ENCM Environmental Impact Assessment and Environmental Monitoring. Determination of nitrate and phosphate levels in well water/surface water

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1 ENCM Environmental Impact Assessment and Environmental Monitoring ntalmonitoringenvironmental Monitoring - Practical Number 4 Determination of nitrate and phosphate levels in well water/surface water Learning outcomes At the end of the practical the learners will be able to gain familiarity with the standard procedures of determining nitrate and phosphate levels in water determine the nitrate and phosphate levels in the water samples provided using the standard analytical procedures present data in a scientific report and comment on the water quality status 1. Introduction 1.1 Nitrate in water: Nitrate is the most highly oxidized form of nitrogen compounds. It is the end product of the decomposition of organic nitrogenous matter under aerobic conditions. Unpolluted natural waters usually contain only trace levels of nitrate. Main sources of nitrate in surface and ground water are chemical fertilizers from cultivated land and drainage from livestock feedlots, as well as domestic and some industrial waters. The nitrate determination helps the assessment of the character and degree of oxidation in surface waters, in groundwater penetrating through soil layers, in biological processes and in the advanced treatment of wastewater. In surface water, nitrate is a nutrient taken up by plants and assimilated into cell protein. Stimulation of plant growth, especially of algae, may cause water quality problems associated with eutrophication. The subsequent death and decay of algae produces undesirable effects on water quality. High levels of nitrate in drinking water may be a risk to bottle-fed babies (under three months of age) because the low acidity of their stomachs favours the reduction of nitrates to nitrites by microbial action. Nitrite is absorbed into the blood where it combines irreversibly with haemoglobin to form methaemoglobin. This results in reduction in oxygen carrying capacity of blood. In severe cases a condition known as infantile methaemoglobinaemia (blue baby syndrome) may occur which can be fatal for young babies. 1.2 Phosphates in water: Phosphorus compounds are present in fertilizers and in many detergents. Consequently, they are carried into both ground and surface waters with sewage, industrial wastes and storm run-off. Ground waters rarely contain > 0.1 mg L -1 phosphorus unless they have passed through soil containing phosphate or have been polluted by organic matter. High concentrations of phosphorus compounds may produce a secondary problem in water bodies where algal growth is normally limited by phosphorus. In such situations the presence of additional phosphorus compounds can stimulate algal productivity and enhance eutrophication. In natural waters/wastewaters, phosphorus occurs almost as phosphates. These are classified as orthophosphates, condensed phosphates (e.g. polyphosphates), and organically bound phosphates. Phosphates also occur in bottom sediments and in biological sludges, both as precipitated inorganic 1

2 forms and incorporated into organic compounds. The determination of phosphorous/different phosphate forms in water helps to assess the ground water/surface water quality status and associated pollution sources. 2. Nitrate analysis in water Several standard analytical methods are available for nitrate determinations. Ultra violet spectrophotometric method and cadmium reduction methods are two such methods. The determination of nitrate in water is difficult because of interferences, and much more difficult in wastewaters because of higher concentrations of numerous interfering substances. 2.1 Sample storage for Nitrate analysis : If possible do the NO3 - determinations in water just after sampling. If water samples have to be stored it can be done only for 2 d at 4 C. Disinfected samples (eg. Chlorinated effluents) are stable much longer without acid preservation. For longer storage of unchlorinated samples is needed, preserve with concentrated H2SO4 (2 ml/l of water) and store at 4 C. Note: When sample is preserved with acid, NO3 - and NO2 - cannot be determined as individual species. 2.2 Nitrate Analysis using cadmium reduction method: The method is recommended especially for NO3 - levels below 0.1 mg N/L where other methods lack adequate sensitivity. In this method, NO3 - is reduced almost quantitatively to nitrite (NO2 - ) in the presence of cadmium. This method uses commercially available Cd granules treated with copper sulfate and packed in a glass column. The NO2 - produced by this is determined by diazotizing with sulfanilamide and coupling with N-(1-naphthyl)-ethylenediamine dihydrochloride to form a highly colored azo dye that is measured colourimetrically. A correction may be made for any NO2 - present in the sample by analyzing without the reduction step. The applicable range of this method is 0.01 to 1.0 mg NO3 - N/L. The results are reported as milligrams oxidized N per liter (the sum of NO3 - -N plus NO2 - N) unless the concentration of NO2 - N is separately determined and subtracted. Note: Suspended matter in the column will restrict sample flow. High concentrations of iron, copper, or other metals lower reduction efficiency. EDTA is added to samples to eliminate this interference. Oil and grease will coat the Cd surface. This is removed by pre-extraction with an organic solvent. Residual chlorine can interfere by oxidizing the Cd column, reducing its efficiency. If the samples contain residual chlorine, residual chlorine can be removed by adding sodium thiosulphate (Na 2 S 2 O 3 ) solution. Turbid sample color that absorbs at about 540 nm also interferes and corrective action needs to be done for the turbid samples. 2.3 Nitrate analysis using ultraviolet spectrophotometric screening method : This method is recommended only for screening samples that have low organic matter contents such as uncontaminated natural waters and potable water supplies. Nitrate levels in such water samples can be rapidly measured by UV absorption at 220 nm (The NO 3 calibration curve follows Beer s law up to 11 mg N/L). However, dissolved organic matter also may absorb at 220 nm. Therefore to correct the interference of dissolved organic matter, a second absorption measurement at 275 nm is used. Dissolved organic matter absorb at 275 nm but nitrate does not absorb at 275 nm. Therefore correction can be made. Sample filtration is needed to remove 2

