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 ALTERNATIVE 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 Implementation ............................................................................................................. 14 

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

3.5 Results, Data Analysis, and Discussions ................................................................................. 17 

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

4.0 NCASI METHOD 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, Preservation, 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, Flowcharts, and Validation Data ............................................................... 34 

REFERENCES ........................................................................................................................................... 36
TABLES 

Table 2.1. Method Comparison for Total Nitrogen by EPA Method 351.2 vs. NCASI Method 

TNTP-W10900 .......................................................................................................................... 4 

Table 2.2. Method Comparison for Total Phosphorus 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 Testing Procedure Study Plan, NCASI Method 

TNTP-W10900 ........................................................................................................................ 15 

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

Table 3.4. Determinative Methods and Equipment 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 Precision and Recovery Results ............................................................................... 18 

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

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

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

Table 3.12. Accuracy Expressed as Percent Recovery 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 Check Initial Precision and Recovery ......................................... 36 

Table 5. Digested Sample Holding Time Study .................................................................................... 36 
NCASI TNTP-W10900 as an Alternative 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 nitrogen and phosphorus levels in biologically treated 

wastewater in part using the Kjeldahl digestion method set forth in EPA Methods 351.2 (USEPA 1993a) 

and 365.4 (USEPA 1974). NCASI proposes that Kjeldahl 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 digestion reduces labor and hazardous waste costs and 

enhances safety. These benefits are detailed herein. 

1. Lowers detection levels and improves precision and accuracy – Nitrogen and phosphorus 

concentrations in pulp and paper effluent are typically 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 promulgated over three decades ago the industry has been 

reducing nutrient loads in watersheds, in particular where total daily maximum loads (TMDLs) are 

being implemented. Single-laboratory and Tier 2 evaluations bear out the advantages of the proposed 

digestion in terms of lowered detection limits, greater 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 modified 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 acidic 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 immediately 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 preparations (nitrite/nitrate and total Kjeldahl 

nitrogen (TKN)) followed by three individual nitrogen determinations (nitrite, nitrate, and ammonia). 

In contrast, the proposed alternative test procedure (ATP) uses a single sample preparation followed 

by a single determination to derive TN. Total phosphorus (TP) is concurrently developed during 

sample preparation and is individually analyzed. Furthermore, Kjeldahl digestion times are on the 

order of two to three hours, whereas the modified process 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, NCASI is seeking EPA approval of TNTP-W10900 for 

monitoring of pulp, paper, and paperboard biologically treated wastewater (PP-BTWW) at the Tier 2 

level. The experimentally-based justification of the proposed method changes can be found in Section 3. 

2.0 METHOD COMPARISON INFORMATION 

EPA Method 351.2 is approved for simultaneous digestion of both nitrogen and phosphorus using the 

Kjeldahl method. However, EPA 351.2 provides quality metrics and analysis instructions only for 

nitrogen and thus necessitates that EPA Method 365.4 be used to address phosphorus analysis after the 

Kjeldahl digestion is completed. The substantial 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 factors contributing to these differences. A point by 
NCASI TNTP-W10900 as an Alternative 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 format. 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 methods 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% K2S2O8 in 0.12 M NaOH solution to 

simultaneously digest TN and TP. This transformation does not materially alter the analytical scope of 

the approved method. To wit: the approved reference 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 proportional to the nitrogen concentration in the digested 

samples. Both the modified and reference methods convert phosphorus forms to orthophosphatephosphorus. Orthophosphate-phosphorus is determined colorimetrically by formation of a molybdenum 

blue complex. The equivalencies of the nitrogen and phosphorus analytes produced by the modified 

process were demonstrated in both single-laboratory 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 fraction of its original volume. Samples are reconstituted 

with water to their initial quantity before commencing 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-bound nitrogen and phosphorus for different matrices. 

The rationale, frequency, and instructions for these 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 and 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 identification of nitrate during analysis. 

Both reference methods include comprehensive lists of analytical reagents. These methods also bestow 

equipment diagrams and setup instructions for colorimetric determination of final products by means of 

Technicon fluid injected auto-analyzers. However, 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 intentionally 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 and coupling with N-(1-napthyl)ethylenediamine 

dihydrochloride (NED) to form a highly colored azo dye. This automated colorimetric procedure is the 

same as performed in EPA Method 353.2 (USEPA 1993b). The corresponding manual method is 
NCASI TNTP-W10900 as an Alternative to EPA 351.2 and 365.4 3 

recounted in Standard Method 4500-NO3

-

 -E (APHA 2005). Analysis of phosphorus in both EPA 365.4 

and TPTN-W10900 is performed by forming an antimony-phospho-molybdate complex in an acidic 

medium, which is reduced to an intensely blue colored 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). Similar methods published by other 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 recommends automated methods 

for TN and TP determinations due to the low volume (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 used in the digestion solution. Chloride at 

>10,000 mg/L can interfere with colorimetric analyses of both nitrate and orthophosphate, but this 

situation is highly unlikely to occur in final effluents. 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 improbable 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 waste 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 metal-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 neutralized and can immediately be disposed of using nonspecialized means. The higher volumes of acidic wastes 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 available 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 the same for both reference and modified methods 

