Methods of Analysis by the U.S. Geological Survey National Water 

Quality Laboratory—Determination of Ammonium Plus Organic Nitrogen 

by a Kjeldahl Digestion Method and an Automated Photometric Finish 

that Includes Digest Cleanup by Gas Diffusion 

Open-File Report 00–170 

U.S. Department of the Interior 

U.S. Geological Survey 
Methods of Analysis by the U.S. Geological Survey National Water 

Quality Laboratory—Determination of Ammonium Plus Organic Nitrogen 

by a Kjeldahl Digestion Method and an Automated Photometric Finish 

that Includes Digest Cleanup by Gas Diffusion

By Charles J. Patton and Earl P. Truitt 

U.S. Geological Survey 

Open-File Report 00–170 

Denver, Colorado 

2000 
U.S. DEPARTMENT OF THE INTERIOR 

BRUCE BABBITT, Secretary 

U.S. GEOLOGICAL SURVEY 

Charles G. Groat, Director

The use of brand, firm, and trade names in this report is for identification 

purposes only and does not constitute endorsement by the U.S. Government. 

For additional information write to: Copies of this report can be purchased from: 

 

U.S. Geological Survey U.S. Geological Survey 

Chief, National Water Quality Laboratory Branch of Information Services 

Box 25046, Mail Stop 407 Box 25286 

Federal Center Federal Center 

Denver, CO 80225-0286 Denver, CO 80225-0286 
Contents III

CONTENTS 

Abstract 1 

Introduction 2 

Analytical method 3 

1. Application 3 

2. Summary of method 4 

3. Interferences 4 

4. Instrumentation 6 

5. Apparatus 7 

6. Reagents 7 

7. Calibrants 9 

8. Sample preparation 10 

9. Instrument performance 11 

10. Calibration 11 

11. Procedure and data evaluation 11 

12. Calculations 13 

13. Reporting results 14 

14. Detection levels, precision, and accuracy 15 

Discussion of results 17 

Analytical methods 17 

Statistical analysis 24 

Conclusions 29 

References cited 30 

FIGURES 

1. Schematic showing analytical cartridge for colorimetric determination 

of Kjeldahl nitrogen in filtered samples (I-2515-91) and whole-water 

samples (I-4515-91) by U.S. Geological Survey methods 5 

2–9. Graphs showing: 

2. Typical calibration plot for determination of ammonium ions in 

Kjeldahl digests prepared by methods I-2515-91/4515-91 12 

3. Duplicate data for 564 randomly selected samples determined at the 

National Water Quality Laboratory by methods I-2515-91/4515-91 

between 2/11/92 and 2/13/93 18 

4. Spike recovery in relation to Kjeldahl nitrogen concentration determined 

by methods I-2515-91/4515-91 during the July –August, 1991 experiment 19 

5. Concentration ranges for Kjeldahl nitrogen blind blank samples 

analyzed at the U.S. Geological Survey National Water Quality 

Laboratory from January 1989 through September 1999 21 

6. Isopleths of nicotinic acid concentration (a), in milligrams nitrogen per 

liter, found in each tube in relation to the position of each tube in the 

block digester (b) 22
Contents IV

7. Kjeldahl nitrogen concentration recovered in digests of a highsuspended-solids sample, an aqueous solution of adenosine 

5’ triphosphate, and an aqueous solution of nicotinic acid 

by methods I-2515-91/4515-91 as a function of high-temperature 

digestion time 23 

8. Relation between Kjeldahl nitrogen concentrations determined 

by methods I-2552-85/4552-85 and I-2515-91/4515-91 in the April and 

July–August experiments for each of the four water types 26 

9. Concentration differences between Kjeldahl nitrogen determined by 

methods I-2515-91/4515-91 and I-2552-85/4552-85 in the April (A) and 

July–August (B) experiments for each of the four water types 27 

TABLES 

1. Kjeldahl nitrogen concentrations determined by methods I-2515-91/ 

4515-91 for resolvated Heidelberg sample digests before and after 

passage through a 0.45-micrometer nylon syringe filter 6 

2. Calibrant preparation protocol 10 

3. Suggested block protocol for determination of Kjeldahl nitrogen by 

methods I-2515-91/4515-91 11 

4. Suggested tray protocol for automated determination of ammonium 

ions in resolvated digests by methods I-2515-91/4515-91 13 

5. Data used to estimate the method detection level (MDL) for Kjeldahl 

nitrogen determination by methods I-2515-91/4515-91 16 

6. Repeatability (within-run precision, May 3, 1991) data for 55 replicate 

determinations of Kjeldahl nitrogen concentration in the Heidelberg 

sample by using methods I-2515-91/4515-91 16 

7. Between-day (April 8–26, 1991) precision of digested calibrants and 

the Heidelberg sample for Kjeldahl nitrogen determination by using 

methods I-2515-91/4515-91 17 

8. Between-day (April 8–26, 1991) accuracy of digested U.S. Environmental 

Protection Agency and U.S. Geological Survey reference samples for 

Kjeldahl nitrogen determination by using methods I-2515-91/4515-91 19 

9. Statistical data and regression analysis results for Kjeldahl nitrogen 

methods I-2515-91/4515-91 (new methods) and I-2552-85/4552-85 

 (former methods) 20 

10. Median difference between paired Kjeldahl nitrogen concentrations 

determined by methods I-2515-91/4515-91 and I-2552-85/4552-85 25 

11. Effect of nitrate plus nitrite concentrations on median differences between 

Kjeldahl nitrogen concentrations determined by U.S. Geological Survey 

methods I-2515-91/4515-91 and I-2552-85/4552-85 (April data set) 28 

12. Effect of nitrate plus nitrite concentrations on median differences between 

Kjeldahl nitrogen concentrations determined by U.S. Geological Survey 

methods I-2515-91/4515-91 and I-2552-85/4552-85 (July–August data set) 28 

13. Average concentrations of nitrate plus nitrite and ammonium for sample 

subsets with nitrate plus nitrite concentrations greater than 1 milligram 

nitrogen per liter 29 
Conversion Factors and Abbreviations V

CONVERSION FACTORS, ABBREVIATED WATER-QUALITY UNITS, AND 

OTHER ABBREVIATIONS 

Multiply By To obtain 

centimeter (cm) 3.94 x 10-1 inch 

gram (g) 3.53 x 10-2 ounce, avoirdupois 

liter (L) 2.64 x 10-1 gallon 

microliter (μL) 3.38 x 10-5 ounce, fluid 

micrometer (μm) 3.94 x 10-5 inch 

milligram (mg) 3.53 x 10-5 ounce, avoirdupois 

milliliter (mL) 3.38 x 10-2 ounce, fluid 

millimeter (mm) 3.94 x 10-2 inch 

nanometer (nm) 3.94 x 10-8 inch 

Degree Celsius (°C) may be converted to degree Fahrenheit (°F) by using the following 

equation: 

°F = 9/5 (°C) + 32. 

Abbreviated water-quality units used in this report are as follows: 

ȝg/L microgram per liter

mg-N/L milligram nitrogen per liter 

Other abbreviations also used in this report: 

A/D analog-to-digital 

ASTM American Society for Testing 

and Materials 

ATP adenosine triphosphate 

FW formula weight 

h hour 

Hz hertz 

ID identification 

LCL lower control limit 

M molarity (moles/liter) 

MDL method detection level 

N normality (equivalents/liter) 

NASQAN National Stream Quality 

Accounting Network 

NWQL National Water Quality 

Laboratory 

PC personal computer 

QC quality control 

RSD relative standard deviation 

s second 

sp gr specific gravity 

TKN total Kjeldahl nitrogen 

TP total phosphorus 

UCL upper control limit 

USEPA U.S. Environmental Protection 

Agency 

USGS U.S. Geological Survey 

v/v volume/volume 

w/w weight/weight 

w/v weight/volume 

§ nearly equal to 

< less than 

 greater than or equal to 

 less than or equal to 

± plus or minus
Abstract 1

Methods of Analysis by the U.S. Geological Survey National 

Water Quality Laboratory—Determination of Ammonium Plus 

Organic Nitrogen by a Kjeldahl Digestion Method and an 

Automated Photometric Finish that Includes Digest Cleanup 

by Gas Diffusion 

By Charles J. Patton and Earl P. Truitt 

ABSTRACT 

The National Water Quality Laboratory 

(NWQL) determined ammonium plus organic 

nitrogen (Kjeldahl nitrogen) by using semiautomated, block digester methods for filtered 

and whole-water samples from 1986 until 

October 1, 1991. During that time, 

phosphorus was determined by a persulfate 

digestion method. In 1991, projected 

increases in demand for both tests by the U.S. 

Geological Survey’s National Water-Quality 

Assessment Program led the NWQL to 

develop and validate methods for determining 

both analytes in a common digest. 

This report describes a rapid and 

accurate method to determine Kjeldahl 

nitrogen. The batch, high-temperature (block 

digester), Hg (II)-catalyzed digestion step 

used in the new methods I-2515-91/4515-91 is 

similar to U.S. Geological Survey methods 

I-2552-85/4552-85 and U.S. Environmental 

Protection Agency method 351.2 except that 

sample and reagent volumes are halved. 

Prepared digests are desolvated at 220 degrees 

Celsius (oC) and digested at 370oC in separate 

block digesters set at these temperatures, 

rather than in a single, temperatureprogrammed block digester. This approach 

permits 40 calibrants, reference materials, and 

samples to be digested and resolvated in about 

an hour. Ammonium ions originally present 

in samples, along with those released during 

the digestion step, are determined photometrically by an automated, salicylatehypochlorite Berthelot reaction procedure at a 

rate of 90 tests per hour. About 100 

microliters of digest are required per 

determination. The upper concentration level 

is 10 milligrams per liter (mg/L) with a 

method detection level of 0.05 mg/L. 

Repeatability for a sample containing about 

4.1 mg/L of Kjeldahl nitrogen in a high 

suspended-solids matrix is 3.1 percent. 

Between-day precision for the same sample is 

4.8 percent. 

A gas diffusion cell in the air-segmented 

continuous flow analyzer eliminates 

particulates and ions that otherwise would 

interfere in the photometric finish. A singlechannel analyzer can process the resolvated 

digests from two pairs of block digesters each 

hour. Statistical analysis of paired data for 

about 1,500 samples determined by U.S. 

