# ncasi

NATIONAL COUNCIL FOR AIR AND STREAM IMPROVEMENT



## NCASI Method TNTP-W10900 as an Alternative Testing Procedure
to EPA Method 351.2 and
EPA Method 365.4

June 2011

### CONTENTS

1.0 JUSTIFICATION FOR ALTER NATIVE TEST PROCEDURE ....................................................... 1

2.0 METHOD COMPARISON INFORMATION .................................................................................... 1

2.1 Overview of Proposed Changes .............................................................................................. .. 2

Method Comparison Tables ...................................................................................................... 4

3.0 VALIDATION STUDY REPORT: NCASI METHOD TNTP-W10900 ......................................... 14

3.1 Background ................................................................................................................ ............. 14

3.2 Study Design and Objective ................................................................................................ .... 14

3.3 Study Impl ementation ...................................................................................................... ....... 14

3.4 Data Reporting and Validation ............................................................................................. ... 16

3.5 Results, Data Analys is, and Discussions ................................................................................. 1 7

3.6 Conclusions ............................................................................................................... .............. 20

4.0 NCASI MET HOD TNTP-W10900 .................................................................................................. . 22

1 Scope and Application ....................................................................................................... ...... 23

2 Summary of Method ........................................................................................................... ..... 23

3 Definitions ................................................................................................................. .............. 23

4 Interferences ............................................................................................................... ............. 24

5 Safety ...................................................................................................................... ................. 25

6 Equipment and Supplies ...................................................................................................... .... 25

7 Reagents and Standards ...................................................................................................... ..... 26

8 Sample Collection, Preser vation, and Storage ........................................................................ 27

9 Quality Control ............................................................................................................. ........... 28

10 Calibration and Standardization ............................................................................................ .. 31

11 Procedure .................................................................................................................. ............... 31

12 Data Analysis and Calculations ............................................................................................. .. 32

13 Method Performance ......................................................................................................... ...... 33

14 Pollution Prevention ....................................................................................................... ......... 33

15 Waste Management ........................................................................................................... ...... 33

16 References ................................................................................................................. .............. 33

17 Tables, Diagrams, Flowchar ts, and Validation Data ............................................................... 34

REFERENCES .................................................................................................................... ....................... 36

### TABLES

Table 2.1. Method Comparison for Total Nitr ogen by EPA Method 351.2 vs. NCASI Method
TNTP-W10900 ................................................................................................................... ....... 4

Table 2.2. Method Comparison for Total Phosph orus by EPA Method 365.4 vs. NCASI Method
TNTP-W10900 ................................................................................................................... ..... 10

Table 3.1. Analytes Determined by NCASI Method TNTP-10900 ......................................................... 14

Table 3.2. Sample Set for Alternative T esting Procedure Study Plan, NCASI Method
TNTP-W10900 ................................................................................................................... ..... 15

Table 3.3. Overview of Study Plan Sample a nd Analysis Scheme, Tier 2 Single Matrix ........................ 16

Table 3.4. Determinative Methods and Equipm ent used in ATP Study Plan Execution ......................... 16

Table 3.5. Data Requirements .................................................................................................. ................ 17

Table 3.6. Calibration Curve Data Summary ..................................................................................... ...... 18

Table 3.7. Method Blank and Continuing Calibration Verification Summary ......................................... 18

Table 3.8. Ongoing Precisi on and Recovery Results ............................................................................. .. 18

Table 3.9. Multi-Laboratory Initial Precision and Recovery .................................................................... 19

Table 3.10. Method Detection Limit Resu lts for the Multi-Laboratory Study ........................................... 19

Table 3.11. Sample Background Level Resu lts from the Multi-Laboratory Study .................................... 20

Table 3.12. Accuracy Expressed as Percent Rec overy of Matrix Spikes and Matrix Spike Duplicates .................................................................................................................... ............ 20



### IN-METHOD TABLES

Table 1. Calibration Standard Solution Makeup Scheme ...................................................................... 34

Table 2. Initial Precision and Recovery ....................................................................................... ......... 35

Table 3. Matrix Spike and Matrix Spike Duplicate Recovery and Precision ........................................ 35

Table 4. Organic Analyte Digestion Ch eck Initial Precision and Recovery ......................................... 36

Table 5. Digested Sample Holding Time Study .................................................................................... 36NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 1



### NCASI METHOD TNTP-W10900 AS AN ALTERNATIVE TESTING PROCEDURE
TO EPA METHOD 351.2 AND EPA METHOD 365.4

1.0 JUSTIFICATION FOR ALTERNATIVE TEST PROCEDURE

The pulp and paper industry has historically tested ni trogen and phosphorus levels in biologically treated wastewater in part using the Kjeldahl digesti on method set forth in EPA Methods 351.2 (USEPA 1993a) and 365.4 (USEPA 1974). NCASI proposes that Kjelda hl digestion be replaced with an alkaline persulfate oxidation digestion that has been demonstrated to exceed quality control acceptance criteria of the reference methods. Moreover, the proposed di gestion reduces labor and hazardous waste costs and enhances safety. These benefits are detailed herein.

1. Lowers detection levels and improves preci sion and accuracy – Nitrogen and phosphorus concentrations in pulp and paper effluent are typical ly lower than in effluents from sewage treatment plants as evinced by the need to add supplemental nutrients to permit normal biomass growth. Also, since the reference methods were initially promul gated over three decades ago the industry has been reducing nutrient loads in waters heds, in particular where total daily maximum loads (TMDLs) are being implemented. Single-laboratory and Tier 2 ev aluations bear out the advantages of the proposed digestion in terms of lowered detection limits, gr eater precision, and diminished bias, which are all desired objectives in characterizing modern effluents, especially where nutrient limits are included in NPDES permits.

2. Reduces hazardous waste and enhances safety – The modi fied digestion is safer to perform because it avoids extreme temperatures and exposure to toxic or hazardous materials. Kjeldahl digestions are carried out in fuming acid at high temperatures (380°C) and are catalyzed by mercury or copper. The proposed digestion transitions from a basic to an aci dic environment at moderate temperatures (120 to 125°C). The Kjeldahl process is more expensive due to overhead and costs associated with retention and disposal of acidified mercury or copper wastes. In contrast, process waste volumes for the modified digestion are 60% lower and can be i mmediately disposed of after neutralization.

3. Decreases labor and overhead costs – Nitrogen load summations are reported as total nitrogen (TN), which requires execution of two distinct sample pr eparations (nitrite/nitrate and total Kjeldahl nitrogen (TKN)) followed by three individual nitrogen determinations (nitrite, ni trate, and ammonia). In contrast, the proposed alternative test proce dure (ATP) uses a single sample preparation followed by a single determination to derive TN. Total phosphorus (TP) is concu rrently developed during sample preparation and is individually analyzed. Furthermore, Kjeldahl digestion times are on the order of two to three hours, whereas the modified pr ocess is carried out in less than an hour. As a result of fewer and shorter processes, the modified method significantly reduces laboratory work load, materials, and related costs while increasing throughput.

Based on these outcomes NCASI is seeking approval of TNTP-W10900 as an alternative test procedure to EPA 351.2 and EPA 365.4. Specifically, NC ASI is seeking EPA approval of TNTP-W10900 for monitoring of pulp, paper, and paperboard biologica lly treated wastewater (PP-BTWW) at the Tier 2 level. The experimentally-based justification of th e proposed method changes can be found in Section 3.

2.0 METHOD COMPARISON INFORMATION

EPA Method 351.2 is approved for si multaneous digestion of both n itrogen and phosphorus using the Kjeldahl method. However, EPA 351.2 provides qual ity metrics and analysis instructions only for nitrogen and thus necessitates that EPA Method 365.4 be used to addr ess phosphorus analysis after the Kjeldahl digestion is completed. The substan tial differences between the modified method, NCASI Method TNTP-W10900, and the reference methods may logically be evaluated using EPA 351.2 as the primary comparison. Section 2.1 elaborates on the f actors contributing to these differences. A point byNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 2

point comparison of TNTP-W10900 to EPA 351.2 and 365.4 can be found in Tables 2.1 and 2.2, respectively, using the standard EPA method form at. Although EPA 365.4 as published contains only eight sections, Table 2.2 incorporates the standard seventeen topics. The two tables focus on the significant differences between TNTP- W10900 and the reference methods.

2.1 Overview of Proposed Changes

The major differences between the EPA reference met hods and the NCASI revision include: (a) smaller sample size; (b) lower method limit for nitrogen; (c) utilization of basic to acidic persulfate digestion; and (d) quantification of TN as nitrate-nitrogen.

The modified digestion uses the oxidizing reagent 2% K 2 S 2 O 8 in 0.12 M NaOH solution to simultaneously digest TN and TP. This transformatio n does not materially alter the analytical scope of the approved method. To wit: the approved refe rence method renders nitrogen forms to ammonianitrogen and the alternative method converts nitrogen forms to nitrate-nitrogen. After digestion the nitrogen analyte is colorimetrically quantified via chemical reactions that form a chromophore. The ammonia-nitrogen chromophore is produced using a salicylate-hypochlorite reaction and the nitratenitrogen chromophore is produced using a diazotization reaction. Both reactions produce highly colored solutions wherein the color intensities are proportiona l to the nitrogen concentration in the digested samples. Both the modified and reference met hods convert phosphorus forms to orthophosphatephosphorus. Orthophosphate- phosphorus is determined colorimetri cally by formation of a molybdenum blue complex. The equivalencies of the nitroge n and phosphorus analytes produced by the modified process were demonstrated in both single-laborat ory and multi-laboratory studies using combined ammonia and orthophosphate standards.

The reference methods decompose a 25 mL sample by means of boiling the sample in concentrated sulfuric acid containing a salt and a mercury or copper catalyst. This is performed in open vessels, resulting in the sample being reduced to a small fracti on of its original volume. Samples are reconstituted with water to their initial quantity before commenc ing analysis. The modified method adds pre-set amounts of sample (7 mL) and digestion solution (3 mL) to closed tubes. No losses occur during digestion and the product can be directly analyzed. Another difference in the modified method is the means to ensure digestion efficiency of organically-b ound nitrogen and phosphorus for different matrices. The rationale, frequency, and instructions for th ese checks are noted in the method (TNTP-W10900, Section 4.0 herein, Part 9.4.8).

Working solutions, calibrants, and quality control samples are made with combined nitrogen and phosphorus standards. These solutions are matrix-matched to the acidified sample pH in order to achieve analytical parity with the samples during digestion a nd quantification. Quality controls are the same as those imposed in the reference methods, with the addition of a nitrate standard. This standard is prepared, digested, and analyzed to assure iden tification of nitrate during analysis.

Both reference methods include comprehensive lists of analytical reagents. These methods also bestow equipment diagrams and setup instructions for colorime tric determination of final products by means of Technicon fluid injected auto-analyzers. Howeve r, since the methods were promulgated several equivalent determinative methods and equipment models that employ corresponding colorimetric chemistries have been approved. In this light , TNTP-W10900 lists the approved methods for nitratenitrogen and orthophosphate-phosphorus determination but intentiona lly excludes reagents, reagent vehicles, proportions, and successions that may be equipment specific.

In TNTP-W10900 the analysis of TN as nitrate-nitrogen occurs via cadmium reduction of nitrate to nitrite followed by diazotization with sulfanilamide a nd coupling with N-(1-napthyl)ethylenediamine dihydrochloride (NED) to form a highly colored azo dy e. This automated colorimetric procedure is the same as performed in EPA Method 353.2 (USEPA 1993b). The corresponding manual method isNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 3

recounted in Standard Method 4500-NO 3 - -E (APHA 2005). Analysis of phosphorus in both EPA 365.4 and TPTN-W10900 is performed by forming an anti mony-phospho-molybdate complex in an acidic medium, which is reduced to an intensely blue colo red complex by ascorbic acid. This same automated colorimetric determination is duplicated in EPA Method 365.1 (USEPA 1993c). The matching manual method is EPA 365.3 (USEPA 1978). Si milar methods published by othe r organizations should provide equivalent performance for both TN and TP as long as they employ the same chemistry and general procedures for the respective determinations. Although TPTN-W10900 recomme nds automated methods
for TN and TP determinations due to the low volum e (10 mL) of digestate, manual analysis is not precluded as long as the quality metrics put forth in TNTP-10W900 are met.