(Federal Register 2007). The acid preservation threshold is designated “pH < 2” in the reference methods 

but constrained to “1.6 < pH < 2” in the modified method. 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 (≤6o

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 digestion 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 digestion. 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 by EPA Method 351.2 vs. NCASI Method TNTP-W

 EPA Method 351.2a NCASI Method TNT

1.0 Scope and 

Application 

Covers TKN determination; applicable range 

0.1-20 mg N/L, may be extended with sample dilution 

Similar aspects; covers TN determ

range 0.05-6.0 mg N/L, may bdilution 

2.0 Summary of Method Digested samples may also be used for phosphorus 

determination 

Stated objective is simultaneous d

TN and TP determination 

 Sample size 25 mL Sample size is 7 mL  Sample is heated in presence of sulfuric acid for 2.5 hr; 

residue is cooled, diluted to 25 mL, and analyzed for ammonia-nitrogen by colorimetry 

Sample is heated at 121oC and 15 

sodium hydroxide and potassiu30 min; product is analyzed focolorimetry 

3.0 Definitions Common terms are defined Similar terms, although more spe Defines general quality control samples (QCS) Discretely defines all QC solution

calibration verification (CCV) 

 Defines MSDS, calibration blank (CB), linear calibration 

range (LCR) 

Does not define ubiquitous terms 

 Defines instrument performance check (IPC) solutions Does not define equipment-speci4.0 Interferences High nitrate concentrations may result in low TKN 

values 

Chloride >10,000 mg/L may depl

in active chlorine species that reactions used to determine nit

 High organic carbon concentratio

persulfate required to oxidize n

 Over-acidification during preserv

recovery of TN 

5.0 Safety Standard disclaimers with specific warnings for three 

potentially highly toxic and hazardous chemicals: mercury, concentrated sulfuric acid, and sodium nitroprusside (used in ammonia analysis) 

Standard disclaimer content is sim

to one reagent, potassium persoxidizer 
Table 2.1. Method Comparison for Total Nitrogen by EPA Method 351.2 vs. NCASI Method TNTP-W

 EPA Method 351.2a NCASI Method TNT

6.0 Equipment and 

Supplies 

Equipment to perform block digestion followed by 

analysis using generic flow injection analysis (FIA) system including peripheral devices such as sampler, manifold, detector, and data recorder 

Block digester is replaced by pre

autoclave; analyst is instructed nitrate-nitrite determinative mcorresponding auto-analyzer minstructions 

7.0 Reagents and 

Standards 

Reagent water conforming to ASTM Type II or 

equivalent 

Requires ASTM Type II or ultrap

as that produced by commerci

 A 10% sulfuric acid solution is p

calibrants and QC elements pr

 Digestion solution is made by dissolving K2SO4 in 20% 

sulfuric acid, this is combined with mercuric sulfate solution made from mercuric oxide (HgO) in 20% sulfuric acid; copper sulfate is offered as a substitute for HgO 

Digestion solution is made by dis

0.12 M NaOH 

 Analysis reagents are defined for colorimetric 

determination of ammonia: sulfuric acid (4%), sodium hydroxide (20%), sodium potassium tartrate (20%), buffer (sodium phosphate+sodium hydroxide), sodium salicylate, sodium nitroprusside, and sodium hypochlorite solutions 

No analysis reagents or recipes ar

analyst is instructed to refer to determinative methods and coranalyzer manufacturer’s instru

 Standard solutions are made from ammonium chloride 

(NH4Cl) 

Separate NH4Cl and KH2PO4 prim

working stocks contain both pr

 Includes nitrate-nitrogen 100 mg 

potassium nitrate for use as a lblank (LFB) for qualitative con

 Includes organic nitrogen and ph

used as digestion efficiency ch

8.0 Sample Collection, 

Preservation, and Storage 

Preservation at pH <2; samples maintained at 4oC and 

held for up to 28 days 

Preservation at 1.6 < pH < 2; sam

assigns 28 day holding time fodigestates 
Table 2.1. Method Comparison for Total Nitrogen by EPA Method 351.2 vs. NCASI Method TNTP-W

 EPA Method 351.2a NCASI Method TNT

9.0 Quality Control Describes formal QC program, requisite laboratory 

performance measures, and frequency 

Same requirements, wording is m

 Rationale, frequency, and assessm

specified for digestion efficienchecks are assessed against est

 Instrument performance verified prior to performing 

analysis 

Instrument performance verified 

manufacturer’s guidelines 

 Analytical performance must be v

QC requirements of determina

 LCR must be determined initially and verified every 

6 months or when a change in instrument response is observed or expected 

Calibration occurs with every dig

 Linearity must be established at ±10% using minimum of 

a blank and three standards 

Linear coefficient of determinatio

used in lieu of ±10% linearity 

 Describes QCS in broad terms to be analyzed when 

beginning use of method, on a quarterly basis, or as required to meet data quality needs 

