potential prevention of metabolic and tumor diseases.

Nutritional deficiency in scarlet eggplant limits its growth by modifying the absorption and use efficiency of macronutrients.

The intensity damages caused by nutritional deficiency in growing plants can vary with nutrients. The effects caused by nutrient omission in the plant nutritional efficiency in relation to the absorption and use of the missing nutrient, and the reasons why these damages reflect in other nutrients have not yet been reported in the culture of scarlet eggplant. A better understanding of the nutritional mechanisms involved may clarify why certain nutrients cause greater limitations than other during plants growth. Thus, this study was designed with the aim of evaluating the damages caused by macronutrients deficiency in the culture of scarlet eggplant in the accumulation of these nutrients, nutritional deficiency, plants growth and in visual symptoms. The experiment was carried out in a controlled environment where plants were cultivated in a hydroponic system. Treatments consisted of supplying a complete Hoagland and Arnon solution (CS), and other nutrient solutions with individual omissions of nitrogen (-N), phosphorus (-P), potassium (-K), calcium (-Ca), magnesium (-Mg) and sulphur (-S). When a nutrient deficiency arose, nutritional analyses, growth and visual symptoms were analyzed. The omissions of N, S and K in the nutrient solution resulted in lower accumulation of all macronutrients in both the above and below ground biomass. Individual omissions resulted in nutritional imbalances with reflexes in the absorption efficiencies and use of the missing nutrient, as well as of other nutrients, revealing that the metabolism involves multiple nutritional interactions. Losses of nutritional efficiencies of macronutrients caused detrimental effects on plants growth, with reduced height, stem diameter, number of leaves, leaf area, and biomass production in above ground and below ground. From the losses in production in above ground biomass, the order of macronutrients limitation was N, S, K, Ca, Mg, and P, with reductions of 99, 96, 94, 76, 51 and 46%, respectively, in comparison to plants cultivated in CS. The most limiting nutrients were N, S, and K, seen that its deficiencies affected the metabolism of all other nutrients. This study demonstrates the importance of an adequate nutritional management of N, S, and K in the cultivation of scarlet eggplant.

Oil biodesulfurization: A review of applied analytical techniques.

The wide use of fossil fuels and their associated environmental concerns, highlighted the importance of affordable and clean energy (goal 7), as adopted by the Sustainable Development Goals of the United Nations for 2030. For years now, the detection of sulfur components in liquid fuels is performed mainly for environmental and health purposes in compliance with the respective legislations. Towards this, the aerobic and anaerobic biodesulfurization (BDS) process, which entails the use of microorganisms to limit the sulfur concentration is followed. To ensure effective BDS, several traditional analytical methods are utilized, although they require bench-top, bulky, costly, and time-consuming instruments along with skilled personnel. The currently employed analytical methods are mostly chromatographic techniques (e.g. liquid and gas) coupled with various detectors. To start with, high-performance liquid chromatography with ultraviolet detector (HPLC-UV), as well as electrospray ionization-LC-mass spectrometry (ESI-LC-MS) were mostly reported. Additionally, many detectors were coupled to gas chromatography (CG) including atomic emission detector (GC-AED), flame ionization detector (GC-FID), flame photometric detector (GC-FPD), sulfur fluorescence detector (GC-SFD), mass selective detector (GC-MS), etc. The solid-phase microextraction (SPME) technique provides extra capabilities when added to the separation techniques. Towards the continuous interest in oil supercomplex synthesis, other atmospheric and surface desorption ionization techniques, as well as the multidimensional 2D chromatographic systems (GC × GC and LC × LC) were also investigated, due to their unsurpassed resolution power. The current review ends with final remarks per applied methodology and the necessity to respect and protect the human environment and life.

Mortality and Morbidity Effects of Long-Term Exposure to Low-Level PM2.5, BC, NO2, and O3: An Analysis of European Cohorts in the ELAPSE Project.