Geological Survey methods I-2552-85/4552-

85 and I-2515-91/4515-91 during method 

validation revealed a median concentration 

difference between the former and the latter 

methods of about 0.1 mg-N/L. This result was 

expected because digestion blank concentrations (nearly equal to 0.1 mg/L) were not 

subtracted from concentrations reported by 

methods I-2552-85/4552-85. A 10-year 

record of National Water Quality Laboratory 

Kjeldahl nitrogen blind blank concentration 

data also supports a step-change decrease in 

Kjeldahl nitrogen concentrations of about 0.1 

mg/L after methods I-2552-85/4552-85 were 

replaced by methods I-2515-91/4515-91 on 

October 1, 1991. Somewhat larger 

concentration differences between the two
2 Determination of Ammonium Plus Organic Nitrogen

methods were observed for a subset of about 

350 samples with nitrate plus nitrite 

concentrations greater than 1 mg-N/L. 

INTRODUCTION 

From 1986 until October 1, 1991, the 

National Water Quality Laboratory (NWQL) 

determined ammonium plus organic 

nitrogen (Kjeldahl nitrogen) by using semiautomated, block digester methods 

I-2252-85 (filtered samples) and I-4551-85 

(whole-water samples). These methods are 

similar to U.S. Environmental Protection 

Agency (USEPA) method 351.2 (U.S. 

Environmental Protection Agency, 1993). 

During that time, phosphorus was 

determined by a persulfate digestion method 

similar to USEPA method 365.1 (U.S. 

Environmental Protection Agency, 1983a). 

In 1991 projected increases in demand for 

both tests, brought on by the National 

Water-Quality Assessment program, led the 

NWQL to develop and validate methods for 

determining both analytes in a common 

digest. Details of the phosphorus method, 

which was developed and validated 

concurrently with the Kjeldahl nitrogen 

method described here, can be found in 

Patton and Truitt (1992). 

Previous work suggested that 

streamlining the Kjeldahl digestion step 

improved data quality for these tests and 

also increased production capacity, 

decreased production costs, and lowered 

analysts' exposure to corrosive and toxic 

chemicals. Jirka and others (1976), for 

example, reported that digestion times for 

Kjeldahl nitrogen could be reduced from 

about 4 hours to 90 minutes by halving 

sample and reagent volumes prescribed in 

USEPA method 351.2. Later, Bowman and 

Delfino (1982) demonstrated that further 

reduction in digestion time could be 

achieved by replacing a single, temperatureprogrammed block digester with a pair of 

block digesters set at the desolvation 

(220C) and digestion (370C) 

temperatures. Methods I-2515-91/4515-91, 

which are described here, have been in 

routine use at the NWQL since October 1, 

1991. They combine the Jirka and others 

(1976) and Bowman and Delfino (1982) 

digestion procedure improvements with a 

novel, highly robust automated colorimetric 

finish (salicylate-hypochlorite reaction) for 

separating ammonia in resolvated Kjeldahl 

digests from ionic and particulate 

interferents by in-line, gas diffusion across a 

microporous, polypropylene membrane. A 

batch of 40 calibrants, reference materials, 

and samples can be digested and made ready 

for colorimetric analysis in about 1 hour by 

using these methods. 

Data in this report result from two 

distinct studies in 1991. The aim of the first 

study, conducted in April, was to assess the 

feasibility of using a common (Kjeldahl) 

digestion procedure for Kjeldahl nitrogen 

and phosphorus determinations and to 

document the performance of updated 

photometric finishes associated with these 

determinations. Analytical performance of 

the newly developed methods was 

documented, and quality-control (QC) 

guidelines for routine operation of the new 

methods were established. In many cases, 

data that appear in this report relating to 

analytical figures of merit—precision and 

accuracy of results, method detection levels, 

and blank concentrations—were collected 

during the April study. At that time, all 

samples received at the NWQL with test 

requests for Kjeldahl nitrogen and 

phosphorus also were analyzed for Kjeldahl 

nitrogen by USGS method numbers I-2515-

91 (filtered samples), I-4515-91 (wholewater samples) and phosphorus by USGS 

method numbers I-2610-91 (filtered 

samples), I-4610-91 (whole-water samples).
Analytical Method 3

At that time, these methods were in the 

initial stages of validation (Patton and Truitt, 

1992). The resulting data set consists of 

about 400 pairs of Kjeldahl nitrogen 

concentrations that had been determined by 

USGS method numbers I-2552-85/4552-85 

and I-2515-91/4515-91, which were used to 

assess relative performance of the two 

methods. 

The second study, conducted in July, 

August, and September, was performed to 

address U.S. Geological Survey (USGS) 

concerns that the full range of water types, 

which are commonly sent to the NWQL for 

nutrient determinations, was not adequately 

represented in the April study. During these 

months, about 1,100 additional samples, 

selected by the USGS, were determined by 

both methods. Results of this comparison 

are included here, but typically QC and 

other analytical performance-related data 

that closely matched those from the April 

study are not. 

This report describes USGS methods 

I-2515-91/4515-91 for determining Kjeldahl 

nitrogen developed for use at the NWQL. 

The method was implemented in the NWQL 

on October 1, 1991. All aspects of methods 

I-2515-91/4515-91 are described from 

sample preparation through calculation and 

reporting of results. Precision and accuracy 

data are included. Method I-2515-91/4515-

91 supplements other methods of the USGS 

for determination of inorganic substances in 

water that are described by Fishman and 

Friedman (1989) and Fishman (1993). 

Several individuals merit special 

thanks for assistance with various aspects of 

work reported here. Andrea M. Jirka (U.S. 

Environmental Protection Agency, Kansas 

City, Kan.), George T. Bowman (State 

Laboratory of Hygiene, Madison, Wis.), and 

Jack W. Kramer (Water Quality Laboratory, 

Heidelberg College, Tiffin, Ohio) are 

gratefully acknowledged for helpful 

discussions during the planning phases. 

Kramer also supplied high-particulate, 

agricultural run-off samples (Heidelberg 

sample) used as a control sample throughout 

this work. Dr. Ivan Sekerka (National 

Water Research Institute, Canada), Bertin 

Francoeur (National Laboratory for 

Environmental Testing, Canada), and 

Andrea Jirka served as colleague reviewers 

for a previous draft of this report. 

The authors especially thank Jeffrey 

W. Pritt (U.S. Geological Survey), who 

performed statistical analysis on surfacewater data included in this report. 

Assistance from several other individuals at 

the National Water Quality Laboratory, the 

Branch of Quality Systems, the Office of 

Water Quality, and the Branch of Systems 

Analysis also is gratefully acknowledged. 

Former USGS chemist Earl P. Truitt 

(deceased) made substantial contributions to 

the development of this analytical method. 

The principal author gratefully acknowledges 

Earl's able assistance and analytical work 

upon which this report is based. 

ANALYTICAL METHOD 

Parameter and Codes: 

Ammonium plus organic nitrogen, 

dissolved, I-2515-91 (mg/L as N): 

00623 Ammonium plus organic 

nitrogen, total, I-4515-91 (mg/L as N): 

00625 

1. Application 

This method is used to determine 

Kjeldahl nitrogen in water, drinking water, 

wastewater, brines, and water-suspended 

sediment. The suitability of this method for 

determination of Kjeldahl nitrogen in 
4 Determination of Ammonium Plus Organic Nitrogen 

bottom materials has not been investigated. 

The analytical range of this method is 0.1 to 

10.0 mg/L of nitrogen. 

2. Summary of Method 

2.1 Organic nitrogen is converted to 

ammonium ions at a temperature of 370oC in a 

reaction medium of sulfuric acid, potassium 

sulfate, and mercury (II). In principle, nitrate 

and nitrite are not reduced to ammonium ions 

under these conditions; USGS nomenclature 

refers to Kjeldahl nitrogen as ammonium plus 

organic nitrogen to emphasize this distinction. 

In practice, however, nitrate and nitrite might 

interfere positively or negatively (see 

section 3, Interferences). 

2.2 The digestion procedure was 

adapted from the method of Jirka and others 

(1976) and Bowman and Delfino (1982), 

which is identical to USEPA method 351.2 

(U.S. Environmental Protection Agency, 

1983b), except that sample and reagent 

volumes are halved, as is the time required for 

digestion. 

2.3 An air-segmented continuous flow 

analyzer is used to automate the photometric 

determination of ammonium ions in resolvated 

Kjeldahl digests by the salicylate analog 

(Reardon and others, 1966) of the Berthelot 

reaction (Patton and Crouch, 1977; Harfmann 

and Crouch, 1989). Resolvated Kjeldahl 

digests often contain suspended particulates 

(clays) and ions that interfere with the 

photometric finish. Both classes of 

interferents are eliminated by means of an online gas diffusion cell, which consists of a 

continuous-flow, parallel-plate dialyzer 

assembly that replaces the dialysis membrane 

with a hydrophobic, microporous, 

polypropylene membrane. Gases pass through 

the microporous polypropylene membrane; 

particles and ions do not. Before passage 

through the gas diffusion cell, ammonium ions 

in acidic resolvated Kjeldahl digests mix with 

the alkaline donor stream and are converted to 

ammonia— 

NH4

+

(aq) plus -

OH NH3 (g) + H2O. 

Inside the diffusion cell, gas-phase ammonia 

in the donor stream passes through the 

polypropylene membrane and is trapped in the 

interferent-free recipient stream (see fig. 1). 

The authors determined ammonium in 

filtered (nominal pore size, 0.45m) and 

unfiltered portions of highly turbid Heidelberg 

sample digests during the April experiments to 

demonstrate that the gas diffusion cell 

effectively removed suspended particles 

(clays) from resolvated Kjeldahl digests. 

(Samples were provided for this study by the 

Water Quality Laboratory at Heidelberg 

College in Tiffin, Ohio.) Equivalent 

ammonium concentrations in unfiltered and 

filtered sample digests are listed in table 1. 

Furthermore, the authors observed no surface 

fouling or mechanical failure of the porous 

polypropylene gas-diffusion membrane during 

the course of this work, and photometer flowcell clogging was never a problem. 

3. Interferences 

3.1 As described in section 2.3, in-line 

digest cleanup by gas diffusion (Seifter and 

others, 1971) eliminates all particulate and 

potential ionic interference in the photometric 

finish. Thus, resolvated digests containing 

suspended particulates (clays) do not require 

filtration prior to analytical determinations. 