Same intent as reference, but term

specific QCS sample sub-type

 At least one LFB must be analyzed with each sample 

batch; recovery must fall within 90-110% 

More detailed descriptions of typ

method performance QC sampnitrate-spiked LFB, and digest

 Digestion efficiency check soluti The method detection limit (MDL) is established at 2-3 

times estimated instrument detection limit in fortified reagent water 

MDL established using 40 CFR 1

reagent grade water, which all1-5 times estimated instrumen

 At least one laboratory reagent blank must be analyzed 

with each batch; value must be less than the MDL or corrective action ensues 

MBs are run: 1 for every 20 sam

sample batch, whichever is moanalyte levels must be less tha

 Laboratory performance assessed using LFB recovery 

performance; data are used to establish ongoing precision statements and control limits 

Same objectives are met using CC
Table 2.1. Method Comparison for Total Nitrogen by EPA Method 351.2 vs. NCASI Method TNTP-W

 EPA Method 351.2a NCASI Method TNT

9.0 (continued) Utilizes laboratory fortified sample matrix (LFM) to 

assess accuracy via analyte recovery 

Precision based on RPD% of sam

duplicate (DUP) and matrix spduplicate (MS/MSD) sets take10% of routine samples 

 Utilizes MS to assess method acc

analyte recovery 

 Reference materials should be analyzed when available Independent calibration verificati

required 

10.0 Calibration and 

Standardization 

Manifold and flow system follow Figure 1 and Table 2 

of method 

Analysis follows approved determ

equipment manufacturer’s inst

 Standards analyzed in order of decreasing concentration Order of standards is not addresse

by equipment vendor) 

 Calibration standards prepared covering desired range Suggested calibration standard le

to Section 17.0 Table 1 of met

 Calibration curve prepared by plotting instrument 

response against concentration; curve fitting may be achieved using computer or calculator based on regression techniques 

Slight difference in wording 

 Calibration periodically verified during analysis Requirement addressed in Section 11.0 Procedure Add 25.0 mL of sample, standard, or blank to digester 

tubes 

Add 7.00 ±0.05 mL of sample, st

15 mL screw-capped culture tu

 Add 5 mL of digestion solution to each tube and vortex Add 3.00 ±0.05 mL of digestion 

cap and mix gently 

 Add boiling chips to tubes and place in block digester 

preheated to 160oC; maintain temperature for 1 hr 

Place tubes into pressure cooker o

heating unit; attain final setting 

relative positive pressure; hold 

 Reset temperature to 380oC, continue to heat for 11⁄2 hr; 

maintain 380oC for at least 30 min during session 

 Cool; dilute to 25 mL with reagent water Cool samples; store tightly cappe
Table 2.1. Method Comparison for Total Nitrogen by EPA Method 351.2 vs. NCASI Method TNTP-W

 EPA Method 351.2a NCASI Method TNT

11.0 (continued) Sections 11.8-11.11 describe reagent manifold 

connections, trouble-shooting, and general operation of a Technicon automated continuous flow system 

Analyze all samples according to 

nitrate analysis and specific eqinstructions 

12.0 Data Analysis and 

Calculations 

Instructs analyst to prepare a calibration curve and 

compute concentrations by comparison; accounts for dilution factors 

Content is similar to reference; ad

provided for reporting QC and review 

 Adds that curve fitting may be ac

or calculator-based regression 

13.0 Method Performance Reports multi-laboratory precision and accuracy from 

varied spike levels in reagent water matrix 

Multi-laboratory Tier 2 study res

accuracy in reagent water and Section 17.0 of method (Sectio

 Single-laboratory accuracy was assessed using two 

sewage samples with concentrations of 4.7 and 8.74 mg N/L, recoveries of 99% and 99%, respectively 

Single-laboratory precision and a

(from NH4Cl) in PP-BTWW; a

98.3%, RPD 11.6% 

 Precision gauged by analyzing three sewage samples at 

concentrations of 1.2, 2.6, and 1.7 mg N/L; precision ±0.07, ±0.03, and ±0.15 mg N/L, respectively 

14.0 Pollution Prevention Asserts pollution prevention as foremost goal; advises 

minimum quantity of chemicals and prepared reagents be purchased 

No special pollution prevention o

requirements exist for material

15.0 Waste Management Wording emphasizes laboratory responsibility to comply 

with hazardous waste disposal regulations 

Omitted; hazardous waste not gen

 Instructions to neutralize all solut

process and to comply with all ordinances 

16.0 References 6 references dated between 1967 and 1978 15 references; other than referen

are dated earlier than 1983 
Table 2.1. Method Comparison for Total Nitrogen by EPA Method 351.2 vs. NCASI Method TNTP-W

 EPA Method 351.2a NCASI Method TNT

17.0 Tables and Figures Table 1. Inter-laboratory Precision and Accuracy; 

provides means, residuals, and SD for multiple analyses performed using reagent water matrix 

Table 1. Calibration Standard Sol

tabulates combined working streagent water for suggested ca

 Table 2. Concentration Ranges; pump parameters and 

buffer volumes during analysis 

Table 2. Single and multi- labora

and Recovery; results for TN awater 

 Table 3. Single and multi- labora

Matrix Spike Duplicate RecovTP MS/MSD in PP-BTWW 

 Table 4. Single and multi- labora

Digestion Check Initial Precisiresults for organic analyte IPR 

 Table 5. Single Laboratory Diges

Time Study; results for TN MS

 Figure 1. Ammonia Manifold; line diagram illustrating 

flow scheme and basic components of auto-analyzer 

No figures included 

a Determination of Total Kjeldahl Nitrogen by Semi-Automated Colorimetry Rev. 2.0 August 1993. United States Environmental Pr