Primary interferences for nitrogen in the modified process are chloride and organic carbon. High levels may compete for the available persulfate required to oxidize nitrogen species to nitrate by forming active chlorine species (e.g., oxychloride) and carbon dioxide, respectively. However, a surplus of persulfate that effectively compensates for excess carbon is u sed in the digestion solution. Chloride at >10,000 mg/L can interfere with colorimetric an alyses of both nitrate and orthophosphate, but this situation is highly unlikely to occur in final effluent s. Other notable interferences for phosphorus in the modified process include calcium, iron, barium, lead, and silver ions, which may form insoluble phosphates (negative interference). Although improba ble in PP-BTWW effluent, arsenate concentrations at >1 mg As/L produce a color similar to phosphate ( positive interference). High nitrate concentration is
the sole interference listed in the reference methods.

Differences in safety, pollution prevention, and wast e management between the reference and modified methods are broader than the comparative statements in Tables 2.1 and 2.2 imply. Overall, the modified digestion contains fewer encounters with hazardous conditions associated with extended process times, extremely high temperatures, and generation of meta l-bearing wastes found in the reference methods. Sample size reduction alone represents a nominal 60% decrease in waste stream volume. The modified digestion produces waste that is easily neutralizedand can immediately be disposed of using nonspecialized means. The higher volumes of acidic wa stes from the reference methods contain mercury or copper. Copper reclamation may be required and mercury waste must be stored and disposed of according to hazardous material protocols. Essential process time is cut from 2.5 hours to 30 minutes. This is accomplished by using a closed, pressurized digestion system that effectively lowers the maximum process temperature from 380°C to 121°C. Energy and labor costs are proportionately reduced as well. The proposed digestion uses readily availabl e and inexpensive items (culture tubes, pressure cooker or autoclave), as opposed to block digesters and peripheral equipment costing several thousand
dollars.

Sample handling, storage, and holding times are th e same for both reference and modified methods (Federal Register 2007). The acid preservation threshol d is designated “pH < 2” in the reference methods but constrained to “1.6 < pH < 2” in the modifiedmethod. The primary reason for this is that overacidification may result in low nitrogen recoveries. Preservation can be controlled by pre-dispensing acid into collection vessels when field sample refrigeration ( ≤ 6 o C) is not available. As written, neither reference method states holding times for digested samples; however, the modified method provides a 28 day window for analysis to occur. Single-laboratory TN holding time studies using PP-BTWW proved that a 28 day hold produced precision and recovery results well within acceptance ranges (TNTP-W10900, Section 17.0). Phosphorus digestates were not tested, but because the method uses combination N and P spikes, a 28 day limit was imposed until further studies can be performed.

In summary, NCASI Method TNTP-W10900 revises the dig estion preparation of samples using a widelyused alkaline persulfate digestion. The procedure is concise, rapid, and effective for PP-BTWW media and has important advantages over Kjeldahl-based diges tion. The proposed method is suitable for treated and untreated pulp and paper mill wastewaters and receiving waters.Table 2.1.Method Comparison for Total Nitrogen byEPA Method 351.2 vs. NCASI Method TNTP-W10900EPA Method 351.2aNCASI Method TNTP-W10900b1.0 Scope andApplicationCovers TKN determination; applicable range0.1-20 mg N/L, may be extended with sample dilutionSimilar aspects; covers TN determination; applicablerange 0.05-6.0 mg N/L, may be extended with sampledilution2.0 Summary of Method Digested samples may also be used for phosphorusdeterminationStated objective is simultaneous digestion of samples forTN and TP determinationSample size 25 mL Sample size is 7 mLSample is heated in presence of sulfuric acid for 2.5 hr;residue is cooled, diluted to 25 mL, and analyzed forammonia-nitrogen by colorimetrySample is heated at 121oC and 15 psi in the presence ofsodium hydroxide and potassium persulfate for30 min; product is analyzed for nitrate-nitrogen bycolorimetry3.0 Definitions Common terms are defined Similarterms, although more specific to applicationDefines general quality control samples (QCS) Discretely defines all QC solutions, e.g. continuingcalibration verification (CCV)Defines MSDS, calibration blank (CB), linear calibrationrange (LCR)Does not define ubiquitous termsDefines instrument performance check (IPC) solutions Does not define equipment-specific terms4.0 Interferences High nitrate concentrations may result in low TKNvaluesChloride >10,000 mg/L may deplete persulfate or resultin active chlorine species thatinterfere in colorimetricreactions used to determine nitrate in digestsHigh organic carbon concentrations may depletepersulfate required to oxidize nitrogenOver-acidification during preservation can result in lowrecovery of TN5.0 Safety Standard disclaimers with specific warnings for threepotentially highly toxic and hazardous chemicals:mercury, concentrated sulfuric acid, and sodiumnitroprusside (used in ammonia analysis)Standard disclaimer content is similar; attention is calledto one reagent, potassium persulfate, that is a strongoxidizerTable 2.1.Method Comparison for Total Nitrogen byEPA Method 351.2 vs. NCASI Method TNTP-W10900EPA Method 351.2aNCASI Method TNTP-W10900b6.0 Equipment andSuppliesEquipment to perform block digestion followed byanalysis using generic flow injection analysis (FIA)system including peripheral devices such as sampler,manifold, detector, and data recorderBlock digester is replaced by pressure cooker orautoclave; analyst is instructed to refer to approvednitrate-nitrite determinative methods andcorresponding auto-analyzer manufacturer’sinstructions7.0 Reagents andStandardsReagent water conforming to ASTM Type II orequivalentRequires ASTM Type II or ultrapure reagent water suchas that produced by commercial purification systemsA 10% sulfuric acid solution is prepared to acidifycalibrants and QC elements prior to digestionDigestion solution is made by dissolving K2SO4in 20%sulfuric acid, this is combined with mercuric sulfatesolution made from mercuric oxide (HgO) in 20%sulfuric acid; copper sulfate is offered as a substitutefor HgODigestion solution is made by dissolving K2S2O8in0.12 M NaOHAnalysis reagents are defined for colorimetricdetermination of ammonia: sulfuric acid (4%),sodium hydroxide (20%), sodium potassium tartrate(20%), buffer (sodium phosphate+sodium hydroxide),sodium salicylate, sodiumnitroprusside, and sodiumhypochlorite solutionsNo analysis reagents or recipes are listed in the method;analyst is instructed torefer to approved nitratedeterminative methodsand corresponding autoanalyzer manufacturer’s instructionsStandard solutions are made from ammonium chloride(NH4Cl)Separate NH4Cl and KH2PO4primary stocks are made;working stocks contain both primary standardsIncludes nitrate-nitrogen 100 mg N/L stock solution ofpotassium nitrate for use as a laboratory fortifiedblank (LFB) for qualitative confirmationIncludes organic nitrogen and phosphorus compoundsused as digestion efficiency checks8.0 Sample Collection,Preservation, andStoragePreservation at pH <2; samples maintained at 4oC andheld for up to 28 daysPreservation at 1.6 < pH < 2; samples maintained at 4oC;assigns 28 day holding time for preserved samples anddigestatesTable 2.1.Method Comparison for Total Nitrogen byEPA Method 351.2 vs. NCASI Method TNTP-W10900EPA Method 351.2aNCASI Method TNTP-W10900b9.0 Quality Control Describes formal QC program, requisite laboratoryperformance measures, and frequencySame requirements, wording is more detailedRationale, frequency, and assessment criteria arespecified for digestion efficiency checks; digestionchecks are assessed against established control limitsInstrument performance verified prior to performinganalysisInstrument performance verified by analyst according tomanufacturer’s guidelinesAnalytical performance must be verified according toQC requirements of determinative methodLCR must be determined initially and verified every6 months or when a change in instrument response isobserved or expectedCalibration occurs with every digestion batchLinearity must be established at ±10% using minimum ofa blank and three standardsLinear coefficient of determination r2at≥0.995 may beused in lieu of ±10% linearityDescribes QCS in broad terms to be analyzed whenbeginning use of method, on a quarterly basis, or asrequired to meet data quality needsSame intent as reference, but terminology identifiesspecific QCS sample sub-types (e.g., IPR)At least one LFB must be analyzed with each samplebatch; recovery must fall within 90-110%More detailed descriptions oftype and frequency ofmethod performance QC samples including ICVs,nitrate-spiked LFB, and digestion efficiency checksDigestion efficiency check solutions run as LFBsThe method detection limit (MDL) is established at 2-3times estimated instrument detection limit in fortifiedreagent waterMDL established using 40 CFR 136 Appendix B inreagent grade water, which allows for fortified level at1-5 times estimated instrument detection limitAt least one laboratory reagent blank must be analyzedwith each batch; value must be less than the MDL orcorrective action ensuesMBs are run: 1 for every 20 samples or 1 for everysample batch, whichever is more frequent; MBanalyte levels must be less than the MDLLaboratory performance assessed using LFB recoveryperformance; data are used to establish ongoingprecision statements and control limitsSame objectives are met using CCV samplesTable 2.1.Method Comparison for Total Nitrogen byEPA Method 351.2 vs. NCASI Method TNTP-W10900EPA Method 351.2aNCASI Method TNTP-W10900b9.0 (continued) Utilizes laboratory fortified sample matrix (LFM) toassess accuracy via analyte recoveryPrecision based on RPD% of sample and sampleduplicate (DUP) and matrix spike/matrix spikeduplicate (MS/MSD) sets taken from minimum of10% of routine samplesUtilizes MS to assess method accuracy in matrix viaanalyte recoveryReference materials should be analyzed when available Independent calibration verification (ICV) samples arerequired10.0 Calibration andStandardizationManifold and flow system follow Figure 1 and Table 2of methodAnalysis follows approved determinative method andequipment manufacturer’s instructionsStandards analyzed in order of decreasing concentration Order of standards is not addressed (parameter is definedby equipment vendor)Calibration standards prepared covering desired range Suggested calibration standard levels prepared accordingto Section 17.0 Table 1 of methodCalibration curve prepared by plotting instrumentresponse against concentration; curve fitting may beachieved using computer or calculator based onregression techniquesSlight difference in wordingCalibration periodically verified during analysisRequirement addressed in Section 9.4.3 (as CCV)11.0 Procedure Add 25.0 mL of sample, standard, or blank to digestertubesAdd 7.00 ±0.05 mL of sample, standard, or blank to15 mL screw-capped culture tubesAdd 5 mL of digestion solution to each tube and vortexAdd 3.00 ±0.05 mL of digestion solution to each tube;cap and mix gentlyAdd boiling chips to tubes and place in block digesterpreheated to 160oC; maintain temperature for 1 hrPlace tubes into pressure cooker or autoclave; beginheating unit; attain final setting of 121oC at 15 psirelative positive pressure; hold for 30 minReset temperature to 380oC, continue to heat for 11⁄2 hr;maintain 380oC for at least 30 min during sessionCool; dilute to 25 mL with reagent water Cool samples; store tightly cappedTable 2.1.Method Comparison for Total Nitrogen byEPA Method 351.2 vs. NCASI Method TNTP-W10900EPA Method 351.2aNCASI Method TNTP-W10900b11.0 (continued) Sections 11.8-11.11 describe reagent manifoldconnections, trouble-shooting, and general operationof a Technicon automated continuous flow systemAnalyze all samples according to approved method fornitrate analysis and specific equipment manufacturer’sinstructions12.0 Data Analysis andCalculationsInstructs analyst to prepare a calibration curve andcompute concentrations by comparison; accounts fordilution factorsContent is similar to reference; additional informationprovided for reporting QC and guidelines for datareviewAdds that curve fitting may be achieved using computeror calculator-based regression techniques13.0 Method Performance Reports multi-laboratory precision and accuracy fromvaried spike levels in reagent water matrixMulti-laboratory Tier 2 study results for precision andaccuracy in reagent water and PP-BTWW matrices,Section 17.0 of method (Section 4)Single-laboratory accuracy was assessed using twosewage samples with concentrations of 4.7 and8.74 mg N/L, recoveries of 99% and 99%,respectivelySingle-laboratory precision and accuracy for nitrogen(from NH4Cl) in PP-BTWW; average recovery98.3%, RPD 11.6%Precision gauged by analyzing three sewage samples atconcentrations of 1.2, 2.6, and 1.7 mg N/L; precision±0.07, ±0.03, and ±0.15 mg N/L, respectively14.0 Pollution Prevention Asserts pollution prevention as foremost goal; advisesminimum quantity of chemicals and prepared reagentsbe purchasedNo special pollution prevention or inventoryrequirements exist for materials used15.0 Waste Management Wording emphasizeslaboratory responsibility to complywith hazardous waste disposal regulationsOmitted; hazardous waste not generatedInstructions to neutralize all solutions resulting fromprocess and to comply with all state, federal, and localordinances16.0 References 6 references dated between 1967 and 1978 15 references; other than reference methods no citationsare dated earlier than 1983Table 2.1.Method Comparison for Total Nitrogen byEPA Method 351.2 vs. NCASI Method TNTP-W10900EPA Method 351.2aNCASI Method TNTP-W10900b17.0 Tables and Figures Table 1. Inter-laboratory Precision and Accuracy;provides means, residuals, and SD for multipleanalyses performed using reagent water matrixTable 1. Calibration Standard Solution Makeup Scheme;tabulates combined working standard, acid, andreagent water for suggested calibration pointsTable 2. Concentration Ranges; pump parameters andbuffer volumes during analysisTable 2. Single and multi- laboratory Initial Precisionand Recovery; results for TN and TP IPR in reagentwaterTable 3. Single and multi- laboratory Matrix Spike andMatrix Spike Duplicate Recovery; results for TN andTP MS/MSD in PP-BTWWTable 4. Single and multi- laboratory Organic AnalyteDigestion Check Initial Precision and Recovery;results for organic analyte IPR in reagent waterTable 5. Single Laboratory Digested Sample HoldingTime Study; results for TN MS/MSD in PP-BTWWFigure 1. Ammonia Manifold; line diagram illustratingflow scheme and basic components of auto-analyzerNo figures includedaDetermination of Total Kjeldahl Nitrogen by Semi-Automated ColorimetryRev. 2.0 August 1993. United States Environmental Protection Agency,Environmental MonitoringSystems Laboratory; http://www.epa.gov/waterscience/methods/method/files/351_2.pdf.bTotal Nitrogen and Total Phosphorus in Pulp and Paper Biologically Treated Effluent by Alkaline Persulfate Digestion, June 2011. National Council forAir and Stream Improvement, Inc.Table 2.2.Method Comparison for Total Phosphorus by EPA Method 365.4 vs. NCASI Method TNTP-W10900EPA Method 365.4aNCASI Method TNTP-W10900b1.0 Scope andApplicationCovers TP determination; applicable range0.01-20 mg P/LSimilar wording; applicable range 0.01-3.0 mg/L, maybe extended with sample dilution2.0 Summary of Method Sample is heated in the presence of sulfuric acid, K2SO4and HgSO4for two and on half hoursSample is heated at 121oC and 15 psi in the presence ofsodium hydroxide and potassium persulfate for30 minThe sample is cooled and diluted to 25 mL then placedon the autoanalyzer for phosphorus determinationSample size is 7 mL; product is analyzed fororthophosphate3.0 Definitions NA; no section titled definitions, no definitions listedelsewhere4.0 Interferences NA; no section titled interferences, no interferenceslisted elsewhereHigh levels of suspended sediment may cause underreporting of phosphorusCalcium, iron, barium, lead, and silver can forminsoluble phosphatesArsenate (AsO43-) forms analogous chromophore toorthophosphate above 1.0mg As/L; arsenite (AsO32-)at levels >2.0 mg As/L may be oxidized to arsenate5.0 Safety NA; no section titled safety, no safety items listedelsewhereCommon disclaimers are given; attention is called to onereagent, potassium persulfate, that is a strong oxidizer6.0 Equipment andSuppliesEquipment is Section 4.0, Apparatus; Technicon blockdigester, Technicon AA11 auto-analyzer withphosphorus manifoldAnalyst is instructed to refer to approved orthophosphatedeterminative methods and corresponding analyzerequipment manufacturer’s instructions7.0 Reagents andStandardsReagents listed in Section 5.0; no standards specifiedSolutions made up in distilled water Requires ASTM Type II or ultrapure reagent water suchas that produced by commercial purification systemsTable 2.2.Method Comparison for Total Phosphorus by EPA Method 365.4 vs. NCASI Method TNTP-W10900EPA Method 365.4aNCASI Method TNTP-W10900b7.0 (continued) Analysis reagentsdefined for colorimetric phosphomolybdenum blue method: sulfuric acid (0.72 N),sodium chloride (0.68 M), ammonium molybdate,antimony potassium tartrate, ascorbic acid solutions;4% sulfuric acid solution used to flush auto-analyzermanifoldNo analysis reagents listed;analyst instructed to followmanufacturer’s instructionsand methods particular toinstrument in forming phospho-molybdenum complex8.0 Sample Collection,Preservation, andStorageSample handling and preservation are listed inSection 3.0; sample should be preserved by theaddition of 2 mL of concentrated sulfuric acid per literof sample then refrigerated at 4oC; written methoddoes not include holding timesPreservation at 1.6 < pH < 2; samples maintained at 4oC;assigns 28 day holding time for preserved samples anddigestates9.0 Quality Control NA; no section titled quality control, no QCrequirements listed elsewhereContains formal and detailed procedures for QC,analytical performance, demonstration of methodproficiency, and QC frequency; laboratories areinstructed to establish QA programs; acceptancecriteria and remedial actions are specifiedLaboratory must demonstrate method proficiency bysuccessful execution of IPR and MS/MSD analysesthat meet or exceed tolerance intervals determinedduring multi-laboratory validation10.0 Calibration andStandardizationNA; no section entitled “Calibration andStandardization”Calibration follows approved method and equipmentmanufacturer’s instructionsSuggested calibration standard levels prepared accordingto Table 1, Section 17.0, of methodCalibration curve is prepared by plotting instrumentresponse against concentration; curve fitting may beachieved using computer or calculator based onregression techniques11.0 Procedure NA; procedures are listed in Section 6.0Add 20.0-25.0 mL of sample, standard, or blank to adigester tubeAdd 7.00 ±0.05 mL of sample, standard, or blank to15 mL screw-capped culture tubesTable 2.2.Method Comparison for Total Phosphorus by EPA Method 365.4 vs. NCASI Method TNTP-W10900EPA Method 365.4aNCASI Method TNTP-W10900b11.0 (continued) Add 5 mL of digestion solution to each tube and vortex Add 3.00 ±0.05 mL of digestion solution to each tube;cap and mix gentlyAdd Teflon boiling chips to tubes and place in blockdigester preheated to 160oC; maintain for 1 hr; resettemperature to 380oC and maintain for 21⁄2 hoursPlace tubes into pressure cooker or autoclave; beginheating unit; attain final setting of 121oC at 15 psirelative positive pressure; hold for 30 minCool; dilute to 25 mL with distilled water; use ammoniafree water if TKN is determinedCool samples; store tightly cappedSections 6.4.1-6.4.5 detailconnection and sequencing ofreagent lines, pump operation and conditioning, andinstrument qualification for a Technicon AA11Analyst is instructed to refer to approved orthophosphatemethods and corresponding instrument or autoanalyzer manufacturer’s instructions12.0 Data Analysis andCalculationsNA; no section entitled “Data Analysis andCalculations”; See Section 10.0 of this table.Instructions are provided for reporting QC metrics andguidelines for data reviewSection 7.0, entitled “Calculations” instructs analyst toprepare standard curve by plotting peak heights ofprocess standards against concentration valuesEquipment specific terms such as “plotting peak height”are excluded13.0 Method Performance NA; there is no section entitled “Method Performance”;Section 8.0 reports single-laboratory precision andaccuracySingle-laboratory accuracy by assessment of two sewagesamples with concentrations of 1.84 and 1.89 mg P/L;recoveries of 95% and 98%, respectivelySingle-laboratory accuracy and precision for phosphorus(from KH2PO4) in PP-BTWW in Section 17.0(Section 4); average recovery 98.8%, RPD 2.5%Single-laboratory precision by analysis of threeconcentration levels (0.23, 1.33, and 2.0 mg P/L);precision ±0.01, ±0.04, and ±0.06 mg P/L,respectively; matrix not givenMulti-laboratory Tier 2 study results for precision andaccuracy in reagent water and BT-PPWW matrixesincluded in Section 17.0 (Section 4)Table 2.2.Method Comparison for Total Phosphorus by EPA Method 365.4 vs. NCASI Method TNTP-W10900EPA Method 365.4aNCASI Method TNTP-W10900b14.0 Pollution Prevention NA; there is no section 14.0 and no section entitled“Pollution Prevention”; no pollution prevention itemsare listed elsewhereNo special pollution prevention or inventoryrequirements exist for the materials used15.0 Waste Management NA; there is no Section 15.0 and no section entitledWaste Management, no waste management topics arelisted elsewhereInstructs users to neutralize all solutions resulting fromprocess and to comply with all state, federal, and localordinances16.0 References NA; there is no Section 16.0; “Bibliography” is foundafter Section 8.0; references are dated 1967 to 1974There are 15 references; no citations are dated earlierthan 198317.0 Tables and Figures NA; there is no Section 17.0; Figure 1 follows the“Bibliography” sectionNo figures are includedFigure 1, Phosphorus Manifold Technicon AA11; linediagram illustrating input and output scheme ofmanifold and basic components of analyzerNo tables included Table 1. Calibration Standard Solution Makeup Scheme;tabulates combined working standard, acid, andreagent water for suggested calibration pointsTable 2. Single and multi- laboratory Initial Precisionand Recovery; results for TN and TP IPR in reagentwaterTable 3. Single and multi- laboratory Matrix Spike andMatrix Spike Duplicate Recovery; results for TN andTP MS/MSD in PP-BTWWTable 4. Single and multi- laboratory Organic AnalyteDigestion Check Initial Precision and Recovery;results for organic analyte IPR in reagent waterTable 5. Single Laboratory Digested Sample HoldingTime Study; results for TN MS/MSD in PP-BTWWaPhosphorus, Total (Colorimetric, Automated, Block Digester AA II)1974. United States Environmental Protection Agency, Environmental MonitoringSystems Laboratory;http://www.epa.gov/waterscience/methods/method/files/365_4.pdf.bTotal Nitrogen and Total Phosphorus in Pulp and Paper Biologically Treated Effluent by Alkaline Persulfate Digestion, June 2011. National Council forAir and Stream Improvement, Inc.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 14