Likewise, colorimetric reagents do not contain 

complexing agents, such as citrate, tartrate, or 

EDTA, that are required to prevent 

precipitation of calcium (II) and magnesium 

(II) in the alkaline analytical stream of 

methods that lack a gas-diffusion cleanup step. 
Analytical Method 5
6 Determination of Ammonium Plus Organic Nitrogen

Table 1. Kjeldahl nitrogen concentrations determined by methods I-2515-91/4515-91 for resolvated 

Heidelberg sample digests before and after passage through a 0.45-micrometer nylon syringe filter 

[μm, micrometer; mg-N/L, milligram nitrogen per liter] 

 Concentration (mg-N/L) Julian Concentration (mg-N/L) 

date Unfiltered Filtered 

Julian 

date Unfiltered Filtered 

 98 3.96 4.03 107 3.78 3.80 

 99 4.28 4.25 107 4.15 4.08 

 99 4.11 4.10 108 4.38 4.46 

 100 3.54 3.62 108 4.44 4.34 

 100 4.26 4.28 109 4.32 4.32 

 101 4.17 4.11 109 4.15 3.99 

 101 4.14 4.07 113 4.12 4.25 

 101 4.09 4.07 113 4.29 4.29 

 101 3.95 4.02 114 3.90 3.71 

 102 3.95 4.05 114 4.11 4.09 

 106 4.33 4.29 115 4.05 4.27 

 106 4.12 4.11 116 4.39 4.30 

 106 4.16 4.20 116 4.38 4.44 

 

 

 

Average concentration (mg-N/L): 

Standard deviation:

Number of samples:

Relative standard deviation (percent): 

4.14 

0.20 

26 

4.88 

4.14 

0.20 

26 

4.83 

3.2 Once samples have been acidified, 

they are subject to contamination by ammonia 

in the laboratory atmosphere. The digestion 

process, therefore, must be performed in a 

hood that is located in an ammonia-free area 

of the laboratory. Other analytical or 

housekeeping procedures with potential to 

contribute ammonia vapor to the laboratory 

atmosphere may not be performed in or near 

this hood. Avoid delays between sample 

preparation, sample digestion, and digest 

analyses to minimize the risk of ammonia 

contamination. 

3.3 Nitrate can exert both a positive 

and negative interference in Kjeldahl nitrogen 

determinations. As stated by the American 

Public Health Association (1992, p. 4–94): 

During [Kjeldahl] digestion, nitrate in 

excess of 10 mg/L can oxidize a portion of 

the ammonia released from the digested 

organic nitrogen, producing N2O and 

resulting in a negative interference. When 

sufficient organic matter in a low state of 

oxidation is present, nitrate can be reduced 

to ammonia, resulting in a positive 

interference. The conditions under which 

significant interferences occur are not well 

defined and there is no proven way to 

eliminate the interference.... 

4. Instrumentation 

4.1 A third-generation, air-segmented 

continuous flow analyzer (Alpkem RFA300TM) was used to automate photometric 

determination of ammonium ions in resolvated 

digests. Modules in this system include a 301 

sampler, 302 peristaltic pump, 313 analytical 

cartridge base, 314 power module, 305A 

photometer, 311 recorder, and a data 

acquisition and processing system for use with 
Analytical Method 7

a personal computer (PC). Alternative 

procedures to automate the photometric 

finish by using flow injection analyzers or 

other second or third generation continuous 

flow analyzers also could be implemented 

(Patton and Wade, 1997). 

4.2 Photometric data from the 

continuous flow analyzer were acquired and 

processed with a software package (Alpkem 

SoftPackTM), operated on a PC equipped with 

a 12-bit, analog-to-digital (A/D) converter 

plug-in card. This A/D converter provided a 

resolution of 1 part in 4,095 ( 0.0012 volts 

when 5 volts is full scale), which is 

conservatively 20 times better than that 

afforded by a 10-inch (26-centimeter) stripchart recorder. The A/D converter must be 

able to acquire data at frequencies in the range 

of 0.5 to 2 Hz—that is, 30 points/min to 120 

points/min. As a general rule, data acquisition 

frequencies for air-segmented continuous flow 

analyzers should match the roller lift-off 

frequency of the peristaltic pump (Patton and 

Wade, 1997)—that is, 0.5 Hz for Technicon 

AutoAnalyzer II TM and 1.5 Hz for Alpkem 

RFA-300 equipment. Most PC-based data 

acquisition and processing systems sold by 

vendors of continuous flow analyzers meet or 

exceed these specifications. 

4.3 Operating characteristics for this 

equipment follow: 

Analytical wavelength 660 nm 

Flow cell path length 10 mm 

Standard calibration control setting 1.4 

Dialyzer1

 4.3 in. (10.9 cm) 

Segmentation frequency 1.5 Hz 

Reaction coil volume 2 mL 

Reaction coil temperature 37oC 

Sample time 20 s 

Wash time 20 s 

Analysis rate 90/h, 1:1 

_______________ 

1

Microporous polypropylene membrane, 0.1 ȝm pore size 

(Gelman Metricel®) 

5. Apparatus 

5.1 Tecator Digestion System 40, 

Model 1016 block digesters or equivalent, 

which accommodate 40, 75-mL tubes, are 

used to desolvate and digest samples. 

5.2 In this procedure, block digesters 

are operated in pairs. Prepared samples are 

desolvated in one block set at 220°C and 

immediately digested in another one set at 

370°C. Time required for desolvation is 30 

minutes and for digestion is 15 minutes. 

6. Reagents 

6.1 Digestion Reagents 

CAUTION: Heat is produced when 

concentrated sulfuric acid is mixed with 

water. Wear protective eyeglasses, gloves, 

and clothing. Hot sulfuric acid solutions are 

hazardous. 

NOTE: All references to deionized water 

shall be understood to mean ASTM Type I 

deionized water (American Society for 

Testing and Materials, 1995). 

6.1.1 Sulfuric acid, 3.6 M: Cautiously 

add 200 mL of concentrated sulfuric acid 

[H2SO4, sp gr 1.84] to 700 mL of deionized 

water contained in a 1-L volumetric flask with 

constant mixing. Allow this solution to cool, 

dilute it to the mark with deionized water, and 

mix it well. Transfer this reagent to a plastic 

bottle where it is stable indefinitely at room 

temperature. 

6.1.2 Mercury (II) sulfate reagent: Add 

25 mL of 3.6 M sulfuric acid to 4.0 g of red 

mercury (II) oxide [HgO, FW = 216.59] 

contained in a 100-mL Griffin beaker. Place 

the beaker in an ultrasonic bath to speed 

dissolution. Use the resulting solution 
8 Determination of Ammonium Plus Organic Nitrogen

immediately to prepare the digestion reagent 

as described in the next paragraph. 

6.1.3 Digestion reagent: Add 268 g of 

potassium sulfate [K2SO4, FW = 174.27] to 

1,300 mL of deionized water contained in a 

2-L volumetric flask. Cautiously add 400 mL 

of concentrated sulfuric acid [H2SO4, sp gr = 

1.84] with constant mixing, and then add the 

mercury (II) sulfate reagent. Stir the mixture 

magnetically or place the flask in an ultrasonic 

bath to speed dissolution. Allow this solution 

to cool, dilute it to the mark with deionized 

water, and mix it well. Transfer this reagent 

to a glass bottle or dispensing apparatus and 

store it at or above 20C to prevent 

precipitation of potassium sulfate. 

6.2 Colorimetric Reagents 

6.2.1 Sampler wash reservoir solution 

(1.3 N sulfuric acid + 0.3 M potassium 

sulfate): Cautiously add 78 mL of 

concentrated sulfuric acid [H2SO4, sp gr 1.84] 

to 1,500 mL of deionized water contained in 

a 2-L volumetric flask with constant mixing. 

Add 54 g of potassium sulfate [K2SO4, FW = 

174.27 g], and after it has dissolved, allow the 

solution to cool. Then dilute it to the mark 

with deionized water, and mix it well. 

Transfer this solution to plastic bottles where 

it is stable indefinitely at room temperature. 

NOTE: This solution has hydronium and 

sulfate ion concentrations similar to those of 

resolvated digests. It can be used as the 

matrix for undigested ammonium ion 

calibrants. 

6.2.2 Sodium hydroxide, 5.0 M (alkaline 

recipient stream reagent): Cautiously add 200 

g of sodium hydroxide pellets [NaOH, FW = 

40.00] to 700 mL of deionized water 

contained in a 1-L volumetric flask. Swirl the 

flask repeatedly to dissolve its contents. 

Allow the resulting solution to cool, dilute it 

to the mark with deionized water, and mix it 

well. Transfer this reagent to a plastic bottle 

where it is stable indefinitely at room 

temperature. 

CAUTION: Heat is produced when sodium 

hydroxide pellets are mixed with water. 

Wear protective eyeglasses, gloves and 

clothing. Hot sodium hydroxide solutions 

are very hazardous. 

6.2.3 Sodium hydroxide, 2.5 M 

(alkaline donor stream reagent): Add 50 

mL of 5 M sodium hydroxide solution to 50 

mL of deionized water contained in a 125-

mL plastic bottle. Swirl the bottle to mix its 

contents. Prepare this reagent daily. 

6.2.4 Brij-35 surfactant (30 percent 

w/w): Most commercially available solutions 

are satisfactory. 

6.2.5 Sodium bicarbonate buffer (0.1 M, 

pH 10.5): Add 16.8 g of sodium 

bicarbonate (NaHCO3, FW = 84.00), and 15 

mL of 5 M sodium hydroxide solution to 

1,500 mL deionized water contained in a 2-L 

volumetric flask. Shake the flask vigorously 

to dissolve its contents. Dilute the resulting 

solution to the mark with deionized water and 

mix it well. Transfer this reagent to plastic 

bottles where it is stable indefinitely at room 

temperature. 

6.2.6 Sodium hypochlorite reagent 

(STOCK, 5.25 percent w/v): Most 

commercially available solutions of household 

chlorine bleach containing 5.25 percent 

available chlorine, Clorox®, for example, are 

satisfactory. 

6.2.7 Sodium hypochlorite reagent 

(WORKING): Add 375 μL of stock sodium 

hypochlorite reagent to 250 mL of bicarbonate 

buffer reagent contained in a plastic bottle. 

Swirl the bottle to mix its contents. Prepare 

this reagent daily. 
Analytical Method 9

6.2.8 Sodium salicylate/sodium 

nitroferricyanide reagent (STOCK): 

Dissolve 150 g sodium salicylate [C7H5O3

-

Na+, FW = 160.10] and 0.30 g sodium nitroferricyanide [Na2Fe(CN)5NO  2H2O, FW = 

297.95] in about 800 mL deionized water. 

Dilute the resulting solution to the mark with 

deionized water and mix it well. Store this 

reagent in an amber-colored glass bottle, 

where it is stable for several months at room 

temperature. 