Environmental Monitoring Systems Laboratory; http://www.epa.gov/waterscience/methods/method/files/351_2.pdf.

b Total Nitrogen and Total Phosphorus in Pulp and Paper Biologically Treated Effluent by Alkaline Persulfate Digestion, June 2011

Air and Stream Improvement, Inc. 
Table 2.2. Method Comparison for Total Phosphorus by EPA Method 365.4 vs. NCASI Method TNT

 EPA Method 365.4a NCASI Method TNT

1.0 Scope and 

Application 

Covers TP determination; applicable range 

0.01-20 mg P/L 

Similar wording; applicable rang

be extended with sample diluti

2.0 Summary of Method Sample is heated in the presence of sulfuric acid, K2SO4

and HgSO4 for two and on half hours 

Sample is heated at 121oC and 15 

sodium hydroxide and potassiu30 min 

 The sample is cooled and diluted to 25 mL then placed 

on the autoanalyzer for phosphorus determination 

Sample size is 7 mL; product is a

orthophosphate 

3.0 Definitions NA; no section titled definitions, no definitions listed 

elsewhere 

4.0 Interferences NA; no section titled interferences, no interferences 

listed elsewhere 

 High levels of suspended sedime

reporting of phosphorus 

 Calcium, iron, barium, lead, and 

insoluble phosphates 

 Arsenate (AsO43-) forms analogou

orthophosphate above 1.0 mg 

at levels >2.0 mg As/L may be 

5.0 Safety NA; no section titled safety, no safety items listed 

elsewhere 

Common disclaimers are given; a

reagent, potassium persulfate, 

6.0 Equipment and 

Supplies 

Equipment is Section 4.0, Apparatus; Technicon block 

digester, Technicon AA11 auto-analyzer with phosphorus manifold 

Analyst is instructed to refer to ap

determinative methods and corequipment manufacturer’s inst

7.0 Reagents and 

Standards 

Reagents listed in Section 5.0; no standards specified 

 Solutions made up in distilled water Requires ASTM Type II or ultrap

as that produced by commerci
Table 2.2. Method Comparison for Total Phosphorus by EPA Method 365.4 vs. NCASI Method TNT

 EPA Method 365.4a NCASI Method TNT

7.0 (continued) Analysis reagents defined 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-analyzer manifold 

No analysis reagents listed; analy

manufacturer’s instructions aninstrument in forming phospho

8.0 Sample Collection, 

Preservation, and Storage 

Sample handling and preservation are listed in 

Section 3.0; sample should be preserved by the addition of 2 mL of concentrated sulfuric acid per liter of sample then refrigerated at 4oC; written method 

does not include holding times 

Preservation at 1.6 < pH < 2; sam

assigns 28 day holding time fodigestates

9.0 Quality Control NA; no section titled quality control, no QC 

requirements listed elsewhere 

Contains formal and detailed pro

analytical performance, demonproficiency, and QC frequencyinstructed to establish QA progcriteria and remedial actions ar

 Laboratory must demonstrate me

successful execution of IPR anthat meet or exceed tolerance iduring multi-laboratory valida

10.0 Calibration and 

Standardization 

NA; no section entitled “Calibration and 

Standardization” 

Calibration follows approved me

manufacturer’s instructions 

 Suggested calibration standard le

to Table 1, Section 17.0, of me

 Calibration curve is prepared by 

response against concentrationachieved using computer or caregression techniques 

11.0 Procedure NA; procedures are listed in Section 6.0  Add 20.0-25.0 mL of sample, standard, or blank to a 

digester tube 

Add 7.00 ±0.05 mL of sample, st

15 mL screw-capped culture tu
Table 2.2. Method Comparison for Total Phosphorus by EPA Method 365.4 vs. NCASI Method TNT

 EPA Method 365.4a NCASI Method TNT

11.0 (continued) Add 5 mL of digestion solution to each tube and vortex Add 3.00 ±0.05 mL of digestion 

cap and mix gently 

 Add Teflon boiling chips to tubes and place in block 

digester preheated to 160oC; maintain for 1 hr; reset 

temperature to 380oC and maintain for 21⁄2 hours 

Place tubes into pressure cooker o

heating unit; attain final setting 

relative positive pressure; hold 

 Cool; dilute to 25 mL with distilled water; use ammoniafree water if TKN is determined Cool samples; store tightly cappe

 Sections 6.4.1-6.4.5 detail connection and sequencing of 

reagent lines, pump operation and conditioning, and instrument qualification for a Technicon AA11 

Analyst is instructed to refer to ap

methods and corresponding inanalyzer manufacturer’s instru

12.0 Data Analysis and 

Calculations 

NA; no section entitled “Data Analysis and 

Calculations”; See Section 10.0 of this table. 