3.0 VALIDATION STUDY REPORT : NCASI METHOD TNTP-W10900

3.1 Background

This section contains a summary of the method, d escriptions of the ATP study design and objective, a summary of data collected during the multi-laboratory study, and resulting conclusions.

TNTP-W10900 was developed to meet the needs of the pulp and paper industry for National Pollutant Discharge Elimination System (NPDES) compliance monitoring of total nitrogen (TN) and total phosphorus (TP). Modifications to the EPA refere nce methods are primarily limited to digestion of samples whereby nitrogen fo rms are converted to nitrate-nitrogen and phosphorus forms are converted to orthophosphate-phosphorus. TN and TP are then determined colorimetr ically from the digestates.
TNTP-W10900 is similar to USGS Method I-4650-3 (USGS 2003) and Standard Method (SM) P-4500J (APHA 2006), which have been approved by EPA for su rface water analysis of TN and TP. The analytes determined in TNTP-W10900 are presented in Table 3.1.

Table 3.1. Analytes Determined by NCASI Method TNTP-10900
Description CAS Number PCS Parameter Code a Nitrogen, total (as N) 7727-37-9 00600 Phosphorus, total (as P) 7723-14-0 00665

a parameter code developed for Permit Compliance Systems (PCS) loads database

Method performances are reported for reagent water a nd the matrix, PP-BTWW final effluent. Results of the multi-laboratory experiments demonstrated that NCASI TNTP-W10900 met or exceeded quality control and performance criteria of the reference me thods in all aspects of performance-based testing.

3.2 Study Design and Objective

The objective of this study was to provide da ta necessary to obtain EPA approval of NCASI Method TNTP-W10900 for use as an ATP to EPA Methods 351.2 and 365.4 for monitoring TN and TP. To reach this objective, a multi-laboratory study w as conducted to collect data to determine if the proposed method could meet the quality control (QC) acceptance and performance criteria put forth in the ATP approval protocol (USEPA 1999) for EPA 351. 2 and 365.4 in reagent water and PP-BTWW
effluents.

3.3 Study Implementation

The study was designed as a Tier 2 ATP for use by all laboratories (nationwide use) for one matrix type (PP-BTWW). The Tier 2 validation study was managed by the NCASI West Coast Regional Center (WCRC) in Corvallis, Oregon. Laboratories were sel ected based on their experience with the reference EPA methods, previous work with pul p and paper industry matrices, a nd previous participation in multilaboratory studies. The study was conducted using three laboratories, Lab I, Lab II, and Lab III.

Table 3.2 shows sample identification numbers and fiel d descriptions from two mill sites (Site A, Site B) and one receiving water (RW) site.

