Characterization of phosphorus in sub-alpine forest and adjacent grassland soils by chemical extraction and phosphorus-31 nuclear magnetic resonance spectroscopy

We used chemical extraction methods and ³¹P-nuclear magnetic resonance (NMR) to investigate the effects of vegetation on the amount and structural composition of phosphorous (P) in the sub-alpine soils of central Taiwan. Chemical extraction methods were used to measure inorganic P (Pi) and organic P (Po) in main soil horizons. The soil P composition was assessed by ³¹P-NMR spectroscopy on alkaline EDTA–NaOH extracts. According to the results of chemical extractions, the forest soil had a higher amount of Pi than the grassland soil, which might be a result of the mineralization of Po. ³¹P-NMR spectra showed inorganic orthophosphate (up to 67%) and orthophosphate monoesters (up to 75%) as the major forms of P extracted in forest and grassland soils, respectively. Smaller proportions of orthophosphate diesters and trace amounts of phosphonates and pyrophosphate were found. With possible hydrolysis of P compounds during chemical extraction and slight systemic error in the processes of extraction with NMR, the results from NMR analysis are, in general, consistent with those of chemical extraction.     

Nucleophilic reactions of phorate and terbufos with reduced sulfur species under anoxic conditions

The reactions of phorate and terbufos with bisulfide (HS-), polysulfide (Sn2-), thiosulfate (S2O32-), and thiophenolate (PhS-) were examined in well-defined aqueous solution under anoxic conditions to investigate their role in the degradations of phorate and terbufos. Reactions were monitored at various concentrations of reduced sulfur species to obtain the second-order rate constants. The reactivity of the reduced sulfur species decreased in the order Sn2- > PhS- > HS- > S2O32-. Hydrolysis products, formaldehyde and diethyl disulfide/di-tert-butyl disulfide, indicated that OH-/H2O attacked the carbon atom between the two sulfur atoms, the so-called thioacetal carbon, which is very reactive due to the presence of the two neighboring sulfur atoms. The reaction of phorate and terbufos with PhS- was investigated to study the transformation products in the reactions with reduced sulfur species. The transformation products demonstrated that the observed increase in rate constants in the reaction with reduced sulfur species compared to hydrolysis could result from the nucleophilic attack of reduced sulfur species at the alpha-carbon of the ethoxy group and at the thioacetal carbon atom. The temperature dependence of measured second-order rate constants of the reaction of phorate and terbufos with HS- over 25-50 degrees C was investigated to explore activation parameters, which are not significantly different for phorate and terbufos. All of the observations may imply similar pathways in the degradation of phorate and terbufos in the presence of reduced sulfur species. Slightly higher hydrolysis rates of terbufos and second-order reaction rate constants for the reactions with sulfur species of terbufos compared with those for phorate are observed, which could be attributed to the slightly different substituents.     

Phytotransformation of Phorate by <Emphasis Type="BoldItalic">Brassica juncea</Emphasis> (Indian Mustard)

Over 5 days, Brassica juncea removed 54% of the highly toxic insecticide phorate from the medium with the formation of phorate sulfoxide in small quantity. The loss of phorate from the medium followed first-order kinetics. The half-life of phorate disappearance from water decreased by ~4.5-fold in the presence of B. juncea. Mild phorate phytotoxicity was evident from the elevated activities of the antioxidative enzymes like glutathione-disulfide reductase, glutathione S-transferase, superoxide dismutase, and catalase in the plants. Nevertheless, the ubiquitous antioxidative peroxidase was not significantly increased, nor the total glutathione content, due to phorate exposure. Phosphotriester bond hydrolysis and glutathione S-transferase-mediated conjugation seemed to be the key reactions for phorate metabolism by B. juncea. From the limited information available, for the first time, a tentative mapping of phytotransformation pathways was performed.     

