Extraction of NOx and Determination of Nitrate by Acid Reduction in Water, Soil, Excreta, Feed, Vegetables and Plant Materials MIR,

Different methods are available for extracting NOx from different samples. A judicious combination of lead acetate, sodium hydroxide and magnesium chloride has been devised to enable extraction of NOx from different samples ensuring removal of potential interfering agents. The method provides over 95 per cent mean recovery with nearly 3 per cent accuracy and precision. Nitrite is determined by Griess reaction, and removed from samples by urea treatment to obviate any interference by nitrite in nitrate determination. Nitrate is determined by acid reduction method with minimum detection limit 0.5 ppm as N. The methods have been applied to selected environmental samples including food materials and excretory products. The average nitrate levels (as ppm N) found in water (0.8), soil (9), human urine (43), sheep excreta (2654), chicken feed (29), radish (270), spinach (222), carrot (194), potato (41), cabbage (11), tomato (2), Bermuda grass (175) and morning-glory leaves (576) have been within safe and documented limits. The average levels of nitrite, as ppm N, have generally ranged from 0.04 to 2.1 with highest content, 13, in sheep fecal matter. The protocol is intended for general use in environmental analysis, toxicological investigations and risk assessments. @JASEM Water, soil, plant materials, feed and excreta are monitored for levels of NOx for their toxicological implications and risk assessments (Schuddeboom, 1993; WHO, 2007). Nitrate is a more stable anion than its toxic metabolite nitrite and is dominant in environmental and biological samples. Methods based on the principle of Griess reaction are widely employed for their determinations as these are simple, specific and sensitive enough for routine use. However, for determination of nitrate, these methods require prior reduction of nitrate to nitrite which is accomplished by cadmium (APHA, 1998; AOAC, 2000; Cortas and Wakid, 1990; Green et al., 1982; Schneider and Yeary, 1973), vanadium (Miranda et al., 2001), zinc (Nelson et al., 1954; Diven et al., 1962; Mir, 2007), hydrazine (Oms et al., 1995), or specific nitrate reductases (Guevara et al., 1998). Of late, an inexpensive, rapid acid reduction method has been optimized for determination of nitrate (Mir, 2008). Standard methods commonly used for determination of nitrate have been rated as “semi quantitative” (Sawyer and McCarty, 1978). A major technical limitation in the methods is that the nitrate is measured indirectly with measurements made twice, before and after reduction, and the nitrate determined as a difference of NOx and NO2 (Sun et al., 2003). This approach has serious limitations while determining nitrate in presence of nitrite (Mir, 2007). Differential abilities of nitrite and nitrate to engage in diazotization of sulfanilamide under different experimental conditions have provided a rationale for their sequential analysis in test samples (Mir, 2008). Urea pretreatment enables direct determination of nitrate in presence of nitrite (Mir, 2007, 2008). The maximum safe and / or regulatory limits for nitrate in drinking water, vegetables, forage/foodstuffs (Bagley et al., 1997; Schuddeboom, 1993; Stallings, 2006; WHO, 2007) or normal levels in human urine (Cortas and Wakid, 1990; Green et al., 1982; Guevara et al., 1998; Tsikas et al, 1994), chicken feeds (Okafor and Okorie, 2006) and soils (Chiroma et al., 2007; Jaiswal, 2003) or for maintaining aquariums (Sharpe, 2008) are well within the detection range, 0.5-10 ppm N, of acid reduction method for nitrate (Mir, 2008). Perusal of literature reveals use of different extractants for extracting NOx from different samples. These include water (Bhargava and Raghupathi, 1993), 0.1M HCl (Krishna and Ranijhan, 1980; Nelson et al., 1954), CdCl2 + BaCl2 (AOAC, 2000), Somogyi reagent (Cortas and Wakid, 1990), CuSO4 + Ca (OH) 2 + MgCO3 (Gupta, 2004), methanol/diethyl ether (Guevara et al., 1998), calcium chloride + charcoal (Chiroma et al., 2007) or Carez reagent (ISO, 1984). A need to develop a uniform protocol for extracting NOx from different samples was mooted on the considerations of ensuring stability of the anions particularly of nitrite, removal of potential interfering agents, minimize losses and obtain colorless transparent extracts. A combination of lead acetate and activated charcoal (Oser, 1976; Pomeranz and Meloan, 1996) coupled to alkaline extraction achieved by sodium hydroxide and magnesium chloride (Mir, 2004) was found to provide an effective alternative extraction method for application to majority of the test samples. Consequently, the investigations were designed to: (1) investigate the efficiency of the extraction method and to apply it to different test samples; (2) employ acid reduction method coupled to urea treatment for determination of nitrate, and (3) compare data generated for nitrate by acid reduction method in selected samples with those obtained by comparable metal reduction method. MATERIALS AND METHODS The experiments were carried out at