6.2.9 Sodium salicylate/sodium 

nitroferricyanide reagent (WORKING): Add 

100 μL of Brij-35 surfactant per 100 mL of 

stock solution and mix it well. 

7. Calibrants 

7.1 Primary calibrant, inorganic 

(1.000 mL = 2.50 mg-N): Dissolve 4.7736 g 

of ammonium chloride [NH4Cl, FW=53.49] 

previously dried at 110C for 2 h and stored 

in a desiccator, in 400 mL of deionized water 

contained in a 500-mL volumetric flask. 

Dilute this solution to the mark with deionized 

water and mix it well. Transfer this calibrant 

to a plastic bottle and store it in a refrigerator 

where it is stable for several months. 

7.2 Primary calibrant, organic (1.000 

mL = 2.50 mg-N): Dissolve 9.9500 g of 

glycine hydrochloride [C2H5NO2  HCl, FW = 

111.5] in 400 mL of deionized water 

contained in a 500-mL volumetric flask. 

Dilute this solution to the mark with deionized 

water and mix it well. Transfer this calibrant 

to a plastic bottle and store it in a refrigerator 

where it is stable for several months. 

7.3 Spike solution, organic (1.000 mL 

= 0.1 mg-N): Use an adjustable pipet (100 to 

1,000 μL) to dispense 1,000 μL of organic 

nitrogen primary calibrant into 20 mL of 

deionized water contained in a 25-mL 

volumetric flask. Dilute the resulting solution 

to the mark with deionized water and mix it 

well. Transfer this solution to a 40-mL amber 

glass vial and store it in a refrigerator where it 

is stable for about a month. 

NOTE: When Kjeldahl nitrogen and 

phosphorus are to be determined in the same 

digest, prepare mixed primary calibrant and 

spike solutions that contain both nitrogen 

and phosphorus. 

7.4 Working calibrants: Use two 

adjustable pipets (ranges 10 to 100 μL and 

100 to 1,000 μL) to dispense the volumes of 

primary calibrant, listed in table 2, and 

1.000 mL of field preservative solution1 (a 

solution containing 1.3 g HgCl2 + 10.0 g NaCl 

in 100 mL of deionized water) into a series of 

250-mL volumetric flasks that each contain 

240 mL of deionized water (U.S. Geological 

Survey Office of Water Quality Technical 

Memorandum No. 94.16, 1996; U.S. 

Geological Survey Office of Water Quality 

Technical Memorandum No. 99.04, 1999). 

Rinse these flasks with a dilute solution of 

hydrochloric acid (5 percent v/v) and 

deionized water just prior to calibrant 

preparation. Dilute the contents of the flasks 

to the mark with deionized water, and shake 

them with repeated inversion to ensure 

thorough mixing. Transfer calibrants to 

plastic bottles and store them in a refrigerator 

when they are not in use. Prepare working 

calibrants as needed or biweekly, whichever 

comes first.

2

 

1The USGS no longer amends nutrient samples with mercuric 

chloride as it did at the time this method was validated. The 

preparation of calibrants, blanks, and check standards in a matrix 

that closely matches that of samples being analyzed is necessary to 

ensure the accuracy of analytical results. Beginning in 1996, some 

nutrient samples received at the NWQL for analysis have been 

amended with sulfuric acid. Since January 1999, all whole-water 

nutrient samples sent to the NWQL require sulfuric acid 

amendment. 

2Undigested calibrants, which are useful for assessing 

instrument function, can be prepared from the primary ammonium 

calibrant by using the sampler wash reservoir solution (see section 

6.2.1) as the matrix.
10 Determination of Ammonium Plus Organic Nitrogen

Table 2. Calibrant preparation protocol 

[μL, microliter; mg-N/L, milligrams of nitrogen per liter; mL, milliliter] 

Calibrant 

identification 

 Primary 

calibrant 

volume (μL) 

 Field 

preservative 

volume (μL) 

 Nominal 

concentration1

(mg-N/L) 

 C1 1,000 1,000 10.0 

 C2 500 1,000 5.0 

 C3 250 1,000 2.50 

 C4 125 1,000 1.25 

 C5 50 1,000 0.50 

 C6 10 1,000 0.10 

 (Blank) 0 1,000 0.00 

1

Based on a final volume of 250 mL. 

8. Sample Preparation 

8.1 Dispense calibrants, blanks 

(generally, ASTM type I deionized water), 

reference materials, and samples into digestion 

tubes as follows. Be sure the matrix of 

calibrants, blanks, and reference materials 

match that of samples amended with 

preservatives—sulfuric acid, for example—at 

collection sites. Note that three positions in 

each block are reserved for duplicate and 

spiked samples. To spike samples at a 

concentration of 1.0 mg-N/L, dispense 100 μL 

of the organic spike solution (see section 7.3) 

directly into appropriate tubes prior to 

digestion. 

8.2 Rinse all glassware first with a 

dilute solution (5 percent v/v) of 

hydrochloric acid and then with deionized 

water before each use. This step is critical to 

achieve consistently low-concentration 

digestion blanks for both nitrogen and 

phosphorus. 

8.3 Use an adjustable (5.0 to 

10.0 mL) pipet to dispense 10.0-mL aliquots 

of calibrant, blank, reference, and sample 

solutions into digestion tubes. The suggested 

block protocol can be found in table 3. 

Vigorously shake sample containers and 

immediately aspirate aliquots to avoid 

sampling errors. 

NOTE: When samples contain large 

quantities of suspended solids, continuous 

stirring during sample aspiration may 

provide the only means of obtaining 

representative aliquots. 

8.4 Use an adjustable (2.0 to 100.0 L) 

pipet to dispense 100 μL of the organic spike 

solution, equivalent to 1 mg-N/L when added 

to 10 mL of sample (see section 7.3), directly 

into samples designated as spikes. 

8.5 Use an adjustable volume, syringebased repetitive dispenser to add 2.00 mL of 

digestion reagent into each tube. Then add 

several acid-rinsed (5 percent HCl), TeflonTM 

boiling chips to each tube. 

8.6 Desolvate prepared digests under a 

hood for 30 minutes in a block digester set at 

220C. At the end of this time, digest 

volumes should be less than 3 mL. 
Analytical Method 11

Table 3. Suggested block protocol for determination of Kjeldahl nitrogen by methods I-2515-91/4515-91 

[ID, identification; mg-N/L, milligrams nitrogen per liter; SRWS, U.S. Geological Survey Standard Reference 

Water Sample] 

Block 

position Sample ID Block 

position Sample ID Block 

position Sample ID 

 1 CALIBRANT 1 15 SAMPLE 29 SAMPLE 

 2 CALIBRANT 2 16 ORG_CHK1 30 SAMPLE 

 3 CALIBRANT 3 17 SAMPLE 31 SAMPLE 

 4 CALIBRANT 4 18 SAMPLE 32 BLANK 

 5 CALIBRANT 5 19 SAMPLE 33 SAMPLE 

 6 CALIBRANT 6 20 SAMPLE 34 SAMPLE 

 7 BLANK 21 SAMPLE 35 SAMPLE 

 8 BLANK 22 SAMPLE 36 SAMPLE 

 9 SAMPLE 23 SAMPLE 37 SAMPLE 

 10 SAMPLE 24 SRWS 38 SPIKE2

 

 11 SAMPLE 25 SAMPLE 39 DUPLICATE2

 

 12 SAMPLE 26 SAMPLE 40 DUPLICATE2

 

 13 SAMPLE 27 SAMPLE 

 14 SAMPLE 28 SAMPLE 

1

A solution of glycine (7.5 mg-N/L) is recommended. 2

Samples for spiking and duplicate determinations should be chosen randomly. 

8.7 Immediately transfer (CAUTION) 

desolvated digests to the block digester set at 

370C, and leave them there for 15 minutes. 

At the end of this time, digest volumes should 

be less than 0.5 mL and appear as a clear, 

syrupy bead at the bottom of each digestion 

tube. Crystallized digests indicate a problem 

in the acid to sulfate ratio of the digestion 

reagent. 

8.8 Cautiously remove digestion tubes 

from the block digester and allow them to cool 

for about 10 minutes in the hood. With 

extreme caution, immediately dispense 

10.0 mL of deionized water into each tube 

with vigorous agitation, by using an adjustable 

volume, syringe-based repetitive dispenser 

and a vortex mixer. 

9. Instrument Performance 

When a pair of block digesters is used 

as described in the Introduction, 40 

calibrants, check standards, and samples can 

be digested and made ready for colorimetric 

analysis in about 1 hour. The air-segmented 

continuous flow analyzer used in this 

method can perform 90 ammonium 

determinations per hour with less than 1 

percent interaction. Thus, a single channel 

analyzer can process the resolvated digests 

from two pairs of block digesters each hour. 

10. Calibration 

With a second-order polynomial leastsquares curve-fitting algorithm (y = 

a+bx+cx2, where y is the corrected peak 

height and x is the concentration), the 

correlation coefficient (r2) of the calibration 

plot should be greater than 0.999. A typical 

calibration plot for Kjeldahl nitrogen 

calibrants in the concentration range of 0.1 

to 10.0 mg-N/L is shown in figure 2. 

11. Procedure and Data Evaluation 

Set up the analytical cartridge of the 

continuous flow analyzer as shown in 

figure 1. Turn on electrical power to all 
12 Determination of Ammonium Plus Organic Nitrogen

0 1 2 3 4 5 6 7 8 9 10

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

 x y

10.0 4.406

 7.5 3.335

 5.0 2.295

 1.25 0.588

 0.5 0.224

 0.1 0.049

y = a + bx + cx2

Parameter Value Error

a -0.0011 0.0130

b 0.4718 0.0081

c -0.0032 0.0008

Corr. Coef.

r

2 0.99994 0.0183

CORRECTED ABSORBANCE, IN VOLTS

NOMINAL AMMONIUM + ORGANIC NITROGEN

CONCENTRATION, IN MILLIGRAMS PER LITER

Figure 2. Typical calibration plot for determination of ammonium ions in Kjeldahl digests prepared 

by methods I-2515-91/4515-91. Corr. Coef = correlation coefficient. 

system modules and put fresh sampler wash 

reservoir solution and reagents on-line. 

After about 10 minutes, verify that the 

output of sample and reference detectors is 

5 volts. A suggested sampler tray protocol 

for automated determination of ammonium 

ions in resolvated digests is provided in 

table 4. 

NOTE: To minimize errors that result from 

contaminated analyzer cups, rinse them 

several times with the solution they are to 

contain before placing them on the analyzer 

sampler tray. 