Site A produces unbleached kraft linerboard mill from a combination of two-thirds virgin softwood and one-third re-pulped old corrugated cont ainers as furnish. Its effluent comprises the composite outflow of two parallel aerated stabilization basi ns (ASBs). Supplemental nitroge n and phosphorus are added to the ASBs as needed to maintain biological activity. Effluent was collected at the NPDES sampling point sited at the receiving water end of the discharge pi peline using an automatic refrigerated compositor. Site B comprises two operations. The first is a bleached kraft mill producing both bleached and unbleached pulps from northern softwood . The wastewater is treated in a primary clarifier followed byNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 15

aerated stabilization basin (ASB) secondary treat ment. The second mill produces tissue and toweling from deinked office wastepaper and purchased kraft pul p. It also produces de-inked pulp. Effluent from this mill undergoes primary clarification followed by s econdary treatment in an activated sludge process. The outflow from both mills is combined and piped un derground to receiving water. Site B effluent was collected from the common pipeline at a port mid-way between Site B and the final discharge point. In addition to the mill effluents, a RW sample was collect ed upstream of Site B to obtain comparable data on method performance in RW matrices. All samples were collected on July 6, 2009, in acid-washed glass containers. The Site A sample was packed in gel- ice and shipped overnight to NCASI WCRC. Site B effluent and RW samples were field refrigerated and brought directly to NCASI WCRC on the day of collection. Preservation to 1.6 < pH < 2 took place the NCASI WCRC laboratory. Samples were then apportioned into three sets of 500 mL aliquots and immediately shipped cold (4 ±2 o C) via overnight service to Laboratory III. During the ensuing week La b II digested a reserved portion from each effluent to obtain approximate TN and TP levels. These estimates were offered to the participating laboratories.
Lab II used this information in orde r to prepare several dilu tion sets needed to facilita te offsite analysis.

Table 3.2. Sample Set for Alternative Testing Pr ocedure Study Plan, NCASI Method TNTP-W10900
Sample ID

Number Field Sample Description a Analysis Requirements
23064 Site A – PPWW Biologically treated effluent Background levels TN, TP MS/MSD dual spike TN, TP

23065 Site B – PPWW and RCY b Biologically treated effluent Background levels TN, TP MS/MSD dual spike TN, TP

23066 Misc-RW c Background levels TN, TP

a initial chain of custody correspondence designated Site A as “Mill A” and Site B as “Mill B”

b recycled furnish (RCY)

c miscellaneous receiving water (RW)

Although only one matrix sample is required for ATP validation each laboratory was asked to digest and analyze both effluents. However, due to an overs ight Lab III subjected only Site A to the matrix spike/matrix spike duplicate (MS/MSD) acceptance testi ng slated to be performed by all laboratories. Nevertheless, Lab I and Lab II MS/MSD results for Site B met the acceptance criteria and are included in the this report. At the outset of the study, La b III erroneously used organic nitrogen and organic phosphorus spiking stocks in executing the initial preci sion and recovery (IPR) validation. The analysis was necessarily repeated to conform to the stud y plan; however, data from these IPR samples met acceptance criteria and are presented as single-laboratory results in Section 17.0 of the method. Despite these inconsistencies, all study requirements were successfully executed and results fully met the quality assurance/quality control (QA/QC) requirements outlined in the ATP protocol for EPA 351.2 and 365.4. Outcomes from earlier NCASI single-laboratory valida tions are presented in Section 17.0, Tables 2 through 5 of TNTP-W10900.

The ATP study plan is summarized in Table 3.3. Ea ch laboratory proved its ability to prepare a linear calibration using the proposed method. Each laboratory then analyzed a method blank (MB) and a continuing calibration verification (CCV) prior to sa mple analysis. Once these requirements were met, each laboratory was obliged to perform initial precision and recovery (IPR) analysis using the procedures specified in the method. Following that, a portion of each effluent was analyzed to determine native levels of the target analytes along with an MS and an MSD to determine precision and accuracy of the
ATP.

Lab I and Lab II digestions were carried out in a 20 quart pressure cooker (Presto mod. 0178005-409A), while Lab III used a Market Forge Sterilmatic autoclav e. All digestions were conducted at 120 to 121°CNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 16

and 15 to 16 psi relative positive pressure. Each partic ipating laboratory performed analytical work using instruments calibrated at a range encompassing the minimum level (ML) and above the highest level detected for target analytes in the matrices tested. The equipment and methods used during the multilaboratory study are listed in Table 3.4. All laborat ories used automated spectroscopic equipment for TN determinations, and automated spectroscopic equi pment was used for TP quantification by two laboratories. Lab III was limited to manual spectro scopic TP determination (EPA Method 365.3). The manual method uses the same colorimetric chemistry and reacted products as the automated technique, but the absorbance range limited the upper end of the calibration scale to 1.50 mg P/L. The acceptance criteria for the automated and manual phosphorus met hods are slightly different (USEPA 1999); however, the more constrained automated acceptance cr iterion was used for both approaches.

Table 3.3. Overview of Study Plan Sample and Analysis Scheme, Tier 2 Single Matrix

Analyses Sample
Count a Combined (all labs)
Calibration blank 1
Calibration curve 4 Method blank (MB) 1 Continuing calibration verification (CCV) 1 Initial precision and recovery (IPR) 4 12 Method detection limit study (MDL) 7 21 Sample set Site A b – background + MS/MSD pair 3 6 MS/MSD c Sample set Site B b – background + MS/MSD pair 3 Sample set RW – background 1 Nitrate laboratory fortified blank – (TN analysis only) 1

Total minimum required analyses per analyte TN

TP 26 (min) 25 (min)

a minimum number of analyses requested

b initial chain of custody correspondence designate d Site A as “Mill A” and Site B as “Mill B”

c one MS/MSD pair from each laboratory is required for Tier 2 ATP

Table 3.4. Determinative Methods and Equipment used in ATP Study Plan Execution

Determination: Total N itrogen Total Phosphorus
Lab Instrument Method Title EPA a Method Title EPA
Lab I B & L – AA3 HR b G-309-04 353.2 e G-146-95 365.1 f Lab II Lachat 8500 QuickChem c 10-107-04-1-C 353.2 10-115-01-4-B 365.1
Lab III B & L – AA CE 6.02 d NO2+NO3 353.2

– – Lab III Beckman DU 520 UV/VIS
– – EPA 365.3 g

a USEPA equivalent methods

b Bran & Luebbe Model Auto-Analy zer 3, segmented flow analyzer

c Lachat Model 8500, fl uid injection analyzer

d Bran & Luebbe Model Auto-Analyzer with control and evaluation software, segmented flow analyzer

e EPA Method 353.2, Nitrogen, Nitrate-Nitrite (Colorimet ric, Automated, Cadmium Reduction)

f EPA Method 365.1, Phosphorus, All Forms (Colorimetric, Automated, Ascorbic Acid)

g EPA Method 365.3, Phosphorus, All Forms (Colorimetri c, Ascorbic Acid, Two Reagent)

3.4 Data Reporting and Validation

Complete data packets were prepared by each participan t and audited by NCASI. The data include all the information listed in Table 3.5. Each particip ating laboratory submitted copies of all records demonstrating how standards were prepared and sa mples were tracked through the process. DigestionNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 17

and analysis records were reviewed and calculations were verified. Satisfactory instrumental sensitivity, as gauged by the slope of the calibration line, was confirmed during data review. Information also included the names of chemical suppliers and the lot numbers of reagents employed during analyses. All data submitted for review met the requirements of the Study Plan and the QA/QC requirements outlined in the ATP protocol for EPA 351.2 and 365.4.

Table 3.5. Data Requirements
General Information Specifics Required Sample numbers or identifiers Unique identi fiers used by laboratory and sample supplier

Laboratory notebook information Sample preparati on dates, sample volumes, dilution information

Instrument information Dates of analyses, r un logs, instrument and operating conditions, programs used, operating conditions

Analysis information Chromatograms, quan titation reports, data system outputs, any other information linking raw data to reported results

3.5 Results, Data Analysis, and Discussions

This section contains summaries and brief discussions of data received from participating laboratories required for the Tier 2 ATP application.

3.5.1 Instrument Calibration

Participating laboratories were required to prepare a multi-point calibration curve along with a calibration blank using the proposed method modifications. Lin earity could be proven either through achieving a 10% relative standard deviation (RSD) using a minimum of three calibration points including a blank, or by linear curve fitting that met a minimum coefficient of determination (r 2 ) of at least 0.995 for each analyte. Each laboratory prepared a calibration curve encompassing the concentration range for each target compound. The lowest calibration points were selected by the laboratories within the constraint that they met or exceeded the required target ML lis ted for the reference methods in the ATP protocol. All laboratories used a TN ML that was below the EPA 353.2 method minimum of 0.10 mg N/L. The ML selected for TP in all laboratories was 0.01 mg P/L, which meets the ATP requirement for EPA 365.4. The highest TP calibration point for Lab III was limited to 1.5 mg P/L due to manual
instrumentation. The calibration criteria data and QA/QC requirements outlined in the ATP protocol for EPA 351.2 and 365.4 were achieved by a ll participating laboratories. A su mmary of calibration data is in
Table 3.6.

Laboratories were required to demonstrate that the MB concentration was less than the ML prior to conducting sample analyses. A CCV sample with a recove ry of ±10% of the theoretical value then had to be confirmed. Each laboratory met the QA/QC accep tance criteria for the MB and CCV as specified in the ATP protocol for EPA 351.2 and 365.4. A summary of MB and CCV data is presented in Table 3.7.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 18

Table 3.6. Calibration Curve Data Summary
Analyte a : Total Nitrogen Total Phosphorus Laboratory
Code Calibration
Points b ML c (mg N/L) r 2 d Calibration
Points b ML c (mg N/L) r 2 d

Lab I 6 0.05 0.999 6 0.01 1.000
Lab II 6 0.02 0.999 6 0.01 0.999
Lab III 5 0.02 1.000 4 0.01 1.000

a determined using combined st andards ammonium chloride (NH 4 Cl) for TN and potassium dihydrogen

orthophosphate (KH 2 PO 4 ) for TP in reagent water

b number of calibration points excluding the calibration blank

c minimum level or lowest calibration point used for calibration curve

d coefficient of determination; pass/fail threshold ≥ 0.995

Table 3.7. Method Blank and Continuing Calibration Verification Summary
Analyte: Total Nitrogen Total Phosphorus Laboratory
Code MB (mg N/L) CCV Recovery (%) MB (mg P/L) CCV Recovery (%)
Lab I 0.04 a 98.7 ND 96.0
Lab II ND 98.2 ND 102
Lab III ND 98 ND 100

ab MB is less than lowest calibration level of 0.05 mg N/L related in Table 3.6
ND = not detected

3.5.2 Ongoing Precision and Recovery (OPR) Results

Instrument performance throughout each analysis was assessed by analyzing one of the initial calibration curve digestates after every tenth sample. An a dditional laboratory fortified blank (LFB) sample consisting of 1.00 mg N/L (N-NO 3 - ) was run during the multi-laboratory study to assure TN analyte identification. As illustrated in Table 3.8, all OPR data obtained during the multi-laboratory study met the QA/QC requirements outlined in the A TP protocol for EPA 351.2 and 365.4.

Table 3.8. Ongoing Precision and Recovery Results
Analyte a : Total Nitrogen Total Phosphor us Total Nitrogen (nitrate) Recovery and

Precision n Results b Acceptance
Criteria c Results b Acceptance
Criteria c n Results b Acceptance
Criteria c Recovery range (%) d 10 94 - 98.7 77 - 121 96.0 - 104 79 - 113 3 92.4 - 107 87 - 111

a determined using combined st andards ammonium chloride (NH 4 Cl) for TN and potassium dihydrogen

orthophosphate (KH 2 PO 4 ) for TP in reagent water; TN (nitrate) determined from potassium nitrate (KNO 3 ) in reagent water

b data from multi-laboratory validation of TNTP-W10900 fr om three laboratories participating in Tier 2 study

c per reference methods EPA 351.2 for TN and 365.4 for TP and per EPA 353.2 for TN (nitrate) in ATP Protocol

for Organic and Inorganic Analytes , Section 10, Appendix E (USEPA 1999)

d range from lowest and highest individual IPR results fr om three laboratories participating in the Tier 2 study

3.5.3 Initial Precision and Recovery (IPR) Results

Each laboratory conducted an IPR study that entailed analysis of spiked reagent water at a combined concentration of 5.0 mg N/L and 1.0 mg P/L in quadruplicate. Table 3.9 contains a summary of results from the three laboratories. Average spike recoveries were calculated for the four replicates from each laboratory in addition to the standa rd deviation of the percent recovery. Initial precision and accuracyNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 19

results were compared to performance criteria specified in the ATP protocol. As illustrated in Table 3.9, all IPR data obtained during the multi-laboratory study met the QA/QC requirements outlined in the ATP protocol for EPA 351.2 and 365.4.