an ambient temperature of 20.6 ± 0.7 C. The chemicals used Extraction of NOx and Determination of Nitrate by Acid Reduction in Water, Soil, Excreta, Feed, Vegetables and ..................... *Corresponding author: Dr S.A. Mir 58 were of analytical grade purity. Appropriate working solutions of nitrate and nitrite were made by dilution in water, respectively, from stock solution of 500 ppm as nitrate N (potassium nitrate 0.361 % in water) and of 700 ppm as nitrite N (sodium nitrite 0.345 % in water) with 0.2 % chloroform as preservative in stock solutions. The strength of HCl used was found to be approximately 11.7 M. The basal diazotization powder contained finely pulverized homogeneous mixture of barium sulfate 10 g, citric acid 7.5 g and sulfanilamide 0.5 g. For reduction purpose, finely pulverized homogenous mixture of zinc dust and manganese sulfate (1:5 by weight) was added @ 30 mg per 500 mg of the diazotization powder (RDP). The other reagents included 1 %( w/v) sulfanilamide in 1 % (v/v) HCl, 1 %( w/v) N-1-(naphthyl)ethylenediamine dihydrochloride in 1% (v/v) HCl (NEDA), 2 % (w/v) urea in water, 20 % (v/v) glacial acetic acid in water, 40% (w/v) lead acetate trihydrate in water, 20 % (w/v) sodium hydroxide in water (ca.5M NaOH) and 50 %( w/v) magnesium chloride hexahydrate in water. The activated charcoal was purified by sequential treatments with 0.1M NaOH, 0.2 M HCl, and deionized water till effluent failed to show any detectable nitrate and nitrite. The purified charcoal was drained off its liquid, dried, cooled to room temperature, and stored till use. The samples included freshly collected local vegetables viz., potatoes, cabbage, radish, carrots, spinach and tomatoes, aerial parts of Cynodon dactylon (Bermuda grass, locally called Dramoon), leaves of Ipomoea purpurea (morning-glory, locally called Ashqpaechan), Formulated chicken feed (labeled composition approximately as maize 62%, ground nut cake 13%, soya meal 13 %, fish meal 10 % and mineral mixture 2 % with crude protein 21.78 %), top soil samples from apparently fertile premises of the Faculty, tap water, fecal pellets from sheep, dominantly Corriedale germplasm maintained at the Faculty Farm, and freshly collected urine samples from apparently healthy young male volunteers of the Faculty. Properly cleansed vegetables were made to pulpy homogenate in electric blender. All solid samples were air-dried and made to fine powder. Powdered soil samples were sieved to remove visible debris. Tap water was directly processed, or following its 5or 10-folds concentration by evaporation. Vegetable homogenates 5g each, tomato pulp homogenate 10 g, chicken feed powder 1.25 g, Bermuda grass powder 0.5 g, morning-glory leaves powder 0.25 g, fecal powder 0.1g and composite soil powder 12.5 g were extracted with 15 ml or 20 ml water with intermittent shaking for about 1 hour at room temperature. Urine samples were diluted with three volumes of water. Urine samples and extracts were added lead acetate to provide 10 % salt concentration, shaken well, centrifuged at 6000 rpm for 10 minutes, and filtered vide Whatman filter paper No.1. Each eight milliliter of the extract was sequentially added 1 mL 5 M NaOH, 1 mL 50% magnesium chloride solution, and purified charcoal @ 10-30 mg mL -1 solution depending upon degree of pigmentation. Water did not require lead acetate and charcoal treatments. Each nine milliliter water sample was added 0.5 mL each of sodium hydroxide and magnesium chloride solutions. All the samples were well shaken, allowed to stand 2-3 minutes following each addition, centrifuged and filtered vide Whatman filter paper No.1. The sample extracts were assayed for NOx. The efficiency of the outlined extraction processes was studied by estimating per cent recovery of the anions from aliquots of water containing nitrate and nitrite, respectively 50 and 2 μg as N. The values obtained from the samples subjected to the extraction processes were compared with the values obtained from unprocessed aliquots of the same cocktail. The reagent blanks for each process were run simultaneously. The purified charcoal was used at maximum concentration limit, 30 mg mL. Appropriate aliquot of assay extract (0.5 to 2 mL), depending upon nitrite concentration as checked by trial tests, was made 2 mL with distilled water, added 0.1 mL sulfanilamide solution and then 0.3 mL HCl, allowed to stand 2-3 minutes, and added 0.2 mL coupling agent. The samples were monitored at 540 nm (UV-Visible Spectrophotometer SL-150, Elico (India) Ltd, Hyderabad) after 20 minutes standing against reagent blank. Standard nitrite 0.1 and 0.2 ppm as N in 2 mL volume were simultaneously processed. Extracts containing excessive nitrate were appropriately diluted with water so that nitrate content remained within the linear detection range of acid reduction method (0.5-10 ppm as N). Urea treatment protocol optimized for determination of nitrate by metal reduction (Mir, 2007) or by acid reduction (Mir, 2008) has been followed in this work. Each milliliter of the sample extract was added 0.1 mL of urea solution and 0.2 mL HCl, and incubated in