NOTE: The full-scale absorbance range 

control (STD CAL) of the photometer 

should not require daily adjustment. 

Between-run/between-day variations in 

baseline-absorbance level and calibration 

curve slope of about ±5 percent are 

acceptable. Adjustment of the STD CAL 

control to compensate for larger variations 

in sensitivity or baseline (reagent blank) 

levels will only mask underlying problems, 

such as incipient light-source failure,

partially clogged flow cells, or contaminated 

or improperly prepared reagents, any of 

which could compromise analytical results. 

Dwell time for the analyzer may increase by 
Analytical Method 13

Table 4. Suggested tray protocol for automated determination of ammonium ions in resolvated digests 

by methods I-2515-91/4515-91

[ID, identification] 

Cup 

number 

Sample 

ID 

Cup 

number 

Sample 

ID 

Cup 

number 

Sample 

ID 

 1 SYNC 15 SAMPLE 29 U_BLANK1 

 2 CALIBRANT 1 16 SAMPLE 30 SAMPLE 

 3 CALIBRANT 2 17 SRWS3

 31 SAMPLE 

 4 CALIBRANT 3 18 SAMPLE 32 SAMPLE 

 5 CALIBRANT 4 19 SAMPLE 33 SAMPLE 

 6 CALIBRANT 5 20 SAMPLE 34 SAMPLE 

 7 CALIBRANT 6 21 SAMPLE 35 SAMPLE 

 8 U_BLANK1

 22 SAMPLE 36 SAMPLE 

 9 BLANK 23 SAMPLE 37 SAMPLE 

 10 BLANK 24 SRWS3

 38 SAMPLE 

 11 ORG_CHK2

 25 SAMPLE 39 SAMPLE 

 12 SAMPLE 26 SAMPLE 40 SPIKE 

 13 SAMPLE 27 SAMPLE 41 DUPLICATE 

 14 SAMPLE 28 SAMPLE 42 DUPLICATE 

1

Undigested blank (sampler wash reservoir solution, see 6.2.1). 

2

Organic nitrogen check sample; see note 2 on table 3. 

3

USGS standard reference water sample. 

a few seconds per day as pump tubes wear, 

but it need not be checked or updated more 

than once per week. 

12. Calculations 

12.1 Instrument calibration requires the 

preparation of a set of solutions (calibrants) in 

which the analyte concentration is known. 

These calibrants are digested along with 

samples and used to establish a calibration 

function that is estimated from a least-squares 

fit of nominal calibrant concentrations (x) in 

relation to peak absorbance (y). A secondorder polynomial function (y = a+bx+cx2) 

usually provides improved concentration 

estimates at the upper end of the calibration 

range than the more conventional linear model 

(y = a+bx). Accuracy is not lost when a 

second-order fit is used, even if the calibration 

function is strictly linear, because, in this case, 

the value estimated for the quadratic 

parameter c will approach zero. 

12.2 Before the calibration function can 

be estimated, the baseline absorbance 

component of measured peak heights, 

including drift (continuous increase or 

decrease in the baseline absorbance during the 

course of an analysis), if present, needs to be 

removed. Baseline absorbance in continuous 

flow analysis is analogous to the reagent blank 

absorbance in batch analysis. Correction for 

baseline absorbance is an automatic function 

of most data acquisition and processing 

software sold by vendors of continuous flow 

analyzers. 

NOTE: These correction algorithms are 

based on linear interpolation between initial 

and intermediate or final baseline 

measurements, and so they do not accurately 

correct for abrupt, step-changes in baseline 

absorbance that usually indicate partial 

blockage of the flow cell. It is prudent, 

therefore, to reestablish baseline absorbance 

at intervals of 20 samples or so. 
14 Determination of Ammonium Plus Organic Nitrogen

12.3 After peaks are baseline corrected, 

they need to be digestion blank corrected. 

This correction can be applied in 

several ways: 

1. Subtract the baseline-corrected 

absorbance of the digestion blank—

compute an average concentration if 

multiple digested blanks are 

included in each block—from the 

baseline-corrected absorbance of all 

calibrants, check standard, and 

samples in the block. Then estimate 

regression parameters (a, b, and c 

terms) for the calibration function 

by using a second-order polynomial 

least-squares algorithm. For 

second- and higher order calibration 

functions, use the Newton-Raphson 

successive approximations 

algorithm (Draper and Smith, 1966; 

Swartz, 1976, 1977, 1979) to 

convert corrected peak heights into 

concentrations. 

2. Designate digestion blanks as a 

calibrant with a nominal 

concentration of zero. The 

calibration function estimated as 

described in section 12.3 then will 

have a positive y-intercept, with a 

magnitude that will approximate the 

baseline-corrected absorbance of the 

digestion blank. If this method is 

used, be sure that the curve-fitting 

algorithm does not force a zero yintercept by including one or more 

"dummy" (0,0) points in the data set 

used for calibration. 

3. Designate digested blanks as baseline 

correction samples. In this case 

initial, intermediate (if included), 

and final baselines are interpolated 

between digested blank peak 

maxima. Thus, baseline and 

digestion blank corrections are 

performed in a single operation. 

NOTE: Digestion blank corrections for 

data in this report were performed by 

method 1. Note, however, that analytical 

results calculated by the other two methods 

should be equivalent, although in the 

authors' opinion the second correction 

method is most sound statistically. In any 

case, choose the digestion blank correction 

algorithm that is most easily implemented 

with the software package available. 

Regardless of the algorithm chosen, make 

sure that it is documented in the standard 

operating procedure (SOP) and that it is 

understood by analysts. This process also 

applies to algorithms used to convert 

corrected peak heights to concentration 

units. The SOP for this method must be 

updated whenever any changes in data 

acquisition and processing software or in 

calculation algorithms are implemented. 

12.4 Most software packages provide a 

data base for entering appropriate dilution 

factors. Usually, these factors can be entered 

before or after analyses are performed. If 

dilution factors are entered, then reported 

concentrations will be compensated 

automatically for the extent of dilution. The 

dilution factor is the number by which a 

measured concentration must be multiplied to 

obtain the analyte concentration in the sample 

before dilution. For example, dilution factors 

of 2, 5, and 10 indicate that sample and diluent 

were combined in proportions of 1+1, 1+4, 

and 1+9, respectively. 

13. Reporting Results 

Report Kjeldahl nitrogen, dissolved (test 

ID = 00623), and total (test ID = 00625) 

concentrations as follows: 0.1 to 0.9 mg-N/L, 

one decimal; 1.0 mg-N/L and greater, two 

significant figures. 
Analytical Method 15

NOTE: The laboratory reporting level 

(LRL)—about twice the method detection 

level (MDL) as defined in section 14.1—for 

this method was reduced from 0.2 mg-N/L 

to 0.1 mg-N/L on November 10, 1997 (U.S. 

Geological Survey National Water Quality 

Laboratory Technical Memorandum 98.07, 

1998). 

14. Detection Levels, Precision, and 

Accuracy 

14.1 A method detection level (MDL) 

of 0.05 mg-N/L was estimated for methods 

I-2515-91/4515-91 by using the protocol set 

forth in the U.S Environmental Protection 

Agency (1990). This MDL estimate was 

obtained with homogenous solutions; 

therefore, the MDL for particulate-laden 

samples might be somewhat higher because of 

the increased difficulty of obtaining a 

representative sample for analysis (see 

table 5). 

14.2 Within-run precision (repeatability) 

for methods I-2515-91/4515-91 on the basis of 

55 replicate determinations of the same high 

suspended-solids sample was 3.1 percent. The 

average concentration and standard deviation 

of these replicates was 4.1  0.1 mg-N/L (see 

table 6). 

Between-day precision based on 26 

replicate determinations of the same sample 

between April 8 and April 26, 1991, was 4.8 

percent. The average concentration and 

standard deviation of between-day replicates 

was 4.1  0.2 mg-N/L (see table 7). Figure 3 

shows duplicate data for 564 randomly 

selected samples determined for Kjeldahl 

nitrogen at the NWQL by method I-2515-

91/4515-91 between the dates of 2/11/92 and 

2/13/93. The top panel of this figure presents 

trial 1 concentra-tion in relation to trial 2 

concentration as a scatter plot about the line of 

equal relation. The bottom panel shows a 

scatter plot of the percent difference between 

duplicates in relation to the trial 1 

concentration. About 90 percent of the 300 

samples with Kjeldahl nitrogen concentrations 

 0.5 mg-N/L (10 MDLs) agreed to within 

25 percent. 

14.3 Between-day accuracy for methods 

I-2515-91/4515-91, which is based on 

concentrations determined for reference 

materials provided by the USEPA and the 

USGS, is listed in table 8. In all cases, 

Kjeldahl nitrogen concentrations determined 

by this method were within published control 

limits for these reference materials. 

14.4 The average recovery for 1.00 mg 

of glycine nitrogen added to 94 randomly 

selected samples analyzed during the July–

August experiment was 1.04  0.10 mg-N/L. 

Figure 4 shows that concentrations of Kjeldahl 

nitrogen in samples did not affect spike 

recovery. 

14.5 When the 1,487 data pairs from 

April and July–August experiments were 

combined, the median difference between 

Kjeldahl nitrogen concentrations determined 

by the present (I-2515-91/4515-91) and old 

(I-2552-85/4552-85) methods was about 0.1 

mg-N/L, which is highly significant 

(p < 0.0001) on the basis of a Wilcoxon 

signed rank test. Linear regression analysis of 

these data pairs shows a statistically 

significant (p < 0.0001), nonzero intercept, 

which also suggests a possible additive 

difference between the former and new 

methods. Table 9 lists mean and median 

Kjeldahl nitrogen concentration differences 

determined for filtered- and whole-water 

samples by the former and the new methods as 

well as the results of the regression of the 

former method in relation to the new methods. 

A concentration difference of this magnitude 

was expected because digestion blank 

concentrations ( 0.1 mg-N/L) were 
16 Determination of Ammonium Plus Organic Nitrogen

Table 5. Data used to estimate the method detection level (MDL) for Kjeldahl nitrogen 

determination by methods I-2515-91/4515-91 

[mg-N/L, milligrams nitrogen per liter. Measured concentrations pertain to eight replicate digestions of 

a 0.25 mg-N/L glycine calibrant that was prepared in deionized water that contained an appropriate volume 

of field preservative solution.] 