Table 3.9. Multi-Laboratory Initial Precision and Recovery
Analyte a : Total Nitrogen Total Phosphorus Recovery and
Precision n Results b Acceptance
Criteria c Results b Acceptance
Criteria c Recovery range (%) d 12 94.4 - 105 79 - 119 93 – 99.8 80 - 112 SD (%) e 12 4.0 20 1.9 16

a determined using combined st andards ammonium chloride (NH 4 Cl) for TN and potassium dihydrogen

orthophosphate (KH 2 PO 4 ) for TP in reagent water

b data from multi-laboratory validation of TNTP-W10900 at the Tier 2 level

c per reference methods EPA 351.2 for TN and 365.4 for TP in ATP Protocol for Organic and Inorganic

Analytes , Section 10, Appendix E (USEPA 1999)

d range of lowest and highest individual IPR results from three laboratories participating in the Tier 2 study

e standard deviation of percent recoveries

3.5.4 Method Detection Limit (MDL) Results

Each participating laboratory conducted an MDL st udy in reagent grade water as specified in 40 CFR Part 136, Appendix B of the Federal Register using the proposed ATP. Spike levels were selected by the
individual laboratories and varied from lab to lab. Spike concentrations ranged from 0.05 to 0.25 mg N/L and 0.01 to 0.10 mg P/L across all laboratories. The approved methods do not specify an MDL however comparison to the prescribed ML thresholds are rele vant. The TN MDL values were below the reference method ML of 0.10 mg N/L. MDLs for phosphorus at Lab I and Lab III were at or below the reference method ML of 0.01 mg P/L. The phosphorus MDL fo r Lab II was 0.03 mg P/L was higher than expected but re-evaluation was not possible as a consequence of having the analysis performed offsite and with limited MDL sample volume. Note that the MDLs de termined for the referenc e methods approached the ML for nitrite/nitrate (0.05 mg N/L) detection us ing EPA 353.2 and on average met or exceeded the ML for phosphorus (0.01 mg/L) using EPA 365.1 and 365. 3. All MDL performance requirements executed during the multi-laboratory study outlined in the ATP protocol for EPA 351.2 and 365.4 (USEPA 1999) were achieved by all participating laboratories. These results are summarized in Table 3.10.

Table 3.10. Method Detection Limit Results for the Multi-Laboratory Study
Analyte a : Total Nitrogen Total Phosphorus Laboratory
Code Spike (mg N/L) MDL (mg N/L) Spike (mg P/L) MDL (mg P/L)
Lab I 0.10 0.03 0.05 0.01
Lab II 0.25 0.07 0.10 0.03
Lab III 0.05 0.01 0.01 0.003

Average 0.04 0.01

a determined using combined st andards ammonium chloride (NH 4 Cl) for TN and potassium dihydrogen

orthophosphate (KH 2 PO 4 ) for TP in reagent water

3.5.5 Sample Results

Each laboratory was required to analyze two effluent s and a receiving water sample to determine native levels of the target analytes. Each participating laborat ory was at liberty to dilute samples either before or after digestion as required to be within the range of the calibration curve. B ackground levels determinedNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 20

by each laboratory are provided in Table 3.11. Th ese results validate that native levels were within method parameters. There are no QA/QC re quirements for this measurement.

Table 3.11. Sample Background Level Resu lts from the Multi-Laboratory Study
Analyte: Total Nitrogen Total Phosphorus Laboratory
Code Site A (mg N/L) Site B (mg N/L) RW (mg N/L) Site A (mg N/L) Site B (mg P/L) RW (mg P/L)
Lab I 4.40 5.88 0.23 0.37 1.22 0.07
Lab II 4.63 6.55 0.20 0.48 1.42 0.20
Lab III 5.00 5.97 a 0.17 0.44 1.25 a 0.04

a single sample; all other tabled values are the av erage of sample and sample duplicate readings

3.5.6 Matrix Spike (MS) and Matrix Spike Duplicate (MSD) Accuracy

In order to assess the precision and accuracy of the met hod in a matrix, effluent from Site A was spiked with the target analytes (MS) then digested and an alyzed using TNTP-W10900. In addition, a matrix spike duplicate (MSD) was processed in the same manner to assess precision in the matrix. Results are shown in Table 3.12. Each laboratory selected MS/M SD spiking levels based on expected outcomes and in consideration of sample dilutions. These levels ranged from 3.00 to 5.00 mg N/L for nitrogen but remained at a constant 1.00 mg P/L for phosphorus . Precision was assessed by calculating the relative percent difference (RPD) between the MS and MSD. RPD ranged from 0.2 to 4% for TN and from 0.0 to
4.2% for TP. Additional MS and MSD data sets for S ite B effluent were analyzed by Lab I and Lab II and demonstrated similar recovery and precision conform ity as the results in Table 3.11. In addition, the RW sample was subjected to MS/MSD testing by Lab I and Lab III. Results embody accepted surface water versions of the modified digestion (USGS 2003; APHA 2006) and demonstrate conformance of the method over a wide range of TN and TP concentra tions. All MS/MSD data obtained during the multilaboratory study met the QA/QC requirements outlined in the ATP protocol for EPA 351.2 and 365.4.

Table 3.12. Accuracy Expressed as Percent Recovery of Matrix Spikes and Matrix Spike Duplicates
Analyte a : Total Nitrogen Total Phosphorus

Recovery and Precision n Results b Acceptance
Criteria c Results b Acceptance
Criteria c Recovery range (%) 6 84.4 - 96.5 77 - 121 97.0 - 105 79 - 113 RPD range d (%) 0.2 - 4 22 0.0 - 4.2 17

a determined using combined st andards ammonium chloride (NH 4 Cl) for TN and potassium dihydrogen

orthophosphate (KH 2 PO 4 ) for TP in PP-BTWW

b data from multi-laboratory validation of TNTP-W10900 at Tier 2 level

c per reference methods EPA 351.2 for TN and 365.4 for TP in ATP Protocol for Organic and Inorganic Analytes

,

Section 10, Appendix E (USEPA 1999)

d relative percent difference between MS and MSD recovery values

3.6 Conclusions

The proposed alternative testing procedure, NCASI Method TNTP-W10900, was found to produce results equivalent to or better than results obtained usi ng the approved reference methods, EPA 351.2 and 365.4 during a multi-laboratory study involving three partic ipating laboratories. TNTP-W10900 is more cost effective and environmentally friendly than the Kjel dahl process for the subject matrix, while improving detection, reducing environmental impacts, incr easing productivity, and subse quently reducing costs.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 21

Reference method acceptance criteria were met for:

• Instrument calibration – linearity was demonstrated by achieving a coefficient of determination of at
least 0.995

• Method blank – no target analytes were detected above the method limit

• Ongoing precision and recovery – ong oing recovery for total nitrogen in reagent grade water ranged from 94 to 98.7% across all laboratories; ongoing r ecovery for total phosphorus ranged from 96.0 to
104 % across all laboratories

• Initial precision and recovery – accuracy for total nitrogen expressed as recove ry ranged from 94.4 to 105% for all laboratories, precision expressed as the st andard deviation of percent recoveries was 4.0; total phosphorus recovery ranged from 93 to 99 .8% for all laboratories, precision was 1.9

• Method detection limit – the standard EPA approach in 40 CFR Part 136 Appendix B was employed to assess MDLs and was successfully executed by all laboratories; MDL thresholds are not specified
in the EPA reference methods

• Matrix spike/matrix spike duplicate – accuracy e xpressed as total nitrogen recovery ranged from 84.4 to 96.5% for all laboratories, precision expressed as relative percent difference (RPD) ranged from 0.2 to 4%; total phosphorus recovery ranged from 97.0 to 105% for all laboratories, precision ranged
from 0.0 to 4.2%

In conclusion, NCASI believes that Method TNTP-W1090 0 should be approved at the Tier 2 level for nationwide use in biologically treated pulp, paper, and paperboard wastewater for monitoring total nitrogen and total phosphorus under 40 CFR Part 136.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 22

4.0 NCASI METHOD TNTP-W10900



### TOTAL NITROGEN AND TOTAL PHOSPHORUS IN
PULP AND PAPER BOLOGICA LLY TREATED EFFLUENT
BY ALKALINE PERSULFATE DIGESTION

NCASI
WEST COAST REGIONAL CENTER

AQUATIC BIOLOGY PROGRAM
JUNE 2011

Acknowledgements

This method was prepared by Diana Cook, Project L eader, and Jan Napack, Rese arch Associate, at the NCASI West Coast Regional Center.

For more information about this method, contact:

Diana Cook Jan Napack NCASI West Coast Regional Center NCASI West Coast Regional Center
PO Box 458 PO Box 458 Corvallis, OR 97339 Corvallis, OR 97339 (541) 752-8801 (541) 752-8801 fax (541) 752-8806 fax (541) 752-8806 DCook@ncasi.org JNapack@ncasi.org

For information about NCASI publications, contact:

Publications Coordinator
NCASI
PO Box 13318 Research Triangle Park, NC 27709-3318 (919) 941-6400

Citation:

National Council for Air and Stream Im provement, Inc. (NCASI). 2011. NCASI Methods Manual

. Method TNTP-W10900: Total nitrogen and total phosphor us in pulp and paper biologically treated effluent by alkaline persulfate digestion. Rese arch Triangle Park, NC: National Council for Air and Stream Improvement, Inc.

© 2011 by the National Council for Air and Stream Improvement, Inc.

NCASI’s Mission:

To serve the forest products industry as a center of ex cellence for providing technical information and scientific research needed to achieve the industry’s environmental goals.

Disclaimer

The mention of trade names or commercial products does not constitute endorsement or recommendation
for use.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 23



### NCASI Method TNTP-W10900

Total Nitrogen and Total Phosphorus in Pulp and Paper Biologically Treated Effluent by Alkaline Persulfate Digestion

1.0 Scope and Application

1.1 This method is applicable to determinations of total nitrogen (TN) and total phosphorus (TP) in acid preserved pulp, paper, and pa perboard mill biologically treated effluent (PP-BTWW). The applicable range of this method is 0.01 to 3.0 mg P/L and 0.1 to 6.0 mg N/L. The range may be extended with sample dilution. Sample size is 7 mL.

1.2 Laboratories intending to use this procedure should establish method detection limits for each sample matrix type. Changes in technology may influence chemical compositions of these matrices, requiring evaluation of samples for di gestion efficiency and method performance. Any modification of the method beyond those expressl y permitted is subject to application and approval of alternative test procedures under 40 CFR Parts 136.4 and 136.5 (Section 16.1).



### 2.0 Summary of Method

2.1 The method simultaneously converts a mmonia and inorganic (excluding N 2 ) and organic nitrogen to nitrate and inorganic and organic forms of phosphorus to orthophosphate by means of an alkaline to acid digestion. Nativ e dissolved nitrate and nitrite ar e unaffected by the digestion.

2.2 Digestion is accomplished by heating acidified, unf iltered samples in the presence of persulfate (a strong oxidizer) at 120 o C and 15 psi positive pressure for 30 mi nutes. The digestion is strongly alkaline at the outset, which promotes oxidation of nitrogen to nitrate. As the persulfate decomposes the solution becomes acidic, allowi ng oxidation of phosphor us to orthophosphate (Section 16.2). Following digestion, separate semi-automated or manual colorimetric determinative methods of the single digestat e are conducted for analysis of TN and TP.

2.3 TN measurement occurs by means of nitrite/nitr ate determinative techniques. TP measurements occur by means of orthophosphate determinative techniques (Section 6.5).



### 3.0 Definitions

3.1 The definitions and purposes herein are specific to this method but conform to common usage as much as possible.

3.1.1 May

− this action, activity, or procedural step is neither required nor prohibited

3.1.2 Must

− this action, activity, or procedural step is required

3.1.3 Must not

− this action, activity, or procedural step is prohibited

3.1.4 Should

− this action, activity, or procedural step is suggested but not required

3.2 Total nitrogen (TN) consists of inorganic (ammonia, nitrite, nitrate, excluding N 2 ) and organic forms of nitrogen. Organic nitrogen may be co mprised of biological, synthetic, or natural nitrogen-containing compounds.

3.3 Total phosphorus (TP) consists of inorganic (orthophosphates, polyphosphates) and organicallybound phosphorous that may consist of biologi cal, synthetic, or natura l phosphorus-containing compounds.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 24

3.4 Calibration standards are made to encompass the app licable range of the analytical method. They must contain both method analytes and be acidified prior to digestion and analysis.