Instructions are provided for repo

guidelines for data review 

 Section 7.0, entitled “Calculations” instructs analyst to 

prepare standard curve by plotting peak heights of process standards against concentration values 

Equipment specific terms such as 

are excluded 

13.0 Method Performance NA; there is no section entitled “Method Performance”; 

Section 8.0 reports single-laboratory precision and accuracy 

 Single-laboratory accuracy by assessment of two sewage 

samples with concentrations of 1.84 and 1.89 mg P/L; recoveries of 95% and 98%, respectively 

Single-laboratory accuracy and p

(from KH2PO4) in PP-BTWW 

(Section 4); average recovery 

 Single-laboratory precision by analysis of three 

concentration 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 given 

 Multi-laboratory Tier 2 study res

accuracy in reagent water and included in Section 17.0 (Secti
Table 2.2. Method Comparison for Total Phosphorus by EPA Method 365.4 vs. NCASI Method TNT

 EPA Method 365.4a NCASI Method TNT

14.0 Pollution Prevention NA; there is no section 14.0 and no section entitled 

“Pollution Prevention”; no pollution prevention items are listed elsewhere 

No special pollution prevention o

requirements exist for the mate

15.0 Waste Management NA; there is no Section 15.0 and no section entitled 

Waste Management, no waste management topics are listed elsewhere 

Instructs users to neutralize all so

process and to comply with all ordinances 

16.0 References NA; there is no Section 16.0; “Bibliography” is found 

after Section 8.0; references are dated 1967 to 1974 

There are 15 references; no citat

than 1983 

17.0 Tables and Figures NA; there is no Section 17.0; Figure 1 follows the 

“Bibliography” section 

No figures are included 

 Figure 1, Phosphorus Manifold Technicon AA11; line 

diagram illustrating input and output scheme of manifold and basic components of analyzer 

 No tables included Table 1. Calibration Standard Sol

tabulates combined working streagent water for suggested ca

 Table 2. Single and multi- labora

and Recovery; results for TN awater 

 Table 3. Single and multi- labora

Matrix Spike Duplicate RecovTP MS/MSD in PP-BTWW 

 Table 4. Single and multi- labora

Digestion Check Initial Precisiresults for organic analyte IPR 

 Table 5. Single Laboratory Diges

Time Study; results for TN MS

a Phosphorus, Total (Colorimetric, Automated, Block Digester AA II) 1974. United States Environmental Protection Agency, Enviro

Systems Laboratory; http://www.epa.gov/waterscience/methods/method/files/365_4.pdf.

b Total Nitrogen and Total Phosphorus in Pulp and Paper Biologically Treated Effluent by Alkaline Persulfate Digestion, June 2011

Air and Stream Improvement, Inc. 
NCASI TNTP-W10900 as an Alternative 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, descriptions 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 reference methods are primarily limited to digestion of 

samples whereby nitrogen forms are converted to nitrate-nitrogen and phosphorus forms are converted to 

orthophosphate-phosphorus. TN and TP are then determined colorimetrically 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 surface 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 Codea

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 and 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 methods in all aspects of performance-based testing. 

3.2 Study Design and Objective 

The objective of this study was to provide data 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 was 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 selected based on their experience with the reference 

EPA methods, previous work with pulp and paper industry matrices, and 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 field 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 containers as furnish. Its effluent comprises the composite outflow of 

two parallel aerated stabilization basins (ASBs). Supplemental nitrogen 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 pipeline 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 by 
NCASI TNTP-W10900 as an Alternative to EPA 351.2 and 365.4 15 

aerated stabilization basin (ASB) secondary treatment. The second mill produces tissue and toweling 

from deinked office wastepaper and purchased kraft pulp. It also produces de-inked pulp. Effluent from 

this mill undergoes primary clarification followed by secondary treatment in an activated sludge process. 

The outflow from both mills is combined and piped underground 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 collected 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 ±2o

C) via overnight 

service to Laboratory III. During the ensuing week Lab 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 order to prepare several dilution sets needed to facilitate offsite analysis. 

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

Sample ID 

Number Field Sample Descriptiona

 Analysis Requirements 

23064 Site A – PPWW 

Biologically treated effluent 

Background levels TN, TP 

MS/MSD dual spike TN, TP 

23065 Site B – PPWW and RCYb

Biologically treated effluent 

Background levels TN, TP 

MS/MSD dual spike TN, TP 

23066 Misc-RWc

 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 oversight Lab III subjected only Site A to the matrix 

spike/matrix spike duplicate (MS/MSD) acceptance testing 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, Lab III erroneously used organic nitrogen and organic 

phosphorus spiking stocks in executing the initial precision and recovery (IPR) validation. The analysis 

was necessarily repeated to conform to the study 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 validations are presented in Section 17.0, Tables 2 

through 5 of TNTP-W10900. 

