Rapid Determination Of Phosphate In Water And Soil And Analysis Of Oxygen Isotope Composition In Soil Phosphate

Phosphorus(P) is one of limiting nutrients to crop production due to its low solubility, easy fixation and relative immobility in soils. Meanwhile P runoff or leaching is the main factor causing eutrophication of water if over P fertilizer application to field soils. The analysis of water and soil phosphate(Pi) concentration is commonly determined in manual by ascorbic acid-molybdenum blue(MA) or malachite green(MG) colorimetric methods with the conventional ultraviolet-visible spectrometer using a 1.0 cm optical path length cuvette. A continuous flow auto-analyzer and a microplate reader combined with MA or MG colorimetric methods were used to rapidly determining Pi in water samples. The proposed automatic methods were adapted to 48- or 96-well microplate in a microplate reader for analysis of soil Olsen-P extracted with 0.5 mol/L Na HCO3(pH=8.5). The ratio between the heaviest and lightest oxygen isotopes(18O and 16O) in phosphate, i.e. the oxygen isotope composition of phosphate(δ18 O-P), can be used to study P cycle and transformations in soils.Fluvo-aquic soil and black soil were sampled from the plots with four treatments including NK, NPK,NPM+straw, NPK+manure in the long-time fertilizer trials. We used 0.5 mol/L NaHCO3(pH 8.5) and1.0 mol/L HCl to extract soil Pi. Pi in the extract solutions was purified with multiple mineral precipitations and finally precipitated as silver phosphate. Silver phosphate samples are prolifically decomposed to carbon monoxide and analyzed for δ18O by elemental analyzer-stable isotope ratio mass spectrometer. The results were showed as following:(1) The MG colorimetric method is a more sensitive method compared to the MA colorimetric method and is often used to determine P at submicromolar concentrations in samples. Optimized experimental conditions with the MG colorimetric method for measuring Pi in water samples were first obtained in this study. The volume ratio of ammonium molybdate: sulfuric acid : malachite green was10:8:9 in chromogenic agent. The range of maximum absorption wavelength was 620~660 nm. The linar curve for P analysis based on Lambert-Beer law was in the concentrations of 0~ 0.5 μg/mL. The recovery rate of standard addition(RRSD) in water samples was 97.0%~108.2%. According to the above experimental conditions, we developed a continuous flow auto-analyzer with the MG colorimetric method to determine Pi in water samples. The standard curve was Y=90872X+1915.2( R2=0.9991**), here Y indicates the peak height obtained by the auto-analyzer, X indicates P concentration. The limit of detection(LOD) was 0.006 mg/L. The RSD was between 95.8 %~99.3 %.The relative standard deviation(RSD) for determining Pi in the lake water samples was less than 2.6%(n=10).The microplate reader is a type of spectrophotometer that consists of a multichannel optical system and quickly reads the absorbance of the wells in a microplate at preset wavelengths.We used the microplate reader coupled with the MG colorimetric method with 48-well or 96-well microplate to determine P in water sample.The standard curve equation of 96 and 48-well microplate method were Y=1.6906 X + 0.13011(R2=0.9948) and Y=3.2637X+ 0.23033(R2=0.9995), respectively, which indicated that the sensitivity were 1.691 and 3.264 mg/L P per absorbance unit, respectively. The RRSDwith the two methods ranged from 98.3 % to 108.9 %. The RSD of 48-well microplate method is less than 96-well microplate method when the P content in water samples is lower than 0.01 mg/L.(2) The analysis of soil Olsen-P extracted with 0.5 mol/L Na HCO3(pH=8.5) is commonly determined in manual by ascorbic acid-molybdenum blue(MA) or malachite green(MG) colorimetric methods.The proposed automatic methods were adapted to 48- or 96-well in a microplate reader. Nine soils sampled from North China were used. For the MA method with the 48- and 96-well microplate, the sensitivity was 2.271 and 3.891 mg L-1 P per absorbance unit; the limit of detection(LOD) was 0.015 and 0.029 mg/L, respectively. For the MG method, the sensitivity was 0.551 and 0.623 mg/L P per absorbance unit; LOD was 0.002 and 0.007 mg L-1 with 48- and 96- well, respectively. Soil Olsen-P levels by the proposed methods significantly correlated with the data by the conventional spectrometer method with R2 >0.99 and linear slope near 1.0. The relative standard deviation(RSD) of the MA method was 1.1%~9.7% and 4.2%~15.1% with the 48- and 96-well, respectively. For the MG method,RSD values of the 48 and 96-well were 2.3%~14.8% and 8.5%~37.6%, respectively. The MA colorimetric technique with 48-well microplate adaptation could be used for testing soil Olsen-P in laboratories requiring high throughput and strict quality.(3) The NaHCO3-Pi and HCl-Pi were enriched and purified through co-precipitating with magnesium hydroxide and ammonium molybdate along with amine phosphate magnesium, respectively.The further purified procedures were through cerium phosphate precipitation and resin separation. It is ultimately converted to silver phosphate. Silver phosphate samples were prolifically decomposed to carbon monoxide and analyzed for δ18O by elemental analyzer-stable isotope ratio mass spectrometer.The P recovery rates in the process of three key purification precipitations were more than 92%,indicting less Pi loss. There was no isotope fractionation during extraction and purification. The obtained silver phosphate samples had high purity according to X-ray diffraction and oxygen content determination. These results indicated that the procedures for oxygen isotope analysis of Na HCO3-Pi and HCl-Pi were feasible. The δ18O-P(NaHCO3-Pi) levels under NK treatment were 16.32‰ of fluvo-aquic soil and 16.23 ‰ of black soil, which were significantly different from the treatments such as NPK, NPKM and NPKS. The δ18O-P(HCl-Pi) of fluvo-aquic soil and black soil did not changed significantly in two tested soils and four fertilization treatments. The δ18O-P(HCl-Pi) values were17.21‰ ~17.95‰. The δ18O-P(NaHCO3-Pi) of fluvo-aquic soil and black soil obviously decreased under no Pi fertilization.In concluded, the continuous flow auto-analyzer and the microplate reader with the MG or MA colorimetric methods could be used for rapid testing Pi in soil and water samples in laboratories requiring high throughput and strict quality. The analysis of the oxygen isotopic composition of inorganic phosphate extracted from soils with Na HCO3 or HCl provided a potential tool to trace P sources and to determine the extent of P cycling in the soil.