3.5 Continuing calibration verifications (CCV) consist of calibration points periodically reanalyzed within the test cycle to verify that the initial ca libration is applicable throughout the sample run. Calibration verifications are required after every te nth sample and at the end of the analytical

sequence.

3.6 The independent calibration verification (ICV) is a solution containing a known concentration of each method analyte derived from externally prepared test materials and is used to verify the quality of the calibration curve. Standards ar e purchased with certifications of purity, concentration, and authenticity sourced from cer tified NIST-traceable references. Two separate ICV solutions, one for each target analyte, are mi xed and acidified to produce a working standard (Section 7.2.6).

3.7 A laboratory fortified blank (LFB) consists of an aliquot of reagent water to which a known quantity of method analyte has been added in the laboratory. The LFB is processed and analyzed exactly as a sample. Its purpose is to determine if the method is within accepted control limits. In this method, a nitrate LFB is processed to confirm that there is no analytical bias between nitratesourced and ammonia-sourced digestates (Section 7.2.8).

3.8 Method blanks (MB) consist of volumes of reag ent water that are carried through the entire preparation and analysis exactly as a sample, including exposure to all glassware, equipment, and reagents. They are used to document and correct for any systematic interference from reagents, containers, or the laboratory environment.

3.9 A sample duplicate (DUP) consists of a second aliquot of a sample, identical to the first and processed through the entire method. Samp le duplicates are used to assess process reproducibility and method precision (Section 9.4.5).

3.10 A matrix spike (MS) consists of an aliquot of sample to which a known quantity of each method analyte is added in the laboratory. It is then processed through the entire method. The purpose of the MS is to determine whether the sample matrix contributes bias to the analytical results. The bias, or accuracy, is expressed as percent recove ry (%R) and is calculated using Equation 4 (Section 9.4.6).

3.11 A matrix spike duplicate (MSD) is prepared usi ng a second aliquot of the same sample used for the MS. The relative percent difference (RPD ) between the MS and MSD is used to assess analytical precision and is calculated as shown in Equation 3 (Section 9.4.5).

3.12 A digestion check consists of an aliquot of reag ent water or sample to which a known quantity of organic method analyte has been added in the labor atory. Digestion checks may be used in this method and are useful in validating digestion of organic nitrogen and phosphorus in complex matrices (Section 9.4.8). Digestion check soluti ons can be added to reagent water (LFB) or samples (MS).



### 4.0 Interferences

4.1 Persulfate digestion for nitroge n species may be less effective in water samples with high organic or nitrogenous compound loading (>20 mg N/L). For these matrices it is recommended that the oxidative capacity of the digestion be confirmed by completing digestion of a spiked sample according to Sections 9.4.6 and 9.4.8.

4.2 Over-acidification of samples at collection sites can result in low recoveries of inorganic and organic nitrogen levels (Section 16.3). This can be avoided by judicial addition of dilute acid andNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 25

use of narrow-range pH indicator strips at the tim e of collection. Other methods employed in the field include sample refrigeration, addition of pre-measured acid aliquots in collection vessels,
and methods with similar effect.

4.3 Chloride concentrations greater than 10,000 mg/L react with persulfate to form oxychlorides or chlorine that might deplete persulfate required to oxidize inorganic and organic nitrogen species
to nitrate. Results will be biased low as a c onsequence (Section 16.3); however, these levels of
chloride have not been encountered in PP-BTWW matrices.

4.4 Phosphorus concentrations may be underestimated under high susp ended sediment concentrations (>150 mg/L). The method is only suitable for very turbid samples when turbidity is due to organic matter (algal cells, plant detritus) (Section 16.4).

4.5 Ionic forms of calcium, iron, barium, lead, a nd silver in the presence of sufficiently high concentrations of phosphorus can interfere by fo rming phosphate precipitates that may lead to under-reporting of TP (Section 16.3).

4.6 Arsenate [AsO 4 3- ] forms a color similar to that of phos phate above 1.0 mg As/L and may produce a positive interference. Arsenite [AsO 3 2- ] at levels >2.0 mg As/L may be oxidized to arsenate (Section 16.3).

4.7 Method interferences may be caused by contaminan ts in reagents, glassware, and other sample processing equipment. All process materials must be routinely demonstrated to be free from interferences under the conditions of the analy ses by running method blanks as described in
Section 3.8.



### 5.0 Safety

5.1 Potassium persulfate must be kept separate fro m flammable and easily oxidizable substances such as combustibles and many solvents. It is harmfu l when swallowed and may be harmful if inhaled or absorbed through the skin. It is irritating to mucous membranes, skin, and eyes. Fume hood, eye protection, chemical-resist ant gloves, and protective clothing are strongly recommended when using this compound.

5.2 This method does not address all safety issues a ssociated with its use. The laboratory is responsible for maintaining a safe work e nvironment and a current awareness of OSHA regulations regarding safe handling of chemicals us ed in this method. Material safety data sheets (MSDS) should be available to all personnel invo lved in these analyses. Prudent and vigilant practices for handling chemicals must be employed at all times.



### 6.0 Equipment and Supplies

6.1 Autoclave or pressure cooker

6.1.1 Operating and maintenance instructions fo r high-pressure equipment and vessels should
be read and understood before use. Inspect seals, vents, and tubes before use.

6.1.2 Autoclaves and pressure cookers must be cap able of developing a steady, relative positive pressure of 15 psi. If using a pressu re cooker, models with built-in pressure gauges are recommended. Ensure that the hot plate used with the pressure cooker has adequate heating surface area.

6.2 Heat-resistant and pressure-resistant test tube racks

6.3 GlasswareNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 26

6.3.1 Class-A volumetric flasks, precision pipettes and micro-volume syringes as required

6.3.2 Borosilicate screw-top glass culture tubes, 16 x125 mm

6.3.3 Polyphenolic tube caps with cement ed-in PTFE-faced 14B rubber liners

6.3.4 Glassware should be washed with hot ta p water followed by copious rinsing with de-ionized water, then undergo a thorough 5% or 10% (v/v) HCl soak before a final reagent water rinse. Store glassware inverted after air or oven drying. Commercial detergents must never be used for cl eaning glassware utilized in this method.

6.4 Top-loading balance capable of weighing to 10 ± 0.1 mg

6.5 Analytical equipment and supplies

6.5.1 Instrumentation, equipment, and supplies for determination of nitrite/nitrate and orthophosphate are found within the approved methods for wastewater analysis (Section 16.5). Due to sample volume constraints, manual techniques are not recommended; however, they are permitted.

6.5.1.1 Approved methods for nitrite/nitrate analyses consist of EPA 353.2 (Section 16.6), ASTM D3867-90 (A and B) (Section 16.7), Standard Method
4500-NO 3 - (E and F) (Section 16.8), and USGS I-4545-85 (Section 16.9).

6.5.1.2 Approved methods for orthophosphor us detection include EPA 365.1 (Section 16.10), EPA 365.3 (Section 16.11) , Standard Methods 4500-P (E and F) (Section 16.8), and USGS I-4601-85 (Section 16.9).



### 7.0 Reagents and Standards

7.1 Reagents

7.1.1 Ultrapure reagent water (UPW) such as that produced by commercial purification systems or deionized water (DIW) conforming to Type II reagent water (Section 16.12) and free of the analytes of interest.

7.1.2 1.84 M sulfuric acid solution (10% v/v) – Slowly add 10 mL of concentrated sulfuric acid (H 2 SO 4 , CAS 7664-93-9) to approximately 50 mL of cold reagent water. Cool and dilute
to 100 mL.

7.1.3 Persulfate digestion solution – Dissolve 5. 0 g of reagent grade potassium persulfate (K 2 S 2 O 8 , CAS 7727-21-1, FW 270.32) and 1.2 g of reagent grade sodium hydroxide (NaOH, CAS 1310-73-2, FW 40.00) in approximately 200 mL of reagent water. Once fully dissolved, transfer to a 250 mL volumetric flask with a reagent water rinse and bring to volume. Reagents with known low nitrogen assays must be used.

7.1.4 Refer to the determinative methods (Secti on 6.5) for reagent lists and preparation instructions used in analyzing nitrite-nitrate and orthophosphate.

7.2 Standards

7.2.1 Ammonium primary stock, 1000 mg N/L – Add 3.818 g of reagent grade anhydrous ammonium chloride (NH 4 Cl, CAS 12125-02-9, FW 53.49) to about 500 mL of reagent water. Quantitatively transfer to a 1 L volumetric flask and dilute to the mark.

7.2.2 Phosphorus primary stock, 500 mg P/L – Add 2.197 g of reagent grade anhydrous potassium dihydrogen orthophosphate (KH 2 PO 4 , CAS 7778-77-0, FW 136.09) to aboutNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 27

500 mL of reagent water. Quantitatively transfer to a 1 L volumetric flask and dilute to
the mark.

7.2.3 Ammonium working stock, 100 mg N/L – Dilute a portion of the ammonium primary stock (Section 7.2.1) by a factor of 10 using reagent water. Use this solution for spiking samples or quality control solutions at discrete analyte levels.

7.2.4 Phosphorus working stock, 100 mg P/L – D ilute a portion of the phosphorus primary stock (Section 7.2.2) by a factor of 10 using reagent water. Use this solution for spiking samples or quality control solutions at discrete analyte levels.

7.2.5 Combined nitrogen/phosphorus working stock solution – Add equal amounts of ammonium primary stock (Section 7.2.1) and phosphorus primary stock (Section 7.2.2) to an acid-washed beaker. One milliliter (1 mL) of solution is equal to 0.50 mg N + 0.25 mg P. Prepare at least 20 mL of combin ed solution to reduce volumetric errors. Use this solution for making up the calibration curve (Section 7.3).

7.2.6 ICV combined nitrogen/phosphorus stoc k solution – Combine purchased ammonia and phosphate reference standards in appropriate amounts to achieve a final concentration of 1.00 mg N/L + 1.00 mg P/L in reagent water. Acidify the solution by incorporating 1.0 mL of 10% sulfuric acid (Section 7.1.2) into every 100 mL of final volume.

7.2.7 Nitrate primary stock, 100 mg N/L – Dissolve 0.072 g of reagent grade potassium nitrate (KNO 3, CAS 7757-79-1, FW 101.1) in about 50 mL of reagent water. Quantitatively
transfer to a 100 mL volumetric flask and dilute to the mark.

7.2.8 Nitrate LFB, 1.00 mg N/L – Dilute a portion of nitrate primary stock (Section 7.2.7) by a factor of 10 using reagent water. Acidify the solution by adding 1 mL of 10% sulfuric acid (Section 7.1.2) into every 100 mL of final volume.

7.2.9 Digestion check stock solution – Prepare digestion check solutions and add to reagent water or samples in the TN and TP ranges expected for BT-PPWW. Several organic compounds are suitable for preparing digestion check solutions, including urea (CAS 57- 13-6), nicotinic acid, (CAS 59-67-6), sodi um phenyl phosphate (CAS 66778-08-03), and others (Section 16.2).

7.3 Calibration standards

Use Table 1 (Section 17.0) as a guide to prepare a dual calibration curve. Spike reagent water with the working combined nitrogen/phosphorus working stock (Section 7.2.5), then acidify with 10% sulfuric acid (Section 7.1.2). The calibration set must include a calibration blank.



### 8.0 Sample Collection, Preservation, and Storage

8.1 Collection

Samples should be collected in glass or HDPE c ontainers that have been acid washed and thoroughly rinsed (Section 6.3.4).

8.2 Preservation

Samples should be preserved at the time of collec tion by adding dilute sulfuric acid to attain 1.6 < pH < 2. Ensure that sample pH is with in the correct range using narrow-range pH color strips or a pH meter to avoid over-acidification (Section 4.2).NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 28

8.3 Storage

Refrigerate samples at 4 ±2 o C until digested. Maximum holding time is 28 days after collection and preservation. Digested samples may be stored at room temperature for up to 28 days (Section 17.0, Table 5). If either holding time is exceeded a new sample must be collected and digested.



### 9.0 Quality Control

9.1 Each laboratory that uses this method should operate a formal quality assurance program and maintain records that define the quality of the data that are generated. Data should be compared
to established criteria in order to determine if the results of analyses meet the performance characteristics of the method. The requiremen ts of this program consist of an initial demonstration of laboratory capability (Secti on 9.3) and routine analysis of quality assurance/quality control (QA/QC) solutions as a continuing check on method performance (Section 9.4).

9.2 Analytical performance

9.2.1 Prior to analyzing any samples or standards, equipment performance must be verified by means appropriate to the determinative met hod (Section 6.5). All QA/QC specifications
listed in the associated determinative method must be met.