Identification of soil organic phosphorus by 31P nuclear magnetic resonance spectroscopy

Abstract

Soil samples from different land use systems were collected before cropping (in spring) and after harvest (in fall) for organic phosphorus (P) extractions by 0.4M sodium hydroxide (NaOH) and characterization by ³¹P nuclear magnetic resonance spectroscopy. To prevent hydrolysis of organic P compounds prior to sample concentration, NaOH was removed from the NaOH soil extracts using a G‐25 Sephadex column. The ³¹P NMR spectra in the NaOH soil extracts showed the presence of glucose‐6 phosphate (up to 64%), glycerophosphate (up to 45%), nucleoside monophosphates (up to 91%), and polynucleotides (up to 58%) as the major forms of organic P in soils. The relative concentration of nucleoside monophosphates and polynucleotides decreased in some of the soils after harvest. The ³¹P NMR spectra of the extracts also revealed the presence of phosphoenolpyruvates, a previously unreported form of soil organic P.     

Comparison of methods for the quantitative determination of phospholipids in lecithins and flour improvers

Phospholipid classes were determined qualitatively and quantitatively in eight commercial lecithins and three flour improvers by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and (31)P nuclear magnetic resonance spectroscopy ((31)P NMR). The total amounts of phospholipids as well as the amounts of phospholipid classes in the samples were comparable but depended on the method used for quantification. Highest selectivity was provided by (31)P NMR as all phospholipids and lysophospholipids could easily be quantified. By TLC only lysophosphatidylcholine could not be quantified, whereas HPLC was the method with the lowest selectivity, because lysophospholipids, except lysophosphatidylethanolamine, could not be determined. Sensitivity was best for HPLC and TLC with detection limits of 20-170 mug/mL. By means of (31)P NMR these figures increased by a factor of 10-70. The coefficients of variation were 5.5, 6.8, and 12.8% for quantification by TLC, HPLC, and (31)P NMR, respectively, showing that TLC was the method with the best reproducibility. Altogether, (31)P NMR can be recommended for the quantification of phospholipids, because it is easy to perform and results can be obtained quickly. As it requires minimum instrumental equipment, TLC is a good alternative to (31)P NMR. If high sensitivity is required, HPLC is the best method.     

固体废弃物及土壤中磷的形态分析技术

畜禽粪便等固体废弃物及土壤中磷的形态分析对于管理固体废弃物、提高磷素利用率、减少磷素流失风险具有重要意义。自20世纪初以来,已提出多种固体废弃物及土壤中磷素形态分析的方法,包括化学分级、酶水解法、红外光谱(FT-IR)、X射线衍射(XRD)、31P核磁共振(31P NMR)、X射线吸收近边结构(XANES)等。本文系统总结了一些固体废弃物及土壤中磷素形态分析的方法,比较了其特点,指出只靠单一的方法并不能很好的鉴定固体废弃物及土壤中磷素的形态,必须综合考虑运用多种方法,从而为磷素形态分析提供方法选择的依据,同时为进一步发展固体废弃物及土壤中磷素形态分析新技术奠定基础。     

Application of P and Al MAS NMR for phosphate speciation studies in soil and aluminium hydroxides: promises and constraints

Magic angle spinning (MAS) nuclear magnetic resonance (NMR) was used to investigate the chemical environment of P in soil and soil components. ³¹P and ²⁷Al MAS NMR spectra are presented of synthetic aluminium hydroxides (amorphous aluminium hydroxide and gibbsite), reacted with P under different conditions of P concentration, temperature and pH. The reaction product is amorphous octahedral aluminium phosphate, which transforms (partly reversibly) to tetrahedral aluminium phosphate upon drying. Results of several experiments on excessively fertilized sandy soil material are discussed. The soil particle fraction smaller than 50 μm was used for NMR analysis. ³¹P and ²⁷Al MAS NMR spectra show a Ca---P pool and an Al---P pool. A six-fold water extraction removes part of both P pools. Oxalate extraction removes all Ca---P and Al---P from the sample. Removal of the labile P pool by the HFO-DMT long-term P desorption technique, does not drastically change the ³¹P MAS NMR spectrum. The formerly mentioned Ca---P and Al---P are thus stable P pools. The ¹H---³¹P cross-polarization (CP) spectrum of the original soil sample revealed a third chemical environment, which was identified as labile Ca---P: this P pool does not appear in the ¹H---³¹P CP spectrum of the soil sample from which the fast P pool had been removed. The combination of MAS and CP MAS NMR can thus reveal at least three different P species in soil, of which two pools were identified as stable, and one as labile.     