INTRODUCTION: Epidemiological cohort studies have consistently found associations between long-term exposure to outdoor air pollution and a range of morbidity and mortality endpoints. Recent evaluations by the World Health Organization and the Global Burden of Disease study have suggested that these associations may be nonlinear and may persist at very low concentrations. Studies conducted in North America in particular have suggested that associations with mortality persisted at concentrations of particulate matter with an aerodynamic diameter of less than 2.5 µm (PM2.5) well below current air quality standards and guidelines. The uncertainty about the shape of the concentration-response function at the low end of the concentration distribution, related to the scarcity of observations in the lowest range, was the basis of the current project. Previous studies have focused on PM2.5, but increasingly associations with nitrogen dioxide (NO2) are being reported, particularly in studies that accounted for the fine spatial scale variation of NO2. Very few studies have evaluated the effects of long-term exposure to low concentrations of ozone (O3). Health effects of black carbon (BC), representing primary combustion particles, have not been studied in most large cohort studies of PM2.5. Cohort studies assessing health effects of particle composition, including elements from nontailpipe traffic emissions (iron, copper, and zinc) and secondary aerosol (sulfur) have been few in number and reported inconsistent results. The overall objective of our study was to investigate the shape of the relationship between long-term exposure to four pollutants (PM2.5, NO2, BC, and O3) and four broad health effect categories using a number of different methods to characterize the concentration-response function (i.e., linear, nonlinear, or threshold). The four health effect categories were (1) natural- and cause-specific mortality including cardiovascular and nonmalignant as well as malignant respiratory and diabetes mortality; and morbidity measured as (2) coronary and cerebrovascular events; (3) lung cancer incidence; and (4) asthma and chronic obstructive pulmonary disease (COPD) incidence. We additionally assessed health effects of PM2.5 composition, specifically the copper, iron, zinc, and sulfur content of PM2,5. METHODS: We focused on analyses of health effects of air pollutants at low concentrations, defined as less than current European Union (EU) Limit Values, U.S. Environmental Protection Agency (U.S. EPA), National Ambient Air Quality Standards (NAAQS), and/or World Health Organization (WHO) Air Quality Guideline values for PM2.5, NO2, and O3. We address the health effects at low air pollution levels by performing new analyses within selected cohorts of the ESCAPE study (European Study of Cohorts for Air Pollution Effects; Beelen et al. 2014a) and within seven very large European administrative cohorts. By combining well-characterized ESCAPE cohorts and large administrative cohorts in one study the strengths and weaknesses of each approach can be addressed. The large administrative cohorts are more representative of national or citywide populations, have higher statistical power, and can efficiently control for area-level confounders, but have fewer possibilities to control for individual-level confounders. The ESCAPE cohorts have detailed information on individual confounders, as well as country-specific information on area-level confounding. The data from the seven included ESCAPE cohorts and one additional non-ESCAPE cohort have been pooled and analyzed centrally. More than 300,000 adults were included in the pooled cohort from existing cohorts in Sweden, Denmark, Germany, the Netherlands, Austria, France, and Italy. Data from the administrative cohorts have been analyzed locally, without transfer to a central database. Privacy regulations prevented transfer of data from administrative cohorts to a central database. More than 28 million adults were included from national administrative cohorts in Belgium, Denmark, England, the Netherlands, Norway, and Switzerland as well as an administrative cohort in Rome, Italy. We developed central exposure assessment using Europewide hybrid land use regression (LUR) models, which incorporated European routine monitoring data for PM2.5, NO2, and O3, and ESCAPE monitoring data for BC and PM2.5 composition, land use, and traffic data supplemented with satellite observations and chemical transport model estimates. For all pollutants, we assessed exposure at a fine spatial scale, 100 × 100 m grids. These models have been applied to individual addresses of all cohorts including the administrative cohorts. In sensitivity analyses, we applied the PM2.5 models developed within the companion HEI-funded Canadian MAPLE study (Brauer et al. 2019) and O3 exposures on a larger spatial scale for comparison with previous studies. Identification of outcomes included linkage with mortality, cancer incidence, hospital discharge registries, and physician-based adjudication of cases. We analyzed natural-cause, cardiovascular, ischemic heart disease, stroke, diabetes, cardiometabolic, respiratory, and COPD mortality. We also analyzed lung cancer incidence, incidence of coronary and cerebrovascular events, and incidence of asthma and COPD (pooled cohort only). We applied the Cox proportional hazard model with increasing control for individual- and area-level covariates to analyze the associations between air pollution and mortality and/or morbidity for both the pooled cohort and the individual administrative cohorts. Age was used as the timescale because of evidence that this results in better adjustment for potential confounding by age. Censoring occurred at the time of the event of interest, death from other causes, emigration, loss to follow-up for other reasons, or at the end of follow-up, whichever came first. A priori we specified three confounder models, following the modeling methods of the ESCAPE study. Model 1 included only age (time axis), sex (as strata), and calendar year of enrollment. Model 2 added individual-level variables that were consistently available in the cohorts contributing to the pooled cohort or all variables available in the administrative cohorts, respectively. Model 3 further added area-level socioeconomic status (SES) variables. A priori model 3 was selected as the main model. All analyses in the pooled cohort were stratified by subcohort. All analyses in the administrative cohorts accounted for clustering of the data in neighborhoods by adjusting the variance of the effect estimates. The main exposure variable we analyzed was derived from the Europewide hybrid models based on 2010 monitoring data. Sensitivity analyses were conducted using earlier time periods, time-varying exposure analyses, local exposure models, and the PM2.5 models from the Canadian MAPLE project. We first specified linear single-pollutant models. Two-pollutant models were specified for all combinations of the four main pollutants. Two-pollutant models for particle composition were analyzed with PM2.5 and NO2 as the second pollutant. We then investigated the shape of the concentration-response function using natural splines with two, three, and four degrees of freedom; penalized