The ATP study plan is summarized in Table 3.3. Each 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 sample 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 autoclave. All digestions were conducted at 120 to 121°C 
NCASI TNTP-W10900 as an Alternative to EPA 351.2 and 365.4 16 

and 15 to 16 psi relative positive pressure. Each participating 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 laboratories used automated spectroscopic equipment for TN 

determinations, and automated spectroscopic equipment was used for TP quantification by two 

laboratories. Lab III was limited to manual spectroscopic 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 methods are slightly different (USEPA 1999); however, 

the more constrained automated acceptance criterion 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 Ab

 – background + MS/MSD pair 3 6 MS/MSDc 

Sample set Site Bb

 – 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 designated 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 Nitrogen Total Phosphorus 

Lab Instrument Method Title EPAa Method Title EPA 

Lab I B & L – AA3 HRb G-309-04 353.2 e G-146-95 365.1f

Lab II Lachat 8500 QuickChemc 10-107-04-1-C 353.2 10-115-01-4-B 365.1

Lab III B & L – AA CE 6.02d NO2+NO3 353.2 – –

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

a

 USEPA equivalent methods 

b

 Bran & Luebbe Model Auto-Analyzer 3, segmented flow analyzer c

 Lachat Model 8500, fluid injection analyzer 

d

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

e

 EPA Method 353.2, Nitrogen, Nitrate-Nitrite (Colorimetric, Automated, Cadmium Reduction) f

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

 EPA Method 365.3, Phosphorus, All Forms (Colorimetric, Ascorbic Acid, Two Reagent)

3.4 Data Reporting and Validation 

Complete data packets were prepared by each participant and audited by NCASI. The data include all the 

information listed in Table 3.5. Each participating laboratory submitted copies of all records 

demonstrating how standards were prepared and samples were tracked through the process. Digestion 
NCASI TNTP-W10900 as an Alternative 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 identifiers used by laboratory and sample supplier 

Laboratory notebook information Sample preparation dates, sample volumes, dilution information 

Instrument information Dates of analyses, run logs, instrument and operating conditions, 

programs used, operating conditions 

Analysis information Chromatograms, quantitation 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. Linearity 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 (r2

) 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 listed 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 all participating laboratories. A summary 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 recovery of ±10% of the theoretical value then had to 

be confirmed. Each laboratory met the QA/QC acceptance 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 Alternative to EPA 351.2 and 365.4 18 

Table 3.6. Calibration Curve Data Summary 

Analytea

: Total Nitrogen Total Phosphorus 

Laboratory 

Code 

Calibration 

Pointsb

MLc

(mg N/L) r2 d

Calibration 

Pointsb

MLc

(mg N/L) r2 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 standards ammonium chloride (NH4Cl) for TN and potassium dihydrogen 

orthophosphate (KH2PO4) 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 0.04a 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 additional laboratory fortified blank (LFB) sample 

consisting of 1.00 mg N/L (N-NO3

-

) 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 ATP protocol for EPA 351.2 and 365.4. 

Table 3.8. Ongoing Precision and Recovery Results 

Analytea

: Total Nitrogen Total Phosphorus Total Nitrogen (nitrate) 

Recovery and 

Precision n Resultsb

Acceptance 

Criteriac Resultsb

Acceptance 

Criteriac n Resultsb

Acceptance 

Criteriac

Recovery range (%)d 10 94 - 98.7 77 - 121 96.0 - 104 79 - 113 3 92.4 - 107 87 - 111 a

 determined using combined standards ammonium chloride (NH4Cl) for TN and potassium dihydrogen 

orthophosphate (KH2PO4) for TP in reagent water; TN (nitrate) determined from potassium nitrate (KNO3) in 

reagent water 

b

 data from multi-laboratory validation of TNTP-W10900 from 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 from 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 standard deviation of the percent recovery. Initial precision and accuracy 
NCASI TNTP-W10900 as an Alternative 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 

Analytea

 : Total Nitrogen Total Phosphorus 

Recovery and 

Precision n Resultsb

Acceptance 

Criteriac Resultsb

Acceptance 

Criteriac

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 standards ammonium chloride (NH4Cl) for TN and potassium dihydrogen 

orthophosphate (KH2PO4) 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 study 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 relevant. 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 for 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 determined for the reference methods approached the 

ML for nitrite/nitrate (0.05 mg N/L) detection using 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 

Analytea

: 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 standards ammonium chloride (NH4Cl) for TN and potassium dihydrogen 

orthophosphate (KH2PO4) for TP in reagent water 

3.5.5 Sample Results

Each laboratory was required to analyze two effluents and a receiving water sample to determine native 

levels of the target analytes. Each participating laboratory was at liberty to dilute samples either before or 

after digestion as required to be within the range of the calibration curve. Background levels determined 
NCASI TNTP-W10900 as an Alternative to EPA 351.2 and 365.4 20 

by each laboratory are provided in Table 3.11. These results validate that native levels were within 

method parameters. There are no QA/QC requirements for this measurement. 

Table 3.11. Sample Background Level Results 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 5.97a

 0.17 0.44 1.25a 0.04 a single sample; all other tabled values are the average 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 method in a matrix, effluent from Site A was spiked 

with the target analytes (MS) then digested and analyzed 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/MSD 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 Site B effluent were analyzed by Lab I and Lab II 

and demonstrated similar recovery and precision conformity 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 concentrations. 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 