固体废弃物及土壤中磷的形态分析技术

畜禽粪便等固体废弃物及土壤中磷的形态分析对于管理固体废弃物、提高磷素利用率、减少磷素流失风险具有重要意义。自20世纪初以来,已提出多种固体废弃物及土壤中磷素形态分析的方法,包括化学分级、酶水解法、X射线衍射(XRD)、红外光谱(FT-IR)3、1P核磁共振(31P NMR)、X射线吸收近边结构(XANES)等。本文系统总结了一些固体废弃物及土壤中磷素形态分析的方法,比较了其特点,指出只靠单一的方法并不能很好地鉴定固体废弃物及土壤中磷素的形态,必须综合考虑运用多种方法,从而为磷素形态分析提供方法选择的依据,同时为进一步发展固体废弃物及土壤中磷素形态分析新技术奠定基础。     

Influence of insecticides on microbial transformation of nitrogen and phosphorus in Typic Orchragualf soil

Four insecticides, viz., BHC, phorate, carbofuran, and fenvalerate, were applied at the rate of 7.5, 1.5, 1.0, and 0.35 kg a.i. ha(-)(1), respectively, to investigate their effects on the growth and activities of N(2)-fixing and phosphate-solubilizing microorganisms in relation to the availability of N and P in laterite (Typic Orchragualf) soil. Insecticides in general, and BHC and phorate in particular, stimulated the proliferation of aerobic nonsymbiotic N(2)-fixing bacteria and phosphate-solubilizing microorganisms and also their biochemical activities, such as nonsymbiotic N(2)-fixing and phosphate-solubilizing capacities, which resulted in greater release of available N (NH(4)(+) and NO(3)(-)) and P in soil. All the insecticides were persistent in soil for a short period of time, and the rate of dissipation was highest for fenvalerate followed by phorate, carbofuran, and BHC, depicting the half-lives (T(1/2)) 8.8, 9.7, 16.9, and 20.6 days, respectively. The insecticides followed first-order reaction kinetics during their dissipation in soil.     

Improving Sensitivity of Solution 31P NMR Analysis in Australian Xeralfs

Phosphorus-31 nuclear magnetic resonance (³¹P NMR) spectroscopy is widely used to identify and quantify phosphorus (P) forms in soil. This study aimed to determine whether narrowing the soil to extractant sodium hydroxide–ethylenediaminetetraacetic acid (NaOH-EDTA) ratio from 1:20 to values as low as 1:4 would improve sensitivity of solution ³¹P NMR spectroscopy without degrading resolution or quantitation. Four Australian soils were tested using four ratios. The narrowest ratio of 1:4 gave the best quality NMR spectra in terms of signal-to-noise ratio. Peak resolution was not degraded on narrowing the ratio. There was no clear effect of narrowing the extraction ratio on extraction efficiency or the distribution of signal among chemical shift regions (orthophosphate, monoester P, diester P, and pyrophosphate). We conclude that a ratio of 1:4 improved NMR analysis for these particular soils and should be considered for other soils, particularly low-P soils, where NMR sensitivity is limiting.     

Hydrolysis of terbufos using simulated environmental conditions: rates, mechanisms, and product analysis.