splines with the degrees of freedom determined by the algorithm and shape-constrained health impact functions (SCHIF) using confounder model 3. Additionally, we specified linear models in subsets of the concentration range, defined by removing concentrations above a certain value from the analysis, such as for PM2.5 25 µg/m3 (EU limit value), 20, 15, 12 µg/m3 (U.S. EPA National Ambient Air Quality Standard), and 10 µg/m3 (WHO Air Quality Guideline value). Finally, threshold models were evaluated to investigate whether the associations persisted below specific concentration values. For PM2.5, we evaluated 10, 7.5, and 5 µg/m3 as potential thresholds. Performance of threshold models versus the corresponding no-threshold linear model were evaluated using the Akaike information criterion (AIC). RESULTS: In the pooled cohort, virtually all subjects in 2010 had PM2.5 and NO2 annual average exposures below the EU limit values (25 µg/m3 and 40 µg/m3, respectively). More than 50,000 had a residential PM2.5 exposure below the U.S. EPA NAAQS (12 µg/m3). More than 25,000 subjects had a residential PM2.5 exposure below the WHO guideline (10 µg/m3). We found significant positive associations between PM2.5, NO2, and BC and natural-cause, respiratory, cardiovascular, and diabetes mortality. In our main model, the hazard ratios (HRs) (95% [confidence interval] CI) were 1.13 (CI = 1.11, 1.16) for an increase of 5 µg/m3 PM2.5, 1.09 (CI = 1.07, 1.10) for an increase of 10 µg/m3 NO2, and 1.08 (CI = 1.06, 1.10) for an increase of 0.5 × 10-5/m BC for natural-cause mortality. The highest HRs were found for diabetes mortality. Associations with O3 were negative, both in the fine spatial scale of the main ELAPSE model and in large spatial scale exposure models. For PM2.5, NO2, and BC, we generally observed a supralinear association with steeper slopes at low exposures and no evidence of a concentration below which no association was found. Subset analyses further confirmed that these associations remained at low levels: below 10 µg/m3 for PM2.5 and 20 µg/m3 for NO2. HRs were similar to the full cohort HRs for subjects with exposures below the EU limit values for PM2.5 and NO2, the U.S. NAAQS values for PM2.5, and the WHO guidelines for PM2.5 and NO2. The mortality associations were robust to alternative specifications of exposure, including different time periods, PM2.5 from the MAPLE project, and estimates from the local ESCAPE model. Time-varying exposure natural spline analyses confirmed associations at low pollution levels. HRs in two-pollutant models were attenuated but remained elevated and statistically significant for PM2.5 and NO2. In two-pollutant models of PM2.5 and NO2 HRs for natural-cause mortality were 1.08 (CI = 1.05, 1.11) for PM2.5 and 1.05 (CI = 1.03, 1.07) for NO2. Associations with O3 were attenuated but remained negative in two-pollutant models with NO2, BC, and PM2.5. We found significant positive associations between PM2.5, NO2, and BC and incidence of stroke and asthma and COPD hospital admissions. Furthermore, NO2 was significantly related to acute coronary heart disease and PM2.5 was significantly related to lung cancer incidence. We generally observed linear to supralinear associations with no evidence of a threshold, with the exception of the association between NO2 and acute coronary heart disease, which was sublinear. Subset analyses documented that associations remained even with PM2.5 below 20 µg/m3 and possibly 12 µg/m3. Associations remained even when NO2 was below 30 µg/m3 and in some cases 20 µg/m3. In two-pollutant models, NO2 was most consistently associated with acute coronary heart disease, stroke, asthma, and COPD hospital admissions. PM2.5 was not associated with these outcomes in two-pollutant models with NO2. PM2.5 was the only pollutant that was associated with lung cancer incidence in two-pollutant models. Associations with O3 were negative though generally not statistically significant. In the administrative cohorts, virtually all subjects in 2010 had PM2.5 and NO2 annual average exposures below the EU limit values. More than 3.9 million subjects had a residential PM2.5 exposure below the U.S. EPA NAAQS (12 µg/m3) and more than 1.9 million had residential PM2.5 exposures below the WHO guideline (10 µg/m3). We found significant positive associations between PM2.5, NO2, and BC and natural-cause, respiratory, cardiovascular, and lung cancer mortality, with moderate to high heterogeneity between cohorts. We found positive but statistically nonsignificant associations with diabetes mortality. In our main model meta-analysis, the HRs (95% CI) for natural-cause mortality were 1.05 (CI = 1.02, 1.09) for an increase of 5 µg/m3 PM2.5, 1.04 (CI = 1.02, 1.07) for an increase of 10 µg/m3 NO2, and 1.04 (CI = 1.02, 1.06) for an increase of 0.5 × 10-5/m BC, and 0.95 (CI = 0.93, 0.98) for an increase of 10 µg/m3 O3. The shape of the concentration-response functions differed between cohorts, though the associations were generally linear to supralinear, with no indication of a level below which no associations were found. Subset analyses documented that these associations remained at low levels: below 10 µg/m3 for PM2.5 and 20 µg/m3 for NO2. BC and NO2 remained significantly associated with mortality in two-pollutant models with PM2.5 and O3. The PM2.5 HR attenuated to unity in a two-pollutant model with NO2. The negative O3 association was attenuated to unity and became nonsignificant. The mortality associations were robust to alternative specifications of exposure, including time-varying exposure analyses. Time-varying exposure natural spline analyses confirmed associations at low pollution levels. Effect estimates in the youngest participants (<65 years at baseline) were much larger than in the elderly (>65 years at baseline). Effect estimates obtained with the ELAPSE PM2.5 model did not differ from the MAPLE PM2.5 model on average, but in individual cohorts, substantial differences were found. CONCLUSIONS: Long-term exposure to PM2.5, NO2, and BC was positively associated with natural-cause and cause-specific mortality in the pooled cohort and the administrative cohorts. Associations were found well below current limit values and guidelines for PM2.5 and NO2. Associations tended to be supralinear, with steeper slopes at low exposures with no indication of a threshold. Two-pollutant models documented the importance of characterizing the ambient mixture with both NO2 and PM2.5. We mostly found negative associations with O3. In two-pollutant models with NO2, the negative associations with O3 were attenuated to essentially unity in the mortality analysis of the administrative cohorts and the incidence analyses in the pooled cohort. In the mortality analysis of the pooled cohort, significant negative associations with O3 remained in two-pollutant models. Long-term exposure to PM2.5, NO2, and BC was also positively associated with morbidity outcomes in the pooled cohort. For stroke, asthma, and COPD, positive associations were found for PM2.5, NO2, and BC. For acute coronary heart disease, an increased HR was observed for NO2. For lung cancer, an increased HR was found only for PM2.5. Associations mostly showed steeper slopes at low exposures with no indication of a threshold.