3 possible interference from suspended particles. Note: Turbid sample should be filtered through 0.45-µm-pore-diameter membrane filter. The samples are acidified with 1N HCl to prevent interference from hydroxide or carbonate concentrations up to 1000 mg CaCO 3 /L. Dissolved organic matter, surfactants, NO 2, Cr 6+. various inorganic ions not normally found in natural water, such as chlorite and chlorate, may interfere the UV absorption. Chloride has no effect on the determination Equipment and glassware (for ultraviolet spectrophotometric screening method) Spectrophotometer (UV-visible) 2 matched Quarts Cuvettes for nitrate measurements(note: for absorption measurements at UV range, Quarts cuvettes should be used) Wash bottles with distilled water, clean soft tissues Pipets, conical flasks, volumetric flasks Chemicals/Reagents (for ultraviolet spectrophotometric screening method) (Note to the technical officers: Reagents should be prepared only the required amounts for the practical based on the following proportions to minimize wastage of chemicals: Consult the lecturer in charge). High purity distilled water to prepare all solutions and dilutions. Stock standard Nitrate solution: Dry potassium nitrate (KNO3) in an oven at 105 C for 24 h. In a 500 ml volumetric flask, dissolve g in 400 ml of distilled water, preserve with 1 ml CHCl3/L and bring the volume to 500 ml with distilled water (1.00 ml = 100 µgno3 -N). This solution is stable for at least 6 months. Working Nitrate solution: In a 1000 ml volumetric flask, dilute 100 ml stock standard nitrate solution to 1000 ml with water; 1.00 ml = 10.0 µg NO 3 -N. Preserve with 2 ml CHCl 3 /L. This solution is stable for 6 months. Hydrochloric acid solution, HCl, 1M Procedure (for ultraviolet spectrophotometric screening method) 1. To 50 ml clear sample, (filter if necessary), add 1 ml HCl solution and mix thoroughly. (Note: Turbid sample should be filtered through 0.45-µm-pore-diameter membrane filter). 2. Prepare NO 3 calibration standards by diluting to 50 ml different volumes of nitrate (10 mg/l) solution (select the ranges of standards which cover the concentration ranges of the unknowns, see the table on page 4 for examples ) 3

4 Volume of nitrate standard (ml) Deionized water (ml) NO 3 -N mg /L Concentration 3. To 50 ml of standard, add 1 ml HCl solution and mix thoroughly. 4. Read absorbance at 220 nm against redistilled water set at zero absorbance to obtain NO 3 reading. 5. Read again the absorbance at 275 nm against redistilled water set at zero absorbance to determine interference due to dissolved organic matter. 6. Calculation For samples and standards, subtract two times the absorbance reading at 275 nm from the reading at 220 nm to obtain absorbance due to NO 3. Construct a standard curve by plotting absorbance due to NO 3 against NO3 -N concentration of the standards. Using corrected sample absorbances, obtain sample concentrations directly from standard curve. Note: If correction value is more than 10% of the reading at 220 nm, this method is not recommended. 3. Phosphate analysis: Several standard analytical methods are available for phosphorous determinations. In the phosphorus analyses, first step is to convert the phosphorus form of interest to dissolved orthophosphate through a digestion method and secondly dissolved orthophosphate is determined by a colourimetric method (e.g. Ascobic acid method). 3.1 Sample storage for phosphate analysis: If dissolved phosphorus forms are to be differentiated, filter sample immediately after collection. Preserve by freezing at or below 10 4