9.2.2 Calibrations must demonstrate linearity with in 10%, as defined by the percent relative standard deviation (%RSD) of the response factors (Equation 2). Otherwise, a coefficient of determination (r 2 ) equal to or better than 0.995 is required. If the calibration fails, instrument solutions and/or digestion systems should be examined prior to preparation of new calibration and digestion solutions.

9.3 Initial demonstration of method proficiency

9.3.1 Initial precision and recovery (IPR)

9.3.1.1 The laboratory shall make an initial dem onstration of its ability to generate acceptable results with this method. Conduct an IPR assessment by digesting and analyzing four aliquots of the combined nitrogen and phosphorus working stock solution (Section 7.2.5) in the range of expected BT-PPWW process
effluents.

9.3.1.2 Using the results of the IPR analyses, compute average %R (Equation 1) and %RSD (Equation 2) of the recoveries for each analyte. Compare the %R and %RSD to the corresponding ranges determined during the multi-laboratory validation of the method (Section 17.0, Tabl e 2). Laboratories should be able to achieve IPR values within the ranges stat ed prior to performing this method.

Equation 1

% R = 100 x Concentration Measured Concentration Spiked

Equation 2

% RSD = 100 x Standard Deviation of Measured Average of MeasuredNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 29

9.3.2 Matrix spike precision and recovery

Each laboratory should establish quality control charts and recovery criteria for specific matrices to prove method performance. This is accomplished by documenting %R (Equation 1) of MS samples and %RPD (Equation 3) for DUP and MS/MSD pairs. These results should be compared to the %R and %RPD values determined during the multi-laboratory study (Section 17.0, Table 3). If laboratory results for both TP and TN meet the criteria, method performance is verified.

9.3.3 Method detection limit (MDL)

To establish the capability to detect the anal ytes in this method, the laboratory should determine the MDL in accordance with the pr ocedure in 40 CFR Part 136, Appendix B (Section 16.5) using the apparatus, reagents , and standards used in practice of this method. Prior to performing this method la boratories should be able to achieve MDLs equal to or less than 0.05 mg N/L for TN and 0.01 mg P/L for TP.

9.4 Method performance

9.4.1 Frequency

The laboratory must prepare DUP and MS sets from a minimum of 10% of routine samples. In addition, representative sampl es from each new or untested source or sample matrix should be allocated for DUP and MS quality control samples.

9.4.2 Method blank (MB)

One reagent blank should be prepared for ev ery sample batch or for every 20 samples, whichever is more frequent. If any of th e compounds of interest or any potentially interfering compounds are found in a blank at greater than the MDL determined in Section 9.3.3, the analysis should be ha lted until the source of contamination is eliminated. Blanks should be analyzed fo llowing initial calibration and prior to any sample analyses. Results should be kept on file with sample analysis data.

9.4.3 Continuing calibration verification (CCV)

CCV solutions must be run before sample an alyses, after every tenth sample, and at the end of the sample sequence. The measured concentration for each CCV point must be
within ± 10% of the expected concentration. If the calibration cannot be verified within the specified limits, reanaly ze the CCV. If the second analysis is still outside the limits, discontinue analyses and correct the source of the problem before resuming. All samples following the last acceptable CCV solution must be reanalyzed. Recovery data for all CCV measurements must be kept on file with sample analysis data.

9.4.4 Independent calibrati on verification (ICV)

One ICV standard solution consisting of mid-range concentrations of N and P (Section 7.2.6) should be digested with each di gestion batch. Recovery must be within ±10% of expected concentrations. If it is not, reanalyze the solution. If the second analysis is still outside the limits, discontinue analyses and correct the source of the problem before resuming. Analyze an ICV at least once during sample analysis following calibration. Recovery data for a ll ICV measurements s hould be kept on file with sample analysis data.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 30

9.4.5 Sample duplicate (DUP)

One DUP must be digested with every batch or one for every ten samples from the same sample group or matrix, whichever is more frequent. Precision is documented by %RPD of duplicate measurements as calculated using Equation 3.

Equation 3

% RPD = 100 x (S1 - S2)

Mean concentration

where: S1 = Sample 1 concentration S2 = Sample 2 concentration

9.4.6 Matrix spike (MS)

One MS sample must be prepared and di gested for every ten samples from the same sample matrix or one per digestion batch, whichever is more frequent. Using the estimated background concentration, use the appropriate stock solutions (Sections 7.2.3 and 7.2.4) to spike the sample at a level th at will give one to five times the background concentration. Digest the MS along with an aliquot of unspiked sample. Calculate %R for the MS using Equation 4. If an MSD was also analyzed calculate the %RPD of the recoveries according to Equation 3. Limits for MS and MSD %R and %RPD should be within those determined during the multi-la boratory study (Section 17.0, Table 3) until sufficient internal performance data a nd control limits can be established.

Equation 4

% R = 100 x (Cs – Cn)
S

where: R = MS or MSD sample recovery
C

s = MS or MSD sample concentration
C

n = native sample concentration S = concentration equivalent of analyte added to sample

9.4.7 Laboratory fortified blank (LFB)

A nitrate LFB must be digested and analyzed at least once during TN sample analysis. Calculate %R using Equation 1. Recovery must be within ±10% of the expected value. If it is not, reanalyze; if the measurement is still outside the limit, discontinue analyses and determine the source of error. Correct before continuing.

9.4.8 Digestion checks

Digestion checks are not required in the norma l operation of this method. However, if they are employed, performance criteria are assessed against the required recovery of ±10% until sufficient internal performance data and control limits can be established. If
%R of the checks falls outside establishe d warning limits, method performance is unacceptable for the compound in the matrix. Limits for digestion check solutions using 2-phenyl glycine (organic nitrogen) and ca lcium glycerylphosphate (organic phosphorus) were determined during single- and multi- laboratory validations of this method (Section 17.0, Tables 4 and 5).NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 31



### 10.0 Calibration and Standardization

10.1 Analytical equipment operating conditions

Analyze sample digestates using EPA-approved de terminative methods for nitrite/nitrate and orthophosphate (Section 6.5). If an auto-analyzer is used follow the manufacturer’s instructions to establish operating conditions. Once the syst em is optimized the same operating conditions must be used to analyze all samples. In strument response checks (e.g. carryover) may be performed using unacidified, undigested nitrate a nd orthophosphate standards prior to analysis of digested samples.

10.2 Prepare a digested calibration curve by plotting instrument response against concentration values (Section 9.2.2). The calibration curve may be fitted to calibration solutions concentration/response data us ing automated operating software or calculator-based regression curve fitting techniques.



### 11.0 Procedure

11.1 Carry out the digestion in a pressure cooker or autoclave at 121 o C and 15 psi positive pressure. All solutions used in a single determinative anal ysis must be digested in the same batch. The instructions herein are written for digestion usin g a pressure cooker. If using an autoclave, follow manufacturer’s operating instructions to affect the same operating conditions.

11.1.1 Remove samples and standards from refrigerated storage and allow them to come to room temperature. Gather and label a sufficient number of culture tubes and caps.

11.1.2 Add enough deionized water to the pressure cooker to provide a water level roughly equal to a 10 mL volume inside the digesti on tubes. Place the uncovered pressure cooker on a hot plate and set the temperature control to low.

11.1.3 Make up acidified calibration solutions (Secti on 17.0, Table 1) and all required acidified quality control solutions according to Sections 7.2 and 9.4. Select and prepare DUP, MS, and MSD quality control samples as directed in Sections 9.4.5 and 9.4.6. Ensure that calibration solution quantities are adequate fo r the number of CCV measurements that must be analyzed for the batch size. Because sample size is limited to 10 mL, additional MB and CCV quantities may be prepared as contingent samples in case reanalysis becomes necessary.

11.1.4 Transfer 7.00 ±0.05 mL of all samples into assigned culture tubes. Shake the sample containers vigorously before tran sferring to ensure homogeneity.

11.1.5 Dispense 3.00 ±0.05 mL of potassium persulfate digestion solution (Section 7.1.3) into each sample tube. Cap and mix gently. Repeat for each tube.

11.1.6 Load the tubes into a heat-proof rack and securely place the rack in the pressure cooker. Ensure that the water level is approximately th e same as the sample level inside the tubes. Secure the cover and set the hot plate temperature control to high. After heating for several minutes a steady stream of steam will begin to escape from the top vent. Allow the steam to escape for an additional 5 minutes to affect a complete seal.

11.1.7 Place the regulator on the vent pipe and monitor pressure. After several minutes the pressure will begin to rise, ultimately reaching 15 psi. Once the pressure is stable begin timing the 30 minute digestion. Pressure may slowly increase a few psi during this time
without adverse effects.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 32

11.1.8 Turn off the heat after 30 minutes has elapsed . Do not remove the pressure cooker from the hot plate nor remove the regulator or c over until the pressure cooker has returned to atmospheric pressure and is sufficiently cool ed to allow safe handling, after which time the racks may be removed and caps may be tightened.

11.1.8.1 The digestion reaction effectively progr esses during the pre-heating and cooling phases. Autoclave settings may or may not automatically include these intervals. Thus the 30 minute digestion timing should begin only after the temperature and pressure parameters are reached. Other means of applying thermal energy to the digestion have also been described (Section 16.4).



### 12.0 Data Analysis and Calculations

12.1 Sample analysis

Analyze the samples using the appropriate determinative method (Section 6.5) and specific instructions for assembly, analytical technique, r eagent preparation, step succession, and manifold proportionalities provided by equipment manufacturers.

12.1.1 If suspended particles remain in the digestat es they must be removed by sedimentation and decantation or filtration prior to TN an alysis to avoid possible clogging of the
cadmium column.

12.1.2 Calculate sample concentrations based on the calibration curve. When a calibration blank is used as a zero-point, the value of the MB must not be subtracted from experimental results.

12.1.2 Calculate %R for ICV, CCV, and LFB quality control samples (Equation 1). MS and
MSD %R must be corrected for native concentrations as measured in an unfortified sample (Equation 4).

12.1.3 Calculate %RPD (Equation 3) for DUPs and for MS/MSD pairs.

12.2 Data review requirements

12.2.1 Ensure that all sample concentrations are w ithin the calibration range of the instrument. Samples exceeding the highest standard should be quantitatively diluted to fall within the range of the calibration curve and reanalyzed.

12.2.2 Manually review all results to confirm that %R and %RPD of spiked samples are within established control boundaries. If MS or MSD recoveries fall outside the expected recovery range (Section 9.3.2) but laboratory performance for that analyte is shown to be in control, the recovery problem encountered w ith the spike is judged to be either matrixor solution-related, not system-related.

12.2.3 Sample concentrations determined for each analyte, range of concentrations, and %RPD determined by DUPs should be tabulated in a method precision log. Record MS %R along with %RPD of all MS/MSD pairs.

12.2.4 Generate a report that includes calculated con centrations of TN in mg N/L and TP in mg P/L and for all samples. Report MB levels and recovery values for CCV, ICV, and LFB samples.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 33



### 13.0 Method Performance

13.1 Verify that MB levels are below the MDL specified in Section 9.3.3 to confirm that laboratory processes are in control.

13.2 If matrix interference and bias are significan t, samples should be assessed for interfering compounds (Section 4) and re-evaluated accordingly.



### 14.0 Pollution Prevention

No federal or state requirements are known to exist for hazard containment or remediation of laboratory quantities of reagents and solutions used in this method.



### 15.0 Waste Management

15.1 It is the laboratory’s responsibility to comply with all federal, state, and local regulations governing waste management, and to protect the ai r, water, and land by minimizing releases into the environment. Compliance with all sewage disch arge permits and regulati ons is also required.

15.2 Instructions for sample and waste handling and disposal

15.2.1 All samples and solutions, including process waste and excess reagent solutions, must be neutralized and disposed of in accordance w ith applicable federal, state, and local regulations.

15.2.2 For further information on waste ma nagement, EPA recommends consulting The waste management manual for laboratory personnel and Less is better: Laboratory chemical management for waste reduction . Both are available from the American Chemical Society’s Department of Government Relations and Science Policy, 1155 16 th Street NW, Washington DC, 20036.



### 16.0 References

16.1 United States Environmental Protection Agency (USEPA). 1999. Protocol for EPA approval of alternate test procedures for organic and inor ganic analytes in wastewater and drinking water

. United States Environmental Protection Agency, Office of Water, Engineering and Analysis Division. http://www.epa.gov/waterscience/methods/atp/EPA821B98002.pdf.