Assessment of selenium spatial distribution using µ-XFR in cowpea (Vigna unguiculata (L.) Walp.) plants: Integration of physiological and biochemical responses.

Low concentrations of selenium (Se) are beneficial for plant growth. Foliar Se application at high concentrations is toxic to plants due to the formation of reactive oxygen species (ROS). This study characterized Se toxicity symptoms using X-ray fluorescence (XRF) technique in response to foliar Se application in cowpea plants. Five Se concentrations (0, 10, 25, 50, 100 e 150 g ha-1) were sprayed on leaves as sodium selenate. The visual symptoms of Se toxicity in cowpea leaves were separated into two stages: I) necrotic points with an irregular distribution and internerval chlorosis at the leaf limb border (50-100 g ha-1); II) total chlorosis with the formation of dark brown necrotic lesions (150 g ha-1). Foliar Se application at 50 g ha-1 increased photosynthetic pigments and yield. Ultrastructural analyses showed that Se foliar application above 50 g ha-1 disarranged the upper epidermis of cowpea leaves. Furthermore, Se application above 100 g ha-1 significantly increased the hydrogen peroxide concentration and lipid peroxidation inducing necrotic leaf lesions. Mapping of the elements in leaves using the XRF revealed high Se intensity, specifically in leaf necrotic lesions accompanied by calcium (Ca) as a possible attenuating mechanism of plant stress. The distribution of Se intensities in the seeds was homogeneous, without specific accumulation sites. Phosphorus (P) and sulfur (S) were found primarily located in the embryonic region. Understanding the factors involved in Se accumulation and its interaction with Ca support new preventive measurement technologies to prevent Se toxicity in plants.