5 C. Do not add either acid or CHCl 3 as a preservative when phosphorus forms are to be determined. If total phosphorus alone is to be determined, add H 2 SO 4 or HCl to ph<2 and cool to 4 C, or freeze without any additions. Do not store samples containing low concentrations of phosphorus in plastic bottles unless kept in a frozen state because phosphates may be adsorbed onto the walls of plastic bottles. Rinse all glass containers with hot dilute HCl, then rinse several times in reagent water. Never use commercial detergents containing phosphate for cleaning glassware used in phosphate analysis. 3.2 Phosphate analysis using persulphate digestion and ascorbic acid method. Because phosphorus may occur in combination with organic matter, persulphate oxidation technique can be used as a digestion method to oxidize organic matter effectively to release phosphorus as orthophosphate to determine total phosphorus. Phosphates that respond to colourimetric tests without preliminary hydrolysis or oxidative digestion of the sample are termed reactive phosphorus. Reactive phosphorus is largely a measure of orthophosphate. But, a small fraction of any condensed phosphate present in the sample is hydrolyzed unavoidably in the procedure. Reactive phosphorus occurs in both dissolved and suspended forms. Filtration through a 0.45µm pore diameter membrane filter separates dissolved from suspended forms of phosphorus. Prefiltration through a glass fiber filter may be used to increase the filtration rate. Total phosphorous levels in the water samples can be determined using persulphate digestion and ascorbic acid colorimetric method. In this colorimetric method, ammonium molybdate and potassium antimonyl tartrate react in acid medium with orthophosphate to form a heteropoly acid-phosphomolybdic acid that is reduced to intensely colored molybdenum blue by ascorbic acid. Colour absorption at the blue range can be detected by the visible range (880 nm) of the spectrophotometer Equipment and glassware ( persulphate digestion and ascorbic acid method). Hot plate: ( cm heating surface ). Autoclave: An autoclave or pressure cooker capable of developing 98 to 137 k Pa may be used in place of a hot plate. Acid-washed glassware. The glassware used, including sample bottles, should be reserved for the determination of phosphate; should not be used for any other purpose and should be left full of sulphuric acid (4.5 mol L -1 ) until required for use. If necessary, glassware may be cleaned with chromic acid, equal mixtures of nitric and hydrochloric acids, or pure sulphuric acid. Detergents containing phosphate compounds must not be used. Spectrophotometer (UV-visible) 2 matched glass cuvettes for phosphate measurements Wash bottles with distilled water, clean soft tissues Pipets, conical flasks, volumetric flasks 5