16.2 Ebina, J., Tsutsui, T., and Shirai, T. 1983. Si multaneous determination of total nitrogen and total phosphorus in water using pe roxodisulfate oxidation. Water Resources 17:1721-1726

.

16.3 United States Geological Survey. 2003. Methods of analysis by the U.S. Geological Survey National Water Quality Laboratory – Evaluation of alkaline persulfate digestion as an alternative to Kjeldahl digestion for determination of total and dissolved nitrogen and phosphorus in water . Patton, C. J., and Kryskalla, J.R. Open File Report (OFR) 03-4174. United States Geological Survey.

16.4 Maher, W., Krikowa, F., Wruck, D., Louie, H., Nguyen, T., and Huang, W.Y. 2002. Determination of total phosphor us and nitrogen in turbid waters by oxidation with alkaline potassium peroxodisulfate and low pressure microwave digestion, autoclave heating or the use of
closed vessels in a hot water bath: Co mparison with Kjeldahl digestion. Analytica Chimica Acta
463:283-293.

16.5 United States Environmental Protection Agency. 2007. 40 CFR Parts 122, 136, et al. Guidelines establishing test procedures for the analysis of pollutants under the Clean Water Act. Federal Register 72(47), March 12, 2007, Rules and Regulations.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 34

16.6 United States Environmental Protection Agency. 1993. Determination of nitrate-nitrite nitrogen by automated colorimetry , Rev. 2. United States Environmental Protection Agency, Environmental Monitoring Systems Laboratory. http://www.epa.gov/waterscience/methods/method/files/353_2.pdf

16.7 American Society for Testing and Materials. 1988. Annual book of ASTM standards Section 11, Water and Environmental Technology . Vol. 11.01, Water (I). West Conshohocken, PA: American Society for Testing and Materials.

16.8 American Public Health Association. 2005. Standard methods for the examination of water and

wastewater , 21 st Edition

. Methods 4500-P and 4500-NO 3 - . Washington, DC: American Public
Health Association.

16.9 Fishman, M.J., and Friedman, L.C. 1989. Meth ods for the determination of inorganic substances in water and fluvial sediments. Chapter A1 in Techniques of Water-Resources Investigations of the U.S. Geological Survey , Book 5. Denver, CO: United States Geological Survey.

16.10 United States Environmental Protection Agency. 1993. Determination of phosphorus by semiautomated colorimetry , Rev. 2. United States Environmental Protection Agency, Environmental Monitoring Systems Laboratory. http://www.epa.gov/waterscience/methods/method/files/365_1.pdf.

16.11 United States Environmental Protection Agency. 1978. Phosphorus, all forms (colorimetric, ascorbic acid, two reagent) . United States Environmental Protection Agency, Environmental Monitoring Systems Laboratory. http://www.epa.gov/waterscience/methods/method/files/365_3.pdf.

16.12 American Society for Testing and Materials. 1983. Standard specifications for reagent water.
D 1193-77 in Annual Book of ASTM Standards , Vol. 11.01. West Conshohocken, PA: ASTM
International.



### 17.0 Tables, Diagrams, Flowcharts, and Validation Data

Table 1. Calibration Standard Solution Makeup Scheme

Calibration
Point Standard Volume a ( μ L) Acid Volume b (mL) Final Volume (mL) Nominal Concentrations TN (mg/L) TP (mg/L)
Cal 0 0 2.5 250 0.00 0.00
Cal 1 20 5.0 500 0.02 0.01
Cal 2 50 2.5 250 0.10 0.05
Cal 3 250 2.5 250 0.50 0.25 Cal 4 1,500 2.5 250 3.00 1.50 Cal 5 3,000 2.5 250 6.00 3.00

a standard contains 0.50 mg N/L and 0.25 mg P/L as described in Section 7.2.5

b 10% sulfuric acid; constitutes 1% of final volume to achieve required pHNCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 35

Table 2. Initial Precision and Recovery a

Recovery and Precision (%) Study n Average b Range c SD d Total Nitrogen Single laboratory 4 98.9 97.9 - 100 0.2 Multi-laboratory 12 98.8 94.4 - 105 4.0

Total Phosphorus Single laboratory 4 93.0 91.1 - 94.9 1.6 Multi-laboratory 12 97.1 93.0 – 99.8 1.9

a determined using combined standards 5.00 mg N/L ammonium chloride (NH 4 Cl) for TN and 1.00 mg P/L

potassium dihydrogen ortho-phosphate (KH 2 PO 4 ) for TP in reagent water

b average of IPR recoveries from four replicates in reag ent water analyzed during single laboratory validation

of NCASI Method TNTP-W10900 July 23, 2008, at NCASI West Coast Regional Laboratory, Corvallis, Oregon; multi-laboratory average from pooled IPR resu lts obtained during multi- laboratory validation of NCASI Method TNTP-W10900 July 2009.

c range taken from lowest and highest individual IPR recoveries from laboratory validations

d precision expressed as standard deviation of pooled percent recoveries

Table 3 . Matrix Spike and Matrix Spike Duplicate Recovery and Precision a

Recovery and Precision (%) Study n Average b Rangec c RPD d Total Nitrogen Single laboratory 2 98.3 92.6 - 104 11.6 Multi-laboratory 6 90.8 84.4 - 96.5 0.2 – 4.4

Total Phosphorus Single laboratory 2 98.8 97.5 - 100 2.5 Multi-laboratory 6 101 97.0 - 105 0.0 – 4.2

a determined using combined standards 5.00 mg N/L ammonium chloride (NH 4 Cl) for TN and 1.00 mg P/L

potassium dihydrogen ortho-phosphate (KH 2 PO 4 ) for TP in PP-BTWW

b average of recoveries from MS/MSD pairs in PP-BTWW analyzed during single laboratory validation of

NCASI Method TNTP-W10900 July 23, 2008, at NCASI West Coast Regional Laboratory, Corvallis, Oregon; multi-laboratory average fr om pooled MS/MSD results obtained during multi-laboratory validation of NCASI Method TNTP-W10900 July, 2009

c range taken from lowest and highest individual MS /MSD pair results from laboratory validations

d precision expressed as percent relative standard devia tion of percent recoveries; RPD is absolute value of

difference between MS and MSD expressed as a percent: %RPD = 100 x [|MS-MSD| / 1⁄2 x (MS+MSD)]NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 36

Table 4. Organic Analyte Digestion Check Initial Precision and Recovery a

Recovery and Precision (%) Study b n Average c Range d SD e Total Nitrogen Single laboratory A 4 93.3 92.2 - 94.7 1.2 Single laboratory B 4 99.8 98 - 101 2

Total Phosphorus Single laboratory A 4 85.2 84.7 - 85.6 0.7 Single laboratory B 4 91 90 - 92 1

a determined using combined standards 1.00 mg N/L 2-phenyl glycine (C 8 H 9 NO 2 ) for TN and 1.00 mg P/L

calcium glycerophosphate (C 3 H 7 O 6 P

∙ Ca) for TP combined standards in reagent grade water

b data from two single laboratory validations of NC ASI Method TNTP-W10900: Lab A NCASI West Coast

Regional Laboratory, Corvallis, Or egon, July 23, 2008; Lab B Columbia Analytical Services, Kelso, Washington, July 24, 2009

c average of IPR recoveries from four replicates in reag ent water analyzed during single laboratory validations

d range of lowest and highest IPR replicate recoveries

e precision expressed as standard deviation of percent recoveries

Table 5. Digested Sample Holding Time Study a

Recovery and Precision (%)

Holding Time b n Average c Range d RPD e Total Nitrogen 4 days 2 98.4 92.8 - 104 5.7 28 days 2 104 103 - 105 1.9

a determined using combined standards 5.00 mg N/L ammonium chloride (NH 4 Cl) for TN and 1.00 mg P/L potassium dihydrogen ortho-phosphate (KH 2 PO 4 ) for TP in PP-BTWW; TP determination not performed

b holding time is number of days elapsed since digestion

c average of recoveries from MS/MSD pair in PP-BTWW on September 22, 2008 and October 16,

2008 at NCASI West Coast Regional Laboratory, Corvallis, Oregon

d range of lowest and highest MS/MSD recoveries

e relative percent difference; RPD is absolute value of difference between MS and MSD expressed as a

percent: %RPD = 100 x [|MS-MSD| / 1⁄2 x (MS+MSD)]

REFERENCES

American Public Health Association (APHA). 2005. Standard methods for the examination of water and

wastewater , 18th, 19th, 20th, 21st Ed. Methods 4500-P (E and F) and 4500-NO 3 - (E and F). Washington, DC: American Public Health Association.

American Public Health Association (APHA). 2006. Standard methods for the examination of water and

wastewater

. Methods 4500-P (J) Persulfate method for si multaneous determination of total nitrogen and total phosphorus. Washington, DC: Am erican Public Health Association.

American Society for Testing and Materials. 1983. Standard specifications for reagent water. D 1193-77
in Annual Book of ASTM Standards , Vol. 11.01. West Conshohocken, PA: American Society for Testing and Materials.

American Society for Testing and Materials (ASTM). 1988. Annual book of ASTM standards Section 11, Water and Environmental Technology . Vol. 11.01, Water (I). West Conshohocken, PA: American Society for Testing and Materials.NCASI TNTP-W10900 as an Alter native to EPA 351.2 and 365.4 37

Ebina, J., Tsutsui, T., and Shirai, T. 1983. Si multaneous determination of total nitrogen and total phosphorus in water using pe roxodisulfate oxidation. Water Resources 17:1721-1726

. Federal Register. 2007. Guidelines establishing test procedures for the analysis of pollutants under the Clean Water Act. 40 CFR Parts 136, 141, and 143. United States Environmental Protection Agency. Federal Register 72(47), March 12, 2007.

Fishman, M.J., and Friedman, L.C. 1989. Method s for the determination of inorganic substances in water and fluvial sediments. Chapter A1 in Techniques of Water-Resources Investigations of the U.S. Geological Survey , Book 5. Denver, CO: United States Geological Survey.

Maher, W., Krikowa, F., Wruck, D., Louie, H., Nguy en, T., and Huang, W.Y. 2002. Determination of total phosphorus and nitrogen in turbid waters by oxidation with alkaline potassium peroxodisulfate and low pressure microwave digestion, autoclave hea ting or the use of closed vessels in a hot water bath: Comparison with Kjeldahl digestion. Analytica Chimica Acta 463:283-293.

United States Environmental Protection Agency (USEPA). 1974. Methods for the chemical analysis of water and wastes; Phosphorus, total (colorimetric, automated, block digester AA II)

. EPA/600/4-79/020. United States Environmental Protection Agency. http://www.epa.gov/waterscience/methods/method/files/365_4.pdf.

United States Environmental Protection Agency (USEPA). 1978. Phosphorus, all forms (colorimetric, ascorbic acid, two reagent) . United States Environmental Protection Agency, Environmental Monitoring Systems Laboratory. http://www.epa. gov/waterscience/methods/method/files/365_3.pdf.

United States Environmental Protection Agency (USEPA). 1993a. Determination of total Kjeldahl nitrogen by semi-automated colorimetry , Rev. 2. United States Environmental Protection Agency, Environmental Monitoring Systems Laboratory. http://www.epa.gov/waterscience/methods/method/files/351_2.pdf.

United States Environmental Protection Agency (USEPA). 1993b. Determination of nitrate-nitrite nitrogen by automated colorimetry , Rev. 2. United States Environmental Protection Agency, Environmental Monitoring Systems Laboratory. http://www.epa.gov/waterscience/methods/method/files/353_2.pdf.

United States Environmental Protection Agency (USEPA). 1993c. Determination of phosphorus by semi-automated colorimetry , Rev. 2. United States Environmental Protection Agency, Environmental Monitoring Systems Laboratory. http://www.epa. gov/waterscience/methods/method/files/365_1.pdf.

United States Environmental Protection Agency (USEPA). 1999. Protocol for approval of alternative test procedures for organic and inorganic analytes in wastewater and drinking water. Washington, DC: United States Environmental Protection Agency, Office of Water, Engineering and Analysis.

United States Environmental Protection Agency (USEPA). 2007. 40 CFR Parts 122, 136, et al. Guidelines establishing test proce dures for the analysis of pollutants under the Clean Water Act. Federal Register 72(47), March 12. Rules and Regulations.

United States Geological Survey (USGS). 2003. Method I-4650-3: Evaluation of alkaline persulfate digestion as an alternative to Kjeldahl digestion for determination of total and dissolved nitrogen and phosphorus in water . Patton, C.J., and Kryskalla, J.R. Water Resources Investigations Report 03-4174. United States Geological Survey National Water Quality Laboratory.