1、Designation: D7781 14Standard Test Method forNitrite-Nitrate in Water by Nitrate Reductase1This standard is issued under the fixed designation D7781; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A numb
2、er in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method is applicable to the determination ofnitrate plus nitrite (as nitrogen) in drinking water, surface,saline, wastewater, and
3、 ground waters. The applicable range ofthis test method is from 0.05 to 5 mg/L of nitrogen. The rangemay be extended upward by dilution of an appropriate aliquot.The 40 CFR Part 136 Method Detection Limit (MDL) is 0.02mg /L.1.2 It is the users responsibility to ensure the validity of thistest method
4、 for waters of untested matrices. The quality controlcriteria in Section 17 for method blanks, laboratory controlsamples, matrix spikes and matrix duplicates must be met.1.3 The values stated in SI units are regarded as standard.No other units of measurement are included in this standard.1.4 This st
5、andard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1
6、 ASTM Standards:2D992D1129 Terminology Relating to WaterD1141 Practice for the Preparation of Substitute OceanWaterD1193 Specification for Reagent WaterD1254 Method of Test for Nitrite Ion in Water (Withdrawn1980)3D3867 Test Methods for Nitrite-Nitrate in WaterD5810 Guide for Spiking into Aqueous Sa
7、mplesD6146 Guide for Monitoring Aqueous Nutrients in Water-sheds3. Terminology3.1 DefinitionsFor definitions of terms used in these testmethods, refer to Terminology D1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 bispecific NaR, nnitrate reductase that can use eitherNADH or NADPH as
8、its electron donor (cofactor).3.2.2 discrete analyzer, na programmable, computer-controlled instrument that automates wet-chemical analysis byusing one or more robotic arms interfaced to high-precisionvolumetric dispensers to aspirate and dispense samples,standards, diluents, and reagents.3.2.3 Grei
9、ss reaction, nchemical formation of an azo dyeby diazotization of nitrite ion with sulfanilamide and subse-quent coupling with N-(1-naphthyl)ethylenediamine hydro-chloride.3.2.4 NADH, nnicotinamide adenine dinucleotide, re-duced form is a coenzyme found in all living cells.3.2.5 NADPH, nnicotinamide
10、 adenine dinucleotidephosphate, reduced form is a coenzyme found in all livingcells; NADP+ is the oxidizing form and NADPH is thereducing form.3.2.6 nitrate reductase (NaR), nNADH:NaR (EC1.7.1.1and CAS 9013-03-0) or bispecific NaR (EC 1.7.1.2 and CAS9029-27-0) with 1 unit of enzyme activity defined
11、as 1micromol nitrite produced per minute at 30C, at pH 7 withNADH (refer to 3.2.4 and 10.2) as an electron donor.4. Summary of Test Method4.1 Nitrite-Nitrate NitrogenThe sample is mixed with abuffered solution containing NAD(P)H: nitrate reductase (EC1.7.1-3) and NADH or NADPH to reduce nitrate ion
12、to nitriteion. The combined nitrite-nitrate (expressed as mg/LNO3+NO2-N) is determined by diazotizing the total nitrite ionwith sulfanilamide and coupling with N-(1-naphthyl) ethylene-diamine dihydrochloride to form a highly colored azo dye thatis measured spectrophotometrically at about 540 nm.4.2
13、Nitrite NitrogenThe nitrite ion (expressed as mg/LNO2-N) originally present in the sample can be determinedseparately by carrying out the procedure and omitting thereduction step.1This test method is under the jurisdiction of ASTM Committee D19 on Waterand is the direct responsibility of Subcommitte
14、e D19.05 on Inorganic Constituentsin Water.Current edition approved April 1, 2014. Published May 2014. DOI: 10.1520/D7781-14.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information,
15、refer to the standards Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States14.3 Nitrate NitrogenThe nitrate ion as
16、 nitrogen can becalculated as the difference between the combined nitrate plusnitrite (NO3+NO2-N) and the nitrite (NO2-N):mgLNO32 N 5 mgLNO31 NO22 N! 2 mgLNO22 N!(1)5. Significance and Use5.1 This test method replaces Test Methods D1254 (Nitrite)and D992 (Nitrate). The nitrite test method (Test Meth
17、odD1254) used a reagent that is considered to be a potentialcarcinogen. The nitrate test method (Test Method D992) hasbeen shown to have relatively large errors when used inwastewaters and also has greater manipulative difficulties thanthe test method described herein.5.2 This test method can be use
18、d in place of Test MethodsD3867 (Nitrite-Nitrate). Test Methods D3867 uses cadmiumfor the reduction of nitrate to nitrite. Cadmium is considered atoxic metal. Also, the heterogeneous cadmium reductant cre-ates greater difficulty than the reduction described in this testmethod.6. Interferences6.1 Tur