boiling water bath for a period of 10 minutes to get rid off any nitrite. The urea treated samples were assayed for nitrate. Nitrate was determined by optimized acid reduction technique employing one milliliter sample extract (Mir, 2008). Nitrite free samples were added each 0.1 mL sulfanilamide solution and 2 mL HCl. The mixture was incubated in boiling water bath for exactly 10 minutes, cooled to room temperature, and added 0.2 mL of coupling agent, and color monitored at 540 nm after 20 minute standing. Water blank and standard nitrate solutions, 1 and 3 ppm as N, were subjected to identical treatments. The extracts from selected samples including soil, carrot, morning-glory, and feed were simultaneously assayed for nitrate by modified (Mir, 2007) metal reduction technique (Nelson et al., 1954).The modifications included: a) substituting sulfanilic acid with a more stable aromatic amine, sulfanilamide, b) substituting 1-naphthylamine with a widely employed safer coupling agent NEDA, c) separately adding coupling agent to avoid adsorption of the azo-dye by the filter paper, and d) using optimized mass of zinc in the reduction powder to curtail furthering of reduction process by the metal (Mir, 2007). UreaExtraction of NOx and Determination of Nitrate by Acid Reduction in Water, Soil, Excreta, Feed, Vegetables and ..................... *Corresponding author: Dr S.A. Mir 59 treated sample extracts were each added 9 mL of acetic acid solution, and a scoopful, ca. 500 mg, of RDP, shaken well for about 1 minute, centrifuged at 6000 rpm for 5-10 minutes and filtered over Whatman No.1 filter paper. Each 5 mL filtrate was added 0.2 mL NEDA, and color monitored at 540 nm after 20 minute standing. Water blank and nitrate standards, 1 and 3 ppm as N, were subjected to identical treatments. The concentrations of nitrate and nitrite in the samples were calculated in terms of simultaneously processed standards: X = (A.B)/ (C.D.E) Where X = Concentration of the analyte in the original sample, ppm N. A = Absorbance of assay extract. B = Concentration standard as μg N. C = Absorbance of standard sample. D = mL sample extract used for the assay. E = Concentration of sample extract, g or mL original sample per mL sample extract RESULTS AND DISCUSSION Lead acetate was chosen for its ability to precipitate proteins, organic acids, and chromogenic phenolics (Pomeranz and Meloan, 1996) that are most likely to be present in plant extracts. Lead acetate has been employed as protein precipitant for clarification of blood (Hoffman, 1925), potato extracts (Kinter, 1972) and for determination of nitrate in rumen liquor (Lewis, 1951). Poor decolorizing ability of lead acetate (Pomeranz and Meloan, 1996) has been compensated by using activated charcoal wherever necessitated. Lead acetate provides additional protection by removing sodium azide, phosphates and, as hydroxide, ascorbate (Oser, 1976). These substances interfere with the Griess technique (Cortas and Wakid, 1990; Miranda et al., 2001). Sodium hydroxide serves to remove lead as hydroxide, as well as to remove other interfering metals including Bi, Ag , Hg, Fe, Au (APHA, 1998), Cu , Zn (Cortas and Wakid, 1990), Al, Fe (author, unpublished) likely to be present in water and soil samples. Magnesium chloride removes residual hydroxide, and prevents any solubilization of lead hydroxide in fixed alkali hydroxide. Combined use of sodium hydroxide and magnesium chloride has been demonstrated to remove various metallic ions including Zn , Fe, Fe and Cu 2+ from samples (Mir, 2004). The protocols ensured high alkalinity (Protocol A, pH 9.9 ± 0.1; protocol B, pH 10.9 ± 0.1, n = 4 each) and high electrolyte concentration in the extracts, maximally ca. 0.42 M CH3COONa and ca. 0.21 M MgCl2. These conditions have been found to prevent any adsorption of nitrate and nitrite to activated charcoal (unpublished data), and to maximize recovery. Insoluble hydroxide matrices provided by lead hydroxide and magnesium hydroxide contribute to clarity of the extracts. The proportions of sodium hydroxide and magnesium chloride can be varied depending upon the metal contents in the test samples. The recovery with protocol A, relevant to majority of the sample, has been 98.8 ± 1.1 % for nitrite nitrogen and 95.4 ± 1.7 % for nitrate nitrogen with per cent accuracy 3.3 ± 1.0 (n = 10). With protocol B, relevant to apparently clean water samples, the recovery has been 99.6 ± 1.1 for nitrite nitrogen and 99.8 ± 1.6 % for nitrate nitrogen with per cent accuracy 2.2 ± 0.4 (n=10) (Table 1). Overall per cent accuracy has been 2.8 ± 0.5 (n=20), and precision as estimated from values of coefficient of variation as 3.0 ± 0.5 for four sets of experiments. Per cent recovery with different extraction procedures have ranged from 85 to 112 (Green et al., 1982; Guevara et al., 1998; Lewis, 1951; Nelson et al., 1954). Reagent blanks did not show any background coloration indicating the reagents were free of any detectable nitrite/nitrate. Table 1: Recovery of nitrate and nitrite with the extraction processes Extraction Extraction efficiency Protocol Relevance Analyte Added Recovered