Analytea

: Total Nitrogen Total Phosphorus 

Recovery and Precision n Resultsb

Acceptance 

Criteriac Resultsb

Acceptance 

Criteriac

Recovery range (%) 6 84.4 - 96.5 77 - 121 97.0 - 105 79 - 113 

RPD ranged (%) 0.2 - 4 22 0.0 - 4.2 17 a

 determined using combined standards ammonium chloride (NH4Cl) for TN and potassium dihydrogen 

orthophosphate (KH2PO4) 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 using the approved reference methods, EPA 351.2 and 365.4 

during a multi-laboratory study involving three participating laboratories. TNTP-W10900 is more cost 

effective and environmentally friendly than the Kjeldahl process for the subject matrix, while improving 

detection, reducing environmental impacts, increasing productivity, and subsequently reducing costs. 
NCASI TNTP-W10900 as an Alternative 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 – ongoing recovery for total nitrogen in reagent grade water ranged 

from 94 to 98.7% across all laboratories; ongoing recovery for total phosphorus ranged from 96.0 to 

104 % across all laboratories 

• Initial precision and recovery – accuracy for total nitrogen expressed as recovery ranged from 94.4 to 

105% for all laboratories, precision expressed as the standard 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 expressed 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-W10900 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 Alternative to EPA 351.2 and 365.4 22 

4.0 NCASI METHOD TNTP-W10900 

TOTAL NITROGEN AND TOTAL PHOSPHORUS IN 

PULP AND PAPER BOLOGICALLY 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 Leader, and Jan Napack, Research 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 Improvement, Inc. (NCASI). 2011. NCASI Methods Manual. 

Method TNTP-W10900: Total nitrogen and total phosphorus in pulp and paper biologically treated 

effluent by alkaline persulfate digestion. Research 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 excellence 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 Alternative 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 paperboard 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 digestion efficiency and method performance. Any 

modification of the method beyond those expressly 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 ammonia and inorganic (excluding N2) and organic nitrogen 

to nitrate and inorganic and organic forms of phosphorus to orthophosphate by means of an 

alkaline to acid digestion. Native dissolved nitrate and nitrite are unaffected by the digestion. 

2.2 Digestion is accomplished by heating acidified, unfiltered samples in the presence of persulfate (a 

strong oxidizer) at 120o

C and 15 psi positive pressure for 30 minutes. The digestion is strongly 

alkaline at the outset, which promotes oxidation of nitrogen to nitrate. As the persulfate 

decomposes the solution becomes acidic, allowing oxidation of phosphorus to orthophosphate 

(Section 16.2). Following digestion, separate semi-automated or manual colorimetric 

determinative methods of the single digestate are conducted for analysis of TN and TP. 

2.3 TN measurement occurs by means of nitrite/nitrate 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 N2) and organic 

forms of nitrogen. Organic nitrogen may be comprised 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 biological, synthetic, or natural phosphorus-containing 

compounds. 
NCASI TNTP-W10900 as an Alternative to EPA 351.2 and 365.4 24 

3.4 Calibration standards are made to encompass the applicable 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 calibration is applicable throughout the sample run. 

Calibration verifications are required after every tenth 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 are purchased with certifications of purity, 

concentration, and authenticity sourced from certified NIST-traceable references. Two separate 

ICV solutions, one for each target analyte, are mixed 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 reagent 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. Sample 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 recovery (%R) and is calculated using Equation 4 

(Section 9.4.6). 

3.11 A matrix spike duplicate (MSD) is prepared using 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 reagent water or sample to which a known quantity of 

organic method analyte has been added in the laboratory. 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 solutions can be added to reagent water (LFB) or 

samples (MS). 

4.0 Interferences 

4.1 Persulfate digestion for nitrogen 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 and 
NCASI TNTP-W10900 as an Alternative to EPA 351.2 and 365.4 25 

use of narrow-range pH indicator strips at the time 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 consequence (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 suspended 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, and silver in the presence of sufficiently high 

concentrations of phosphorus can interfere by forming phosphate precipitates that may lead to 

under-reporting of TP (Section 16.3). 

4.6 Arsenate [AsO4

3-] forms a color similar to that of phosphate above 1.0 mg As/L and may produce 

a positive interference. Arsenite [AsO3

2-] at levels >2.0 mg As/L may be oxidized to arsenate 

(Section 16.3). 

4.7 Method interferences may be caused by contaminants 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 analyses by running method blanks as described in 

Section 3.8. 

5.0 Safety 

5.1 Potassium persulfate must be kept separate from flammable and easily oxidizable substances such 

as combustibles and many solvents. It is harmful 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-resistant gloves, and protective clothing are strongly recommended 

when using this compound. 

5.2 This method does not address all safety issues associated with its use. The laboratory is 

responsible for maintaining a safe work environment and a current awareness of OSHA 

regulations regarding safe handling of chemicals used in this method. Material safety data sheets 

(MSDS) should be available to all personnel involved 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 for 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 capable of developing a steady, relative 

positive pressure of 15 psi. If using a pressure 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 Glassware 
NCASI TNTP-W10900 as an Alternative 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 cemented-in PTFE-faced 14B rubber liners 

6.3.4 Glassware should be washed with hot tap 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 cleaning 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-NO3

-

 (E and F) (Section 16.8), and USGS I-4545-85 (Section 16.9). 

6.5.1.2 Approved methods for orthophosphorus 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 

(H2SO4, 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 

(K2S2O8, 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 (Section 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 (NH4Cl, 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 (KH2PO4, CAS 7778-77-0, FW 136.09) to about 
NCASI TNTP-W10900 as an Alternative 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 – Dilute 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 combined solution to reduce volumetric errors. Use 

this solution for making up the calibration curve (Section 7.3). 