19、bid samples should be filtered prior to analysis toeliminate particulate interference.6.2 Sample color that absorbs at wavelengths between 520and 540 nm interferes with the absorbance measurements.When color is suspect, analyze a sample blank, omitting theN-(1-naphthyl)ethylenediamine dihydrochlorid
20、e from the colorreagent.NOTE 1The instrumentation described in this standard may automati-cally correct for some turbidity and sample color. See the instrumentmanual for further information.6.3 Certain ions may cause interferences. See Table 1.7. Purity of Reagents7.1 Reagent grade chemicals shall b
21、e used in all tests.Unless otherwise indicated, it is intended that all reagents shallconform to the specifications of the Committee on AnalyticalReagents of the American Chemical Society, when suchspecifications are available.4Other grades may be used, pro-vided it is first ascertained that the rea
22、gent is of sufficient highpurity to permit its use without lessening the accuracy of thedetermination.7.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water conformingto Specification D1193, Type I or Type II. Other reagent watertypes may be used,
23、 provided it is first ascertained that the wateris of sufficiently high purity to permit its use without adverselyaffecting the bias and precision of these test methods.8. Sampling and Sample Preservation8.1 Collect the sample in accordance with Guide D6146.8.2 When nitrite ion is to be determined s
24、eparately, analyzewithin 48 hours after sampling. Even when sterile bottles areused, bacteria naturally present in the water may cause con-version of all or part of nitrite ion to other forms such as nitrateor ammonia. Ammonia and natural amines, which are fre-quently present in natural waters, may
25、react with nitrites toform nitrogen. If samples are to be stored for 48 h or less,preserve the sample by refrigeration at 26C. If the samplemust be stored for more than 48 h, preserve it by the additionsulfuric acid to pH 2 in addition to refrigeration at 26C.NOTE 2Use sulfuric acid for preservation
26、 of nitrite-nitrate nitrogenonly. Samples for nitrite must be analyzed within 48 hours.NOTE 3Sulfuric acid does not necessarily inhibit oxidation andmercury compounds should be avoided to prevent environmental pollu-tion.NOTE 4Residual chlorine does not interfere, however, attempts toremove residual
27、 chlorine (such as addition of ascorbic acid) interfere byinhibiting reduction of nitrate to nitrite. Do attempt to remove residualchlorine.9. Apparatus9.1 Automated discrete analysis system (see 3.2.2).10. Reagents10.1 Phosphate Buffer SolutionDissolve 3.75 g of potas-sium dihydrogen phosphate (KH2
28、PO4), 0.01 g of disodiumethylenediaminetetraacetate dehydrate4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC, www.chemistry.org. For suggestions on thetesting of reagents not listed by the American Chemical Society, see the UnitedStates Pharmaco
29、peia and National Formulary, U.S. Pharmacopeial Convention,Inc. (USPC), Rockville, MD, http:/www.usp.org.TABLE 1 Determination of Nitrate in the Presence of PotentialInterferencesSpeciesConcentrationAdded(mg/L)UnspikedSampleResult(mg/L)SpikedSampleResults(mg/L)SpikeAdded(mg/L)%RecoveryCl-500 0.02 0.
30、23 0.200 1050.17 2.54 2.50 95F-500 0.01 0.22 0.200 105Br-500 Miscibility 2 mg dissolved organic carbon /L) have little affect on the NaR activityin the temperature range of 515C, but become increasingly inhibitory inthe temperature range of 2030C. Humic substances at the operationtemperatures specif
31、ied in this standard do not inhibit other forms of NaR.5If humic acids are expected to be present the user must verify reductionefficiency of the NaR is use by analysis of Quality Control checks thatapproximate the sample matrix.10.7 Nitrate Solution, Stock (1000 mg/L NO3-N)Dry po-tassium nitrate (K
32、NO3) in an oven at 105C for 24 h. Dissolve7.218 g in water in about 500 mL reagent water contained in a1000 mL volumetric flask, dilute to the mark and mix. Thissolution is stable for up to 2 months with refrigeration.Alternatively, certified nitrate stock solutions are commerciallyavailable through
33、 chemical supply vendors and may be used.10.8 Nitrate Solution, Standard (10 mg/L NO3-N)Dilute10 mL of stock nitrate solution (10.7) to 1 L with water andstore in a dark bottle. Prepare fresh as needed.10.9 Nitrite Solution, Stock (1000 mg/L NO2-N)Placeabout 7 g of potassium nitrite (KNO2) in a tare
34、d 125-mLbeakerand dry for about 24 h to a constant weight in a desiccatorcontaining a suitable desiccant. Adjust the weight of the drypotassium nitrite to 6.072 g. Add 50 mL of water to the beaker,stir until dissolved, and transfer quantitatively to a 1000-mLvolumetric flask. Dilute to the mark with