Quantitative analysis of phosphorus and sulfur in vegetation samples at minor and trace levels by EDXRF technique.

In this paper, a rapid, simple and reliable quantitative analysis method for Phosphorus and Sulfur in milligram quantities of plant samples by EDXRF has been described. The method uses a thin film sample preparation procedure which includes drying, suspension samples, filtration and pressing of the thin film samples. By measuring four random points of the same thin film sample, the homogenization of thin layer samples was evaluated to ensure the stability of the quantitative analysis results. The calibration curves of P, S, Ca and Fe was established by changing the weight of certified reference materials (CRMs) deposited on the filter. Then, the emission-transmission (E-T) method was used for correcting the matrix absorption effects of phosphorus and sulfur in the thin layer samples. After the correction, the correlation coefficients (R2) of the calibration curves of P and S were higher than 0.99. To evaluate the accuracy of quantitative analysis method, three vegetation verification samples were synthesized by adding the analytical pure powder to CRMs. The quantitative analytical results of EDXRF and ICP-OES were compared to the synthesized value. For P and S elements, the relative error of EDXRF and ICP-OES were 1.2%-6.4% and 4.2 %to 11.4%, respectively.

Investigation of chocolate types on the content of selected metals and non-metals determined by ICP-OES analytical technique.

Chocolate, one of the most popular sweets in the world, is consumed by people of all ages. Available data point to significant increases in consumption and production. However, successful determination of elements in chocolate is still difficult because of the characteristics of the matrix which contains a high content of organic compounds, like hydrogenated vegetable oil, vegetable fats, solids from malt extract, salts, emulsifiers, etc., causing problems with appropriate decomposition or digestion of sample. In this study, chocolate samples were prepared according to two procedures: water bath and microwave-assisted mineralisation. The use of Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) allowed us to determine the elemental composition of dark, milk, and white chocolate bars available on the Polish market as well as a cacao sample (100% cocoa powder). The elements assessed were Al, Ba, Ca, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, Sr, and Zn. The obtained results were used to compare the effectiveness of sample pre-treatment methods and to assess the correlation between the concentrations of specific elements and type of chocolate by the application of chemometric and statistical tools. The research showed that levels of analysed macro- and microelements are directly connected with the type of chocolate, characterised by the variable contents of cocoa paste and added milk. Data for all samples after mineralisation shown that among macroelements P was the most abundant, followed by Mg, Ca, Na, K, and S. The major essential element with the highest level was Fe, followed by Zn and Cu. In the group of toxic metals the highest content was obtained for Ba, then Al and Sr, but none exceeded permissible values prepared by health benefit organisations.

Effects of sulfur application on selenium uptake and seed selenium speciation in soybean (Glycine max L.) grown in different soil types.

AIMS: The objective of the present study was to elucidate the effects of sulfur (S) application on selenium (Se) uptake and seed Se speciation in high-protein soybean (Glycine max L.) grown in different soil types. METHODS: Pot experiments were conducted with soybean plants grown in yellow-brown soil (pH 5.68) and in calcareous alluvial soil (pH 7.87). Sodium selenate (Na2SeO4, 2 mg kg-1) was applied to soil with or without S fertilizer (S, 100 mg kg-1). RESULTS: Soybean grain yield and total biomass in calcareous alluvial soil were both approximately 1.3-fold the levels in yellow-brown soil. Following Se application, seed Se concentration in calcareous alluvial soil was 3.2-fold the concentration in yellow-brown soil, although additional S application reduced the corresponding seed Se concentrations by 55.6% and 38.6%, respectively. Generally, Se application facilitated Se translocation and enrichment in soybean seeds. Organic Se accounted for 92% of seed total Se and Se-methionine (>90%) was always the major Se species. Available Se (soluble and exchangeable fractions) accounted for 50.7% (yellow-brown soil) and 70.1% (calcareous alluvial soil) of soil total Se under Se treatment, while additional S application decreased the corresponding proportion of soluble Se by 12.6% and 14.4%. CONCLUSIONS: The bioavailability of selenate in calcareous alluvial soil was higher than the bioavailability in yellow-brown soil and was more negatively affected by S application. Although S application inhibited Se uptake in soybean plants in both soil types, it did not influence seed Se speciation and Se-methionine was the major Se species.