This study focuses on the hydrolysis of terbufos, an organophosphorus pesticide. Combining GC-MS and wet chemistry methods, di-tert-butyl disulfide and formaldehyde were identified and quantified as major degradation products. Diethyl dithiophosphate was also indirectly identified as a degradation product under alkaline conditions. Hydrolysis rate constants of terbufos under homogeneous conditions were comparable to those of phorate and show relative insensitivity to pH under slightly acidic to neutral pH conditions, as the observed rate constants varied only in the range of (4.5-5.0) x 10(-6) s(-1) between pH 5.7 and 9.4; neutral hydrolysis is thus the most dominant hydrolysis pathway of terbufos in ambient waters. The mechanisms for terbufos hydrolysis and the formation of the major products and their temporal profiles are discussed. To assess the environmental impact of degradation products of this widely used pesticide, Microtox was used to analyze the toxicity of terbufos and two of its degradation products: diethyl dithiophosphate and di-tert-butyl disulfide; the EC(50) of terbufos was found to be >17 microM, whereas the EC(50) of di-tert-butyl disulfide was 1.3 microM.     

Protein analysis by 31p NMR spectroscopy in ionic liquid: quantitative determination of enzymatically created cross-links

Cross-linking of β-casein by Trichoderma reesei tyrosinase (TrTyr) and Streptoverticillium mobaraense transglutaminase (Tgase) was analyzed by (31)P nuclear magnetic resonance (NMR) spectroscopy in ionic liquid (IL). According to (31)P NMR, 91% of the tyrosine side chains were cross-linked by TrTyr at high dosages. When Tgase was used, no changes were observed because a different cross-linking mechanism was operational. However, this verified the success of the phosphitylation of phenolics within the protein matrix in the IL. Atomic force microscopy (AFM) in solid state showed that disk-shaped nanoparticles were formed in the reactions with average diameters of 80 and 20 nm for TrTyr and Tgase, respectively. These data further advance the current understanding of the action of tyrosinases on proteins on molecular and chemical bond levels. Quantitative (31)P NMR in IL was shown to be a simple and efficient method for the study of protein modification.     

Quantification of inositol phosphates using (31)P nuclear magnetic resonance spectroscopy in animal nutrition

A (31)P NMR method for quantitative determination of inositol phosphates in simple incubation samples of sodium phytate and Aspergillus niger phytase and in different types of complex samples, such as diets, digesta, and feces, is described. The inositol phosphates in complex samples were extracted with HCl, concentrated, and purified using freeze-drying and filtration and subsequently determined at pH 12.6 in aqueous solution using a (31)P NMR method. The (31)P NMR technique has as its main advantages over the HPLC techniques that it does not necessitate standards that may cause background matrix effects and that the spectra of inositol phosphates and orthophosphate appear in the same run without further sampling errors. The results of inositol hexaphosphate analysis with HPLC can be confirmed by this (31)P NMR method. Contents of inositol tetra-, tri-, di-, and monophosphate in the biological samples appear to be quantitatively not important. The (31)P NMR method can be applied for use in animal nutrition in general and studies of using phytase in diets for farm animals in particular, by measuring the content of inositol phosphates in feed ingredients, complete feeds, ileal contents, and feces of pigs and poultry.     

Classification of edible oils by employing 31P and 1H NMR spectroscopy in combination with multivariate statistical analysis. A proposal for the detection of seed oil adulteration in virgin olive oils

A combination of (1)H NMR and (31)P NMR spectroscopy and multivariate statistical analysis was used to classify 192 samples from 13 types of vegetable oils, namely, hazelnut, sunflower, corn, soybean, sesame, walnut, rapeseed, almond, palm, groundnut, safflower, coconut, and virgin olive oils from various regions of Greece. 1,2-Diglycerides, 1,3-diglycerides, the ratio of 1,2-diglycerides to total diglycerides, acidity, iodine value, and fatty acid composition determined upon analysis of the respective (1)H NMR and (31)P NMR spectra were selected as variables to establish a classification/prediction model by employing discriminant analysis. This model, obtained from the training set of 128 samples, resulted in a significant discrimination among the different classes of oils, whereas 100% of correct validated assignments for 64 samples were obtained. Different artificial mixtures of olive-hazelnut, olive-corn, olive-sunflower, and olive-soybean oils were prepared and analyzed by (1)H NMR and (31)P NMR spectroscopy. Subsequent discriminant analysis of the data allowed detection of adulteration as low as 5% w/w, provided that fresh virgin olive oil samples were used, as reflected by their high 1,2-diglycerides to total diglycerides ratio (D > or = 0.90).     