6 3.2.2 Chemicals/Reagents (for persulphate digestion and ascorbic acid method) (Note to the technical officers: reagents should be prepared only the required amounts for the practical based on the following proportions to minimize wastage of chemicals: Consult the lecturer in charge). Phenolphthalein indicator solution. Dissolve 0.5 g of phenolphthalein in 50 ml of 95% ethyl alcohol, and add 50 ml of distilled water. Add a dilute (e.g or 0.05 mol L -1 ) carbon dioxide-free solution of sodium hydroxide, a drop at a time, until the indicator turns faintly pink. Potassium peroxydisulphate solution(potassium persulphate). Dissolve 5 g K2S2O8 in 100 ml distilled water. Prepare a fresh solution daily. Sodium hydroxide, 5M. Sulphuric acid, 2.5 M. Add carefully, with mixing, 70 ml conc. sulphuric acid (d=1.84) to 500 ml of distilled water. Potassium antimonyl tartrate solution. In a 500 ml volumetric flask, dissolve g K(SbO)C 4 H 4 O 6. 1 /2 H 2 O in 400 ml of distilled water and make up to 500 ml. Store in a glass-stoppered bottle. Ammonium molybdate tetrahydrate (40 gl -1 ):Dissolve 20 g (NH 4 ) 6 Mo 7 O 24 4H 2 O in 500 ml of distilled water. Store in a glass-stoppered bottle. Ascorbic acid 0.1M: Dissolve 1.76 g ascorbic acid in 100 ml distilled water. Refrigerate. The solution is stable for about 1 week at 4 C. Phosphate stock solution. Dissolve g anhydrous potassium dihydrogen phosphate, KH2PO4, in 1 L of distilled water. (1.0 ml stock solution is equivalent to 50 µg PO4 3- P). (should be freshly prepared when required) Phosphate standard working solution. Dilute 20 ml of stock solution to 1000 ml with distilled water and mix well. (1.0 ml working solution is equivalent to 1.0 μg P). (should be freshly prepared when required) Combined reagent(this should be prepared fresh each day): For 200 ml of the combined reagent, mix together the reagents in the following proportions, mix after addition of each reagent: 100 ml 2.5 M H 2 SO 4, 10 ml potassium antimonyl tartrate solution, 30 ml ammonium molybdate solution, and 60 ml ascorbic acid solution. Let the ascorbic acid solution reach the room temperature before mixing. Mix in the order given. If turbidity forms in the combined reagent, shake and let stand for a few minutes until turbidity disappears before proceeding. The reagent is stable for 4 h. (For each standard and each sample, 8 ml of combined solution is required). 6

7 Procedure(for persulphate digestion and ascorbic acid method) 1. Take 100 ml of thoroughly mixed sample (Note: Dilution of the sample to 100 ml with distilled water may be necessary for the samples with high phosphate levels). 2. Add 1 drop (0.05 ml) of phenolphthalein indicator solution. If a red colour develops, add sulphuric acid solution drop by drop to just discharge the colour. 3. Add 2 ml sulphuric acid solution and 15 ml potassium peroxydisulphate solution. 4. Boil gently for at least 90 minutes, adding distilled water to keep the volume between 25 and 50 ml. Alternatively, heat for 30 minutes in an autoclave orpressure-cooker at kPa cm Cool, add 1 drop (0.05 ml) phenolphthalein indicator solution, and neutralize to a faint pink colour with sodium hydroxide solution. 6. Restore the volume to 100 ml with distilled water and set aside. 7. Prepare a series of standards in 50-mL volumetric flasks as follows (Note: Higher concentrations of the standards may be needed only if the tested samples contain the phosphate levels which exceed the standard concentrations range). Volume of working phosphate standard (ml) Concentration when diluted to 40 ml (µg L -1 of phosphorous) 0 (blank) Place 40 ml of sample in a stoppered 50 ml volumetric flask. 9. Add 8 ml of the combined reagent to the standards and samples, make up to 50 ml with distilled water and mix. Allow to stand for 10 minutes. 7

8 10. Measure the absorbance of the blank at 880 nm and each of the standards and the samples. (Note: The absorbance should be measured within 30 minutes). 11. Prepare a calibration curve from a series of six standards within the phosphate ranges. Use a distilled water blank with the combined reagent to make photometric readings for the calibration curve. Plot absorbance vs. phosphate concentration to give a straight line passing through the origin. 11. From the calibration graph, read the number of μg L -1 of phosphorus in the samples. (Note: Natural color of water generally does not interfere at the high wavelength used. For highly colored or turbid waters, prepare a blank by adding all reagents except ascorbic acid and potassium antimonyl tartrate to the sample. Subtract blank absorbance from absorbance of each sample). LAB EXERCISE You are provided with 4 samples of well water and 4 water samples collected from an urban water body. 1. Using the standard procedures given, analyze the nitrate and total phosphate levels of the well water and surface water samples provided. 2. Comment on the water quality status of the samples analyzed. 3. Prepare a lab report following the instruction given in the practical number 3. ** Due date for submission of the lab report : 12 th June 2015 References used for the preparation of the practical: American Public Health Association, APHA (1999). Standard Methods for the Examination of Water and Wastewater. 20 th ed. American Public Health Association, Washington DC, USA UNEP/WHO (1996).Water Quality Monitoring-A practical guide to the design and implementation of freshwater quality studies and monitoring programmes, Chapman and Hall, UK Prof A Pathiratne/Dept of ZEM/UOK,