35、 water store in asterilized bottle under refrigeration. Prepare fresh as needed.Alternatively, certified nitrite stock solutions are commerciallyavailable through chemical supply vendors and may be used.NOTE 8Potassium nitrite is easily oxidized; use only dry, free flowingwhite, or yellowish white c
36、rystalline powder of this reagent.10.10 Nitrite Solution, Standard (10 mg/L NO2-N)Dilute10 mL of stock nitrite solution (10.9) to 1 L with water. Thissolution is unstable; prepare fresh as needed.11. Hazards11.1 All reagents and standards should be prepared involumes consistent with laboratory use t
37、o minimize the gen-eration of waste.12. Calibration12.1 Using the standard nitrate solution (10.8) preparecalibration standards by using the automated calibration func-tion of the discrete analyzer (3.2.2). Table 2 specifies suggestedcalibration levels.12.2 Prepare at least one calibration standard
38、from thestandard nitrite solution (10.10) at the same concentration asone of the nitrate standards to verify the efficiency of thereduction. Verify that reduction efficiency is between 90 and115 % with each batch of enzyme. See Table 3.NOTE 9When the sample to be analyzed is saline water, use substi
39、tuteocean water (SOW) to prepare the standards (Practice D1141 or a5NaR available from the Nitrate Elimination Company Inc. (NECi),, has been found suitable.TABLE 2 Example Concentrations of Calibration StandardsNO3-N or NO2-N, mg/LmL of 10 mg/L Standard Solution/100 mLfinal volume0.05 0.50.1 1.00.5
40、 5.01.0 102.0 203.0 305.0 50TABLE 3 Reduction EfficiencyNO3-N or NO2-N, mg/LmL of 10 mg/L Standard Solution/100 mLfinal volumeMean (%) 103Standard Deviation 4.14Lower Limit (%) 91Upper Limit (%) 115D7781 143commercially available synthetic seawater). Run a reagent water blank inaddition to a SOW bla
41、nk because the reagents used to prepare SOWfrequently contain nitrite or nitrate, or both. Adjust this curve for thecontaminant level in SOW.NOTE 10Most discrete analyzers generate calibration standards andcalibration curves automatically. Follow the manufacturers instructionsfor calibrating with in
42、dividual calibration standards if an automaticcalibration function is not available.12.3 Develop the color and determine the absorbance ofeach standard as directed in the procedure (13.4.6).12.4 Prepare a standard curve by plotting the absorbance (oroptical density) of each processed calibration sta
43、ndard againstits known concentrations. See Fig. 1 for an example of acalibration curve.12.5 Verify the calibration each day, or before each use witha calibration verification solution (see 17.2.2).13. Conditioning13.1 Removal of InterferencesRemove interferences (Sec-tion 6) by the following procedu
44、res:13.2 For turbidity removal, when suspended solids arepresent, filter the sample through a glass-fiber filter or a0.45-m filter. Centrifugation can be used as an option.13.3 For correction for color interferences, if there is apossibility that the color of the sample might absorb in thephotometri
45、c range from 530 6 10 nm, determine the back-ground absorbance.NOTE 11Many discrete analyzers automatically compensate forbackground absorbance and turbidity on each sample. Follow the manu-facturers instructions.13.4 Prepare a method in the discrete analyzer softwareaccording to the following, or s
46、imilar, conditions as recom-mended by the manufacturer:13.4.1 Dispense 170 microliters of Nitrate Reductase NaR(10.6) plus 10 microliters of sample. Mix.13.4.2 Add 15 microliters of NADH (10.3). Mix and mea-sure the background absorbance (or optical density).13.4.3 Incubate 600 seconds at 37C.13.4.4
47、 Add 25 microliters of SAN reagent (10.4). Mix andincubate 120 seconds at 37C.13.4.5 Add 25 microliters of NED reagent (10.5). Mix andincubate 120 seconds at 37C.13.4.6 Measure absorbance (or optical density) at 540 nm.NOTE 12When determining nitrite alone, replace NaR reagent (10.6)with Phosphate B
48、uffer (10.1).NOTE 13Samples may be reduced manually keeping sample toreagent ratios similar to steps 13.4.1 13.4.5.Analyze the reduced sampleby Test Methods D3867 without the cadmium column inline.14. Calculation14.1 Determine the concentration of nitrate or nitrite nitro-gen in the samples in milli
49、grams per liter using the computerbased data handler provided with the automated discreteanalyzer software.NOTE 14The discrete analyzer will automatically calculate the netabsorbance by subtracting the background absorbance from the measuredabsorbance of the color developed sample. Use the net absorbance todetermine the concentration of nitrogen in the sample.FIG. 1 Example Calibration CurveD7781 14414.2 Where separate values are required for nitrite-nitrogenand nitrate-nitrogen, calculate the nitrate-nitrogen by su