Analysis of monoglycerides, diglycerides, sterols, and free fatty acids in coconut (Cocos nucifera L.) oil by 31P NMR spectroscopy

Phosphorus-31 nuclear magnetic resonance spectroscopy ( (31)P NMR) was used to differentiate virgin coconut oil (VCO) from refined, bleached, deodorized coconut oil (RCO). Monoglycerides (MGs), diglycerides (DGs), sterols, and free fatty acids (FFAs) in VCO and RCO were converted into dioxaphospholane derivatives and analyzed by (31)P NMR. On the average, 1-MG was found to be higher in VCO (0.027%) than RCO (0.019%). 2-MG was not detected in any of the samples down to a detection limit of 0.014%. On the average, total DGs were lower in VCO (1.55%) than RCO (4.10%). When plotted in terms of the ratio [1,2-DG/total DGs] versus total DGs, VCO and RCO samples grouped separately. Total sterols were higher in VCO (0.096%) compared with RCO (0.032%), and the FFA content was 8 times higher in VCO than RCO (0.127% vs 0.015%). FFA determination by (31)P NMR and titration gave comparable results. Principal components analysis shows that the 1,2-DG, 1,3-DG, and FFAs are the most important parameters for differentiating VCO from RCO.     

Determination of phospholipids in olive oil by 31P NMR spectroscopy

A nondestructive analytical method based on NMR spectroscopy was developed for the determination of phospholipids in olive oil. The phospholipids extracted from virgin olive oil with a mixture of ethanol/water (2:1 v/v) were identified and quantified by high resolution (31)P NMR spectroscopy. The main phospholipids found in olive oil were phosphatidic acid, lyso-phosphatidic acid, and phosphatidylinositol. Validation of the (31)P NMR methodology for quantitative analysis of phospholipids in olive oil was performed. Sensitivity was satisfactory with detection limits of 0.25-1.24 mumol /mL. In addition, the composition of fatty acids in phospholipids model compounds and those in olive oil samples was estimated by employing one- and two-dimensional (1)H NMR. The results indicated that the fatty acid composition in phospholipids and triacylglycerols of olive oil was similar.     

Evaluation of the factors affecting direct polarization solid state 31P-NMR spectroscopy of bulk soils

³¹P‐NMR spectroscopy on bulk soils is a powerful tool for the identification of the different phosphorus forms in soils and for the evaluation of the dynamics of soil P. Up to now the majority of the papers dealt with liquid state ³¹P‐NMR spectroscopy on soluble soil organic substances. Only few papers were addressed to the study of the different phosphorus forms directly in bulk soils. In the present paper, some organic and inorganic phosphates of known structures, which are likely to be present in soil systems, were studied by direct polarization (DP) magic angle spinning (MAS) ³¹P‐NMR spectroscopy in order to understand the electronic factors responsible for chemical shifts of the phosphorus (P) nucleus and to serve as guidelines to assign P resonances in soil spectra. Number of hydrating water molecules, type of counter‐cation, degree of covalence, and spatial conformation of P in phosphate structures were found to affect signal positions in ³¹P‐NMR spectra. Both hydrating water and increase in degree of covalence of the X‐O‐P bonds (X=H, Na) enhanced the electronic density (E_D) around P, thereby producing up‐field shifts in ³¹P‐NMR spectra. The exchange of the Na⁺ counter‐cation with NH₄⁺ resulted in an increase of the cation potential (P_C) that is a measure of the cation polarizing power, and induced a down‐field shift of P signals, due to a corresponding reduction in E_D around the P nucleus. Both NMR down‐ and up‐field shifts were observed in organic phosphates, and were dependent on the spatial orientation of the phosphate groups that may have been fixed anisotropically in the solid state. Based on the factors that influence P chemical shifts for standard phosphates, attempts to assign ³¹P‐NMR signals in the spectra of five different unperturbed bulk soils were made.     