Replicate 

number 

 Nominal concentration 

(mg-N/L) 

 Measured concentration 

(mg-N/L) 

1 0.25 0.194 

2 .25 .196 

3 .25 .202 

4 .25 .229 

5 .25 .192 

6 .25 .204 

7 .25 .166 

8 .25 .196 

Average concentration (mg-N/L): 

Standard deviation (mg-N/L): 

Number of samples: 

Degrees of freedom: 

One-sided t value (99 percent confidence): 

Method detection level (MDL): 

.197 

.017 

8 

7 

2.998 

0.052 

Table 6. Repeatability (within-run precision, May 3, 1991) data for 55 replicate determinations 

of Kjeldahl nitrogen concentration in the Heidelberg sample by using methods I-2515-91/4515-91 

[mg-N/L, milligrams nitrogen per liter] 

Trial 

number 

Concentration 

(mg-N/L) 

Trial 

number 

Concentration 

(mg-N/L) 

Trial 

number 

Concentration 

(mg-N/L) 

 1 3.97 20 4.12 38 4.13 

 2 4.36 21 4.29 39 4.06 

 3 4.16 22 4.29 40 4.25 

 4 4.19 23 4.09 41 3.94 

 5 4.36 24 4.20 42 4.06 

 6 4.07 25 4.25 43 4.22 

 7 4.21 26 3.99 44 3.93 

 8 4.37 27 4.13 45 4.11 

 9 4.04 28 4.20 46 4.23 

 10 4.21 29 3.88 47 3.99 

 11 4.26 30 4.03 48 4.18 

 12 4.09 31 4.15 49 4.21 

 13 4.33 32 3.94 50 3.97 

 14 4.22 33 3.98 51 4.04 

 15 4.15 34 4.11 52 4.19 

 16 4.34 35 3.89 53 3.90 

 17 4.11 36 4.06 54 4.11 

 18 4.14 37 4.22 55 4.14 

 19 4.24 

 Average concentration (mg/L):

Standard deviation (mg/L):

Relative standard deviation (percent):

4.13 

0.13 

3.1 
Discussion of Results 17

Table 7. Between-day (April 8–26, 1991) precision of digested calibrants and the Heidelberg sample for 

Kjeldahl nitrogen determination by using methods I-2515-91/4515-91 

[ID, identification; mg-N/L, milligrams nitrogen per liter; std. dev., standard deviation; RSD, relative standard 

deviation; , nearly equal to; μm, micrometer] 

Sample 

ID 

Nominal 

concentration 

(mg-N/L) 

Number

of 

samples

Average 

concentration

(mg-N/L) 

Std. dev. 

(mg-N/L) 

RSD 

(percent)

 10.0 24 9.99 0.04 0.38 

 7.5 25 7.52 .08 1.10 

 5.0 23 4.98 .08 1.65 

 2.5 25 2.50 .06 2.30 

 1.25 25 1.27 .06 4.39 

 .5 25 .48 .06 3.51 3  4.1 26 4.14 .20 4.88 

CALIBRANT 1 

CALIBRANT 2 

CALIBRANT 3 

CALIBRANT 4 

CALIBRANT 5 

CALIBRANT 6 

Heidelberg1

Heidelberg2 3  4.1 26 4.14 .20 4.83 

1

Diluted digest was shaken and poured into analyzer cup. 

2

Diluted digest was dispensed into analyzer cup through a 0.45-μm nylon syringe filter. 3

National Water Quality Laboratory consensus concentration. 

not subtracted from concentrations reported 

by the former methods. Indeed, a step 

change of about 0.1 mg-N/L in NWQL 

Kjeldahl nitrogen blind blank concentrations 

is evident after October 1, 1991, when the 

new methods became operational, as shown 

in figure 5. Complete details of the method 

comparison studies can be found in the 

Discussion of Results section that follows. 

DISCUSSION OF RESULTS 

Analytical Methods 

Before beginning validation studies, the 

authors performed several experiments to 

characterize block digester and continuous 

flow analyzer performance. For example, to 

estimate the extent of temperature variation 

throughout the 40 positions within the block 

digester, a batch of 40 test solutions as 

described in section 8 was prepared and 

digested. The first eight tubes in row 1 of the 

block digester contained calibrants and blanks 

(see table 3 and fig. 6b). The remaining 32 

tubes in rows 2 through 5 contained an 

aqueous solution of nicotinic acid with a 

nominal concentration of 7.5 mg-N/L. 

Nicotinic acid was chosen for this study 

because it is one of the most difficult 

compounds that contain organic nitrogen to 

digest by the Kjeldahl method (Bradstreet, 

1965; Jirka and others, 1976; Bowman and 

Delfino, 1982). Digestion time was limited to 

90 minutes at 370°C—less than the time 

required for 100-percent recovery of nicotinic 

acid. The authors hypothesized that recovery 

of nicotinic acid in each tube would indicate 

the relative temperature at each block position. 

That is, tubes with lower nicotinic acid 

recoveries would be correlated with cooler 

positions in the block. Results of this 

experiment are presented in figure 6a, which 

shows isopleths for nicotinic acid 

concentration found in each tube relative to its 

position in the block digester. This figure 

shows that recovery of nicotinic acid 

approached 100 percent only in tubes near the 

center of the block digester and decreased 

radially from the digester's center to its 

perimeter. 

Next, the authors performed a set of 

experiments to assess how the temperature 

gradient detected within the block digester 

would affect method performance for 
18 Determination of Ammonium Plus Organic Nitrogen

0.1 1 10

-100

-75

-50

-25

0

25

50

75

100

(b)

10 method detection levels (MDLs)

DIFFERENCE BETWEEN

DUPLICATES, IN PERCENT

LOG10 KJELDAHL NITROGEN CONCENTRATION,

IN MILLIGRAMS NITROGEN PER LITER, TRIAL 1

0.1

1

10

(a)

Line of equal relation

LOG10 KJELDAHL NITROGEN

CONCENTRATION, IN MILLIGRAMS

NITROGEN PER LITER, TRIAL 2

Figure 3. Duplicate data for 564 randomly selected samples determined at the National Water Quality 

Laboratory by methods I-2515-91/4515-91 between 2/11/92 and 2/13/93. About 90 percent of the 300 

samples with Kjeldahl nitrogen concentrations  0.5 milligram nitrogen per liter (10 MDLs) agreed to 

within  25 percent. Note that several extreme outliers shown in graph (a) do not appear in graph (b), 

because the y-axis scale is limited to differences between trial 1 and trial 2 of plus or minus 100 percent. 
Discussion of Results 19

Table 8. Between-day (April 8–26, 1991) accuracy of digested U.S. Environmental Protection Agency 

and U.S. Geological Survey reference samples for Kjeldahl nitrogen determination by using methods 

I-2515-91/4515-91 

[ID, identification; mg-N/L, milligrams nitrogen per liter; , plus or minus; std. dev., standard deviation; RSD, 

relative standard deviation; LCL, lower control limit; UCL, upper control limit; μL, microliters; mL, milliliters; 

SRWS, standard reference water sample] 

Sample 

ID 

Nominal 

concentration 

(mg-N/L) 

Number

of 

samples 

Average 

concentration

(mg-N/L) 

Std. dev. 

(mg-N/L) 

RSD 

(percent)

 USEPA LOW1

(LCL - UCL)2

 2.50 ± 0.34 

(1.82 – 3.18) 

 13 2.40 0.19 8 

 USEPA3

(LCL - UCL)2

 4.95 ± 0.44 

(4.07 – 5.83) 

 10 5.00 .20 4 

 SRWS N-28 

(LCL - UCL) 

 0.26 ± 0.17 

(-0.08 – 0.60) 

 11 .15 4

 (0.03 – 0.35)

 .10 67 

 SRWS N-29 

(LCL - UCL) 

 1.21 ± 0.21 

(0.79 – 1.63) 

 13 1.02 4

 (0.88 – 1.17)

 .09 8 

1

A solution containing 500 μL USEPA "Nutrient 2" and 400 μL National Water Quality Laboratory HgCl2/NaCl 

preservative solution diluted to 100 mL with deionized water. 2

Confidence interval of 95 percent. 

3

A 1,000 μL USEPA "Nutrient 2" solution and 400 μL National Water Quality Laboratory HgCl2/NaCl 

preservative solution diluted to 100 mL with deionized water. 4

Concentration range. 

0.1 1

80

100

120

SPIKE RECOVERY, IN PERCENT

KJELDAHL NITROGEN CONCENTRATION,

IN M ILLIGRAMS PER LITER

Figure 4. Spike recovery in relation to Kjeldahl nitrogen concentration determined by methods 

I-2515-91/4515-91 during the July–August, 1991 experiment. Number of samples = 94, nitrogen added 

(as glycine) = 1.0 milligram nitrogen per liter (mg-N/L), average nitrogen found = 1.04  0.10 mg-N/L, and 

average percent recovery = 104  10.
20 Determination of Ammonium Plus Organic Nitrogen

Table 9. Statistical data and regression analysis results for Kjeldahl nitrogen methods 

I-2515-91/4515-91 (new methods) and I-2552-85/4552-85 (former methods)

[Mean and median concentration differences, in milligrams nitrogen per liter, are the result of subtracting formermethod concentrations from new-method concentrations. Slopes and y-intercepts are the result of using new-method 

concentrations as the independent (x) variable. Data for surface-water and ground-water samples are combined.] 

Sample 

type 

Number of 

samples 

Median 

difference 

Mean 

difference Slope y-intercept 

Filtered 508 -0.074 -0.091 0.922 0.159 

Whole-water 979 -0.115 -0.049 0.962 0.096 

several real and synthetic samples that 

contain various forms of organic nitrogen. 

Percent recovery and precision of analytical 

results were used to evaluate method 

performance. In this second set of 

experiments, full blocks for each of three 

different test solutions were prepared and 

digested. As before (see section 8), the first 

eight tubes in row 1 of the block digester 

contained calibrants and blanks. The 

remaining 32 tubes in rows 2 through 5 

contained an aqueous solution of nicotinic 

acid with a nominal concentration of 7.5 

mg-N/L in batch one, an aqueous solution of 

adenosine triphosphate (ATP) with a 

nominal concentration of 1.0 mg-N/L in 

batch 2, and a high suspended-solids sample 

with a consensus concentration of 6.3  0.5 

mg-N/L in batch 3. Each batch was 

desolvated in a block digester set at 220C 

for 30 minutes and then transferred to the 

high-temperature block digester set at 

370C. After 15 minutes, all eight tubes 

were removed from row 1, which contained 

calibrants and blanks, and tubes 1 through 4 

from row 2. At 15-minute intervals 

thereafter, tubes 5 through 8 were removed 

from row 2, tubes 1 through 4 from row 3, 

tubes 5 through 8 from row 3, tubes 1 

through 4 from row 4, tubes 5 through 8 

from row 4, tubes 1 through 4 from row 5, 

and tubes 5 through 8 from row 5 (see fig. 