7.2.6 ICV combined nitrogen/phosphorus stock 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 

(KNO3, 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), sodium 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 containers that have been acid washed and 

thoroughly rinsed (Section 6.3.4). 

8.2 Preservation 

Samples should be preserved at the time of collection by adding dilute sulfuric acid to attain 

1.6 < pH < 2. Ensure that sample pH is within 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 Alternative to EPA 351.2 and 365.4 28 

8.3 Storage 

Refrigerate samples at 4 ±2o

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 requirements of this program consist of an initial 

demonstration of laboratory capability (Section 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 method (Section 6.5). All QA/QC specifications 

listed in the associated determinative method must be met. 

9.2.2 Calibrations must demonstrate linearity within 10%, as defined by the percent relative 

standard deviation (%RSD) of the response factors (Equation 2). Otherwise, a coefficient 

of determination (r2

) 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 demonstration 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, Table 2). Laboratories should be able to 

achieve IPR values within the ranges stated 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 Measured 
NCASI TNTP-W10900 as an Alternative 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 analytes in this method, the laboratory should 

determine the MDL in accordance with the procedure 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 laboratories 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 samples 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 every sample batch or for every 20 samples, 

whichever is more frequent. If any of the 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 halted until the source of contamination is 

eliminated. Blanks should be analyzed following 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 analyses, 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, reanalyze 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 calibration verification (ICV) 

One ICV standard solution consisting of mid-range concentrations of N and P 

(Section 7.2.6) should be digested with each digestion 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 all ICV measurements should be kept on file 

with sample analysis data. 
NCASI TNTP-W10900 as an Alternative 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 digested 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 that 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-laboratory study (Section 17.0, Table 3) until 

sufficient internal performance data and control limits can be established. 

 Equation 4 

% R = 100 x (Cs – Cn)

 S 

where: 

R = MS or MSD sample recovery 

Cs = MS or MSD sample concentration 

Cn = 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 normal 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 established warning limits, method performance is 

unacceptable for the compound in the matrix. Limits for digestion check solutions using 

2-phenyl glycine (organic nitrogen) and calcium 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 Alternative 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 determinative 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 system is optimized the same operating conditions 

must be used to analyze all samples. Instrument response checks (e.g. carryover) may be 

performed using unacidified, undigested nitrate and 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 using 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 121oC and 15 psi positive pressure. 

All solutions used in a single determinative analysis must be digested in the same batch. The 

instructions herein are written for digestion using 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 digestion 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 (Section 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 for 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 transferring 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 the 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 Alternative 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 cover until the pressure cooker has returned to 

atmospheric pressure and is sufficiently cooled 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 progresses 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, reagent preparation, step succession, and manifold 

proportionalities provided by equipment manufacturers. 

12.1.1 If suspended particles remain in the digestates they must be removed by sedimentation 

and decantation or filtration prior to TN analysis 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 within 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 with 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 concentrations 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 Alternative 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 significant, 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 air, water, and land by minimizing releases into 

the environment. Compliance with all sewage discharge permits and regulations 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 with applicable federal, state, and local 

regulations. 

15.2.2 For further information on waste management, 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 16th 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 inorganic 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. Simultaneous determination of total nitrogen and total 

phosphorus in water using peroxodisulfate 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 phosphorus 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: Comparison 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 Alternative 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, 21st Edition. Methods 4500-P and 4500-NO3

-

. Washington, DC: American Public 

Health Association. 

16.9 Fishman, M.J., and Friedman, L.C. 1989. Methods 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 Volumea

(μL) 

Acid Volumeb

(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 pH 
NCASI TNTP-W10900 as an Alternative to EPA 351.2 and 365.4 35 

Table 2. Initial Precision and Recoverya

 Recovery and Precision (%) 

Study n Averageb Rangec SDd

 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 (NH4Cl) for TN and 1.00 mg P/L 

potassium dihydrogen ortho-phosphate (KH2PO4) for TP in reagent water 

b average of IPR recoveries from four replicates in reagent 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 results 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 Precisiona

 Recovery and Precision (%) 

Study n Averageb Rangecc RPDd

 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 (NH4Cl) for TN and 1.00 mg P/L 

potassium dihydrogen ortho-phosphate (KH2PO4) 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 from 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 deviation 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 Alternative to EPA 351.2 and 365.4 36 

Table 4. Organic Analyte Digestion Check Initial Precision and Recoverya 

 Recovery and Precision (%) 

Studyb n Averagec Ranged SDe

 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 (C8H9NO2) for TN and 1.00 mg P/L 

calcium glycerophosphate (C3H7O6P∙Ca) for TP combined standards in reagent grade water 

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

Regional Laboratory, Corvallis, Oregon, July 23, 2008; Lab B Columbia Analytical Services, Kelso, 

Washington, July 24, 2009 c

 average of IPR recoveries from four replicates in reagent 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 Studya

 Recovery and Precision (%) 

Holding Timeb n Averagec Ranged RPDe

 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 (NH4Cl) for TN and 

1.00 mg P/L potassium dihydrogen ortho-phosphate (KH2PO4) 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)] 

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