Confirmation of organothiophosphorus insecticide residues in fruit and vegetables by oxidative derivatization.

Oxidative derivatization of 10 organothiophosphorus insecticides at nanogram levels, with neutralized sodium hypochlorite (NaOCl) produced their respective P=O oxygen analogs, as shown by GC/MS and FT-IR analysis.In addition, phorate resulted in phorate oxon sulfoxide while methidathion gave a mixture of oxidation products. Four macro-scale oxidation products of methidathion were isolated by thin layer chromatography and 3 were identified, namely, methidaoxon, bis(2-methoxy-delta2-1,3,4-thiadiazolin-5-on-4-yl) sulfide, and the corresponding disulfide, by comparison of their infrared and mass spectra with those of authentic samples. The relative response to sulfide and disulfide products of the flame photometric detector in both the P- and S-modes is discussed. This derivatization is applied to the confirmation of chlorpyrifos, ethion, phorate, DMPA (Zytron), leptophos, and methidathion in celery, potatoes, lettuce, tomatoes, and apples at 0.25-0.5 ppm fortification levels.     

Transformations of phosphorus during incubation of beech leaf litter in the presence of oxides

Some minerals have large PO₄^3? sorption capacities. However, we do not know how these mineral phases influence the speciation of organic P when organic matter decomposes. To characterize the interactions between mineral phases and organic P species, we incubated mixtures of beech leaf litter and Fe oxide, Al hydroxide, birnessite, and quartz sand. The samples were extracted with NaOH-NaF and analysed by ³¹P nuclear magnetic resonance (NMR) spectroscopy. Phosphate mono-and diesters gave the most prominent signls in the ³¹P NMR spectra. During incubation, the proportion of the monoester P decreased, whereas that of the diester P increased. Since NMR intensities assigned to teichoic acids increased, it seems that microbial P accumulated during incubation. The synthesis of microbial compounds was strongest in the presence of Fe oxide. All mineral phases investigated gave rise to a resonance in the ³¹P NMR spectra which was not present in the control treatment to which the mineral phases had not been added. The resonance was tentatively assigned to sugar diesters, which probably originated from organo-mineral interactions.     

Kinetics of diglyceride formation and isomerization in virgin olive oils by employing 31P NMR spectroscopy. Formulation of a quantitative measure to assess olive oil storage history

Diacylglycerol isomers and free acidity were determined for five extra virgin olive oils of different initial acidities by employing a facile (31)P NMR methodology as a function of storage time and storage conditions. The kinetic treatment of the hydrolysis of triacylglycerols (TGs) and the isomerization of 1,2-diacylglycerols (1,2-DGs) to 1,3-diacylglycerols (1,3-DGs) during storage of 18 months at ambient temperature in the dark and light and at 5 degrees C in the dark showed that the isomerization is strongly dependent on the rate of the TGs hydrolysis, the initial free acidity (H(0)) of the virgin olive oil samples, and storage conditions. Although the time-evolution of the diacylglycerols (DGs) depends on the TGs hydrolysis, the ratio D of the concentration of 1,2-DGs to the total amount of DGs was found to be independent of this factor. From the kinetic expression of the ratio D, a quantitative measure was formulated that allows the estimation of the storage time or age of virgin olive oils. Application of this quantitative measure to several olive oil samples of known and unknown storage history resulted in a very good agreement with respect to the actual storage time for up to 10-12 months of storage. For a longer storage period, where the isomerization of DGs is close to its equilibrium state, the calculated age index is only indicative.