6b). This procedure yielded 4-tube subsets 

of each test solution that had undergone 

digestion at 370C for 15, 30, 45, 60, 75, 90, 

105, and 120 minutes. 

Results of these experiments are 

summarized in figure 7, where symbols 

plotted as a function of digestion time 

represent the average Kjeldahl nitrogen 

concentration measured in each 4-tube subset 

for each of the three test solutions. Error bars 

associated with the symbols represent 1 

standard deviation. As shown in figure 7, 

recovery of nitrogen from digested ATP 

solutions and high suspended-solids samples 

were  95 percent after 15 minutes and 

remained constant at longer digestion times. 

Standard deviations for these two test 

solutions fluctuated randomly as digestion 

time increased. Typically, however, shorter 

digestion times are desirable because digestion 

blank concentrations and variability tend to 

increase as digestion time increases. In 

contrast, but in keeping with the block digester 

temperature gradient experiment, digestion 

times greater than 90 minutes were required to 

approach nitrogen recoveries  95 percent in 

nicotinic acid solution digests. The authors 

interpret the increased precision of nicotinic 

acid concentrations observed at the longest 

digestion time as an indication that the effect 

of the block digester's temperature gradient on 

nicotinic acid recovery decreased substantially 

as digestion time approached 120 minutes. 
Discussion of Results 21
22 Determination of Ammonium Plus Organic Nitrogen

12345678

1

2

3

4

5 (b)

ROW NUMBER, FROM FRONT TO BACK

TUBE POSITION IN BLOCK DIGESTER,

FROM THE FRONT, LEFT TO RIGHT

4.3

4.7

5.1 4.7

5.5 5.1

5.9 5.5

5.9

6.3 6.3

6.7

7.1

2

3

4

5

(a)

Figure 6. Isopleths of nicotinic acid concentration (a), in milligrams nitrogen per 

liter, found in each tube in relation to the position of each tube in the block digester (b).
Discussion of Results 23

15 30 45 60 75 90 105 120

0

1

2

3

4

5

6

7

8

CONCENTRATION, IN MILLIGRAMS

NITROGEN PER LITER

 Adenosine triphosphate (ATP)

 Nicotinic acid

 High suspended-solids sample

DIGESTION TIME AT 370 DEGREES CELSIUS, IN MINUTES

Figure 7. Kjeldahl nitrogen concentration recovered in digests of a high-suspended-solids sample, an 

aqueous solution of adenosine 5' triphosphate, and an aqueous solution of nicotinic acid by methods 

I-2515-91/4515-91 as a function of high-temperature digestion time. Error bars represent +1 standard 

deviation for the average of four determinations of each sample type at specified digestion times. 

On the basis of these results and statistical 

analysis of Kjeldahl nitrogen concentrations 

determined by USGS methods I-2552-

85/4552-85 (370C digestion time 90 

minutes) and I-2515-91/4515-91 (370C 

digestion time =15 minutes), the authors 

conclude as follows: 

1. Digestion times of 15 minutes at 

370C are adequate for full 

recovery of organic nitrogen 

compounds in natural-water 

samples typically received for 

analyses at the NWQL, and 

2. Longer digestion times required for 

quantitative recovery of nicotinic 

acid might contribute to over- or 

underestimation of Kjeldahl 

nitrogen concentrations in samples 

containing nitrate concentration  1 

mg-N/L (see discussion that 

follows).
24 Determination of Ammonium Plus Organic Nitrogen

Statistical Analysis 

The lack of precision in the former 

USGS methods (I-2552-85/4552-85) for 

several years preceding validation of the new 

methods (I-2515-91/4515-91) is indicated by 

NWQL blind blank data for the years 1989 

through 1999 (see fig. 5). In an effort to 

mitigate errors arising from statistical 

comparison of methods with differing 

precision, the authors used medians and 

median-based, nonparametric statistics 

(Wilcoxon signed rank test) in the discussions 

that follow in preference to means and meansbased, parametric paired t-tests. As listed in 

table 9, when filtered- and whole-watersample data from the April and July–August 

experiments are combined, median Kjeldahl 

nitrogen concentrations determined by the 

new methods are about 0.1 mg-N/L less than 

those determined by the former methods. This 

was expected because Kjeldahl nitrogen 

concentrations reported for the former 

methods were not corrected for digestion 

blank concentrations, which are approximately 

equal to this difference. Median concentration 

differences between the two methods were 

comparable during the April and July–August 

experiments, as listed in table 10. 

When data pairs from the April and 

July–August experiment were grouped into 

the categories filtered and whole surface-water 

samples and filtered and whole ground-water 

samples, similar trends were observed. Data 

for each of these four categories are shown as 

scatter plots in figure 8 and as box plots in 

figure 9. As shown in these figures, the 

deviations of plotted data from the line of 

equal relation, which represent the 

concentration difference between the new and 

former methods, was least for unfiltered 

surface-water samples from both studies. 

Additional trends in the data emerge when 

method median differences are calculated for 

the subset of samples in these four categories 

with nitrate plus nitrite concentrations either 

less than or equal to 1.0 mg-N/L or greater 

than 1.0 mg-N/L. The results of such an 

analysis are presented for data from the April 

experiment (table 11) and July–August 

experiments (table 12). 

Inspection of tables 11 and 12 reveals 

that median concentration differences between 

the two methods in all four categories more 

closely matches the median digestion blank 

concentration in the subset of samples with 

nitrate plus nitrite concentrations less than or 

equal to 1 mg-N/L than for the entire 

population. Likewise, larger concentration 

differences than can be accounted for by the 

lack of blank correction in the former methods 

are observed for the subset of samples with 

nitrate plus nitrite concentrations greater than 

1 mg-N/L. For a substantial number of 

samples in these subsets, the differences 

between Kjeldahl nitrogen concentrations 

determined by the former and new methods 

increases as nitrate plus nitrite concentrations 

increase. The authors hypothesize that this 

effect is caused by complex and poorly 

characterized interactions among nitrate, 

ammonium, and organic matter during 

Kjeldahl digestion as discussed in section 3.3. 

The authors further speculate that the much 

longer 370C digestion time of the former 

methods in relation to the new methods—90 

minutes rather than 15 minutes—intensifies 

these undesirable side reactions. 

The concentration difference between 

the new and former methods was positive in a 

single case in the July–August experiment. 

This difference was observed in the subset of 

whole surface-water samples with nitrate plus 

nitrite concentrations greater than 1 mg-N/L 

(see table 12). 
Discussion of Results 25

Table 10. Median difference between paired Kjeldahl nitrogen concentrations determined by 

methods I-2515-91/4515-91 and I-2552-85/4552-85

[Median differences are the result of subtracting methods I-2552-85/4552-85 concentrations from methods 

I-2515-91/4515-91 concentrations. Data for surface-water and ground-water samples are combined. 

mg–N/L, milligrams nitrogen per liter] 

 Median Kjeldahl nitrogen concentration (mg-N/L) 

Data source Number 

of samples I-2515-91 

I-4515-91 

I-2552-85 

I-4552-85 

Median 

difference 

April 411 0.495 0.566 -0.071 

July–August 1,076 .422 .519 -.097 

Both 1,487 .437 .531 -.094 

Careful inspection of original data 

eliminated inadvertent transposal of data 

columns for the two methods during initial 

stages of data analysis as a possible 

explanation for this apparent anomaly. In a 

further effort to account for the anomaly, the 

ammonium concentrations were examined in 

the subset of samples with nitrate plus nitrite 

concentrations greater than 1 mg/L (see tables 

11 and 12). As indicated in table 13, the 

average nitrate plus nitrite and ammonium 

concentrations in the July–August subset have 

higher than average ammonium concentrations 

and, in general, lower than average nitrate plus 

nitrite concentrations than the April subset. 

Furthermore, the authors found that 14 

samples in the July–August subset (126 

samples)—seven each from two USGS 

stations near minimally treated sewage 

discharge outfalls—were largely responsible 

for the observed positive concentration 

difference between the two methods (table 

12). Average ammonium and nitrate plus 

nitrite concentrations were 11.1 and 2.2 mgN/L for the seven samples from station 

9419700 and 9.4 and 2.6 mg-N/L for the seven 

samples from station 9419753. Samples with 

Kjeldahl nitrogen and ammonium 

concentrations in this range would have 

required dilution before analysis. The authors, 

therefore, can only speculate whether dilution 

errors, the atypically high ammonium to 

nitrate ratio (table 13), other unknown factors, 

or some combination of all three are 

responsible for the positive concentration 

difference between the new and former 

methods in this data set. 

This study provides evidence that nitrate 

plus nitrite concentrations greater than 1 mgN/L might cause positive or negative 

interference in Kjeldahl nitrogen determinations. This information should serve as a 

caution to data users who calculate total 

nitrogen concentrations by summing Kjeldahl 

nitrogen and nitrate plus nitrite concentrations. 

Data analysis in this section also 

provides insight into the difficulties of 

comparing Kjeldahl nitrogen (ammonium plus 

organic nitrogen) concentrations with total 

nitrogen concentrations determined by hightemperature combustion methods (U.S. 

Environmental Protection Agency, 1997) or 

alkaline persulfate digestion (D’Elia and 

others, 1977) methods as discussed in 

previous studies (Ameel and others, 1993; 

Kroon, 1993). 
26 Determination of Ammonium Plus Organic Nitrogen

0.05

0.25

0.5

2.5

5

25

50

APRIL DATA Line of equal relation

SET F9

n = 43

APRIL DATA Line of equal relation

SET R9

n = 317

0.05

0.25

0.5

2.5

5

25

50

APRIL DATA Line of equal relation

SET F6

n = 32

APRIL DATA Line of equal relation

SET R6

n = 19

0.05

0.25

0.5

2.5

5

25

50

JULY DATA Line of equal relation

SET F9

n = 416

JULY DATA Line of equal relation

SET R9

n = 601

0.05 0.25 0.5 2.5 5 25 50

0.05

0.25

0.5

2.5

5

25

50

Line of equal relation JULY DATA

SET F6

n = 17

0.05 0.25 0.5 2.5 5 25 50

LOG10 KJELDAHL NITROGEN CONCENTRATION, IN MILLIGRAMS PER LITER (METHODS I-2515-91/4515-91) 

LOG10 KJELDAHL NITROGEN CONCENTRATION,

IN MILLIGRAMS PER LITER (METHODS I-2252-85/4552-85) 

JULY DATA Line of equal relation

SET R6

n = 42

Figure 8. Relation between Kjeldahl nitrogen concentrations determined by methods I-2552-85/ 

4552-85 and I-2515-91/4515-91 in the April and July–August experiments for each of the four water types. 

Sets F9, R9, F6, and R6 were composed of filtered and whole surface-water samples and filtered and 

whole ground-water samples, respectively. The letter n represents the number of points in each set. 
Discussion of Results 27
28 Determination of Ammonium Plus Organic Nitrogen

Table 11. Effect of nitrate plus nitrite concentrations on median differences between Kjeldahl nitrogen 

concentrations determined by U.S. Geological Survey methods I-2515-91/4515-91 and 

I-2552-85/4552-85 (April data set) 

[mg-N/L, milligrams nitrogen per liter; , less than or equal to; >, greater than] 

All samples 

 Median Kjeldahl nitrogen concentration (mg-N/L) Water type Number of 

samples 

I-2515-91 

I-4515-91 

I-2552-85 

I-4552-85 Difference 

Filtered surface 43 0.475 0.636 -0.161 

Whole surface 317 .561 .592 -.031 

Filtered ground 32 .143 .325 -.182 

Whole ground 19 .144 .281 -.137 

 Samples with NO3

-

 + NO2

-

 concentrations (1 mg-N/L)

Filtered surface 25 .375 .442 -.067 

Whole surface 249 .459 .496 -.037 

Filtered ground 20 .231 .33 -.099 

Whole ground 10 .149 .207 -.058 

 Samples with NO3

-

 + NO2

-

 concentrations (> 1 mg-N/L)

Filtered surface 18 .843 1.281 -.438 

Whole surface 68 1.217 1.48 -.263 

Filtered ground 12 .075 .319 -.244 

Whole ground 9 .144 .451 -.307 

Table 12. Effect of nitrate plus nitrite concentrations on median differences between Kjeldahl nitrogen 

concentrations determined by U.S. Geological Survey methods I-2515-91/4515-91 and 

I-2552-85/4552-85 (July–August data set) 

[mg-N/L, milligrams nitrogen per liter; , less than or equal to; >, greater than]

All samples 

 Median Kjeldahl nitrogen concentration (mg-N/L) Water type Number of 

samples I-2515-91 

I-4515-91 

 I-2552-85 

I-4552-85 

 

Difference 

Filtered surface 416 0.352 0.452 -0.100 

Whole surface 601 .566 .616 -.050 

Filtered ground 17 .138 .579 -.441 

Whole ground 42 .109 .382 -.273 

 Samples with NO3

- + NO2-

 concentrations (1 mg-N/L)

Filtered surface 341 .311 .413 -.102 

Whole surface 475 .455 .53 -.075 

Filtered ground 7 .272 .446 -.174 

Whole ground 15 .064 .172 -.108 

 Samples with NO3

- + NO2-

 concentrations (> 1 mg-N/L)

Filtered surface 75 .582 .713 -.131 

Whole surface 126 1.123 1.017 +.106 

Filtered ground 10 .073 .573 -.5 

Whole ground 27 .122 .493 -.371 

 
Conclusions 29

Table 13. Average concentrations of nitrate plus nitrite and ammonium for sample subsets 

with nitrate plus nitrite concentrations greater than 1 milligram nitrogen per liter 

[mg-N/L, milligrams nitrogen per liter]

Average concentration (mg-N/L)

Water type April July – August 

NO2

- + NO3- NH4+ NO2- + NO3- NH4+

Filtered surface (F9) 11.1 0.12 2.5 2.1 

Whole surface (R9) 6.0 .62 2.4 1.6 

Filtered ground (F6) 5.0 .00 19.6 .04 

Whole ground (R6) 8.3 .00 6.1 .06 

CONCLUSIONS 

U.S. Geological Survey (USGS) 

methods I-2515-91/4515-91 for determining 

Kjeldahl nitrogen in filtered and whole-water 

samples were implemented at the National 

Water Quality Laboratory (NWQL) on 

October 1, 1991. The methods, in which hightemperature block digestion is followed by 

automated, colorimetric ammonium 

determination that includes digest cleanup by 

continuous flow gas diffusion, have method 

detection levels of about 0.05 mg-N/L as 

estimated by using U.S Environmental 

Protection Agency protocols. Within-run 

precision (repeatability) and between-day 

precision for filtered and whole-water samples 

analyzed by methods I-2515-91/4515-91 are 

about 3 percent and 5 percent, respectively. 

Duplicate determinations for these methods 

typically agree to within 25 percent, and spike 

recoveries are typically 100 ± 20 percent. 

Data provided in this report demonstrate that 

digestion times of 15 minutes at 370C are 

adequate for full recovery of organic nitrogen 

compounds in natural-water samples typically 

received for analyses at the NWQL. 

Furthermore, these data suggest that digestion 

times longer than 15 minutes might contribute 

to over- or underestimation of Kjeldahl 

nitrogen concentrations in samples with nitrate 

plus nitrite concentrations  1 mg-N/L. 

Statistical analysis of paired data 

collected during method validation 

experiments and NWQL blind blank data are 

consistent with a step-change decrease of 

about 0.1 mg-N/L in Kjeldahl nitrogen 

concentrations reported by the NWQL 

beginning on October 1, 1991. On that date, 

USGS methods I-5225-85/4525-85 were 

replaced by USGS methods I-2515-91/4515-

91. Generally, this concentration difference 

resulted from the lack of digestion blank 

correction in methods I-5225-85/4525-85. 

This work documents the importance of 

considering factors, such as digestion time and 

whether digestion blank corrections have been 

applied, when Kjeldahl nitrogen data 

produced at different laboratories are 

compared. It also provides insight into the 

difficulties of comparing Kjeldahl nitrogen 

concentration with true total nitrogen 

concentrations. This work also indicates that 

nitrate plus nitrite concentrations greater than 

1 mg-N/L might cause positive or negative 

interference in Kjeldahl nitrogen 

determinations. This information should serve 

as a caution to data users who either combine 

Kjeldahl nitrogen concentrations from 

different laboratories, or who calculate total 

nitrogen concentrations by summing Kjeldahl 

nitrogen and nitrate plus nitrite concentrations, 

or both. 
30 Determination of Ammonium Plus Organic Nitrogen

REFERENCES CITED 

Ameel, J.J., Axler, R.P., and Owen, C.J., 

1993, Persulfate digestion for 

determination of total nitrogen and 

phosphorus in low-nutrient waters: 

American Environmental Laboratory, 

no. 10/93, p. 1, 8, 10, and 11. 

American Public Health Association, 1992, 

Standard methods for the examination 

of water and wastewater (18th ed.): 

Washington, D.C., American Public 

Health Association Inc., p. 4–94. 

American Society for Testing and Materials, 

1995, Annual book of ASTM 

standards, Section 11, Water (D1193, 

Standard specification for reagent 

water): Philadelphia, v. 11.01, 

p. 122–124. 

Bowman, G.T., and Delfino, J.J., 1982, 

Determination of total Kjeldahl 

nitrogen and total phosphorus in 

surface waters and wastewaters: 

Journal of the Water Pollution Control 

Federation, v. 54, no. 9, p. 1324–1330. 

Bradstreet, R.B., 1965, The Kjeldahl method 

for organic nitrogen: New York, 

Academic Press, p. 239. 

D’Elia, C.F., Steudler, P.F., and Corwin, N., 

1977, Determination of total nitrogen 

in aqueous samples using persulfate 

digestion: Limnology and 

Oceanography, v. 22, p. 760–764. 

Draper, N.R., and Smith, Harry, 1966, 

Applied regression analysis: New 

York, Wiley, 407 p. 

Fishman, M.J., ed., 1993, Methods of 

analysis by the U.S. Geological Survey 

National Water Quality Laboratory—

Determination of inorganic and organic 

constituents in water and fluvial 

sediments: U.S. Geological Survey 

Open-File Report 93–125, 217 p. 

Fishman, M.J., and Friedman, L.C., eds., 

1989, Methods for determination of 

inorganic substances in water and 

fluvial sediments: U.S. Geological 

Survey Techniques of WaterResources Investigations, book 5, 

chap. A1, 545 p. 

Harfmann, R.G., and Crouch, S.R., 1989, 

Kinetic study of Berthelot reaction 

steps in the absence and presence of 

coupling reagents: Talanta, v. 36, 

p. 261–269. 

Jirka, A.M., Carter, M.J., May, Dorothy, and 

Fuller, F.D., 1976, Ultramicro 

semiautomated method for 

simultaneous determination of total 

phosphorus and total Kjeldahl nitrogen 

in wastewaters: Environmental 

Science and Technology, v. 10, no. 10, 

p. 1038–1044. 

Kroon, Henie, 1993, Determination of 

nitrogen in water: Comparison of a 

continuous flow method with on-line 

UV digestion with the original 

Kjeldahl method: Analytica Chimica 

Acta, v. 276, p. 278–293. 

Patton, C.J., and Crouch, S.R., 1977, 

Spectrophotometric and kinetics 

investigation of the Berthelot reaction 

for the determination of ammonia: 

Analytical chemistry, v. 49, 

p. 464–469. 

Patton, C.J., and Truitt, E.P., 1992, Methods 

of analysis by the U.S. Geological 

Survey National Water Quality 

Laboratory—Determination of total 

phosphorus by a Kjeldahl digestion 

method and an automated colorimetric 

finish that includes dialysis: U.S. 

Geological Survey Open-File Report 

92-146, 39 p. 

Patton, C.J., and Wade, A.P., 1997, 

Continuous flow analyzers, in Ewing, 

Galen, ed., Analytical instrumentation 

handbook (2d ed.): New York, Marcel 

Dekker, p. 125–220. 
References Cited 31

Reardon, J., Foreman, J.A., and Searcy, 

R.L., 1966, New reactants for 

determination of ammonia: Clinica 

Chimica Acta, v. 14, p. 403–405. 

Seifter, Eli, Kambosos, Demetrios, and 

Chanas, Alexandra, 1971, 

Determination of dissloved CO2 in 

biological fluids, in Advances in 

automated analysis, Technicon 

International Congress, 1970, Volume 

I (Clinical Analysis): Miami, Florida, 

Thurman Associates, p. 509–511. 

Swartz, L.M., 1976, Nonlinear calibration 

curves: Analytical Chemistry, v. 48, 

no. 14, p. 2287–2290. 

________1977, Nonlinear calibration: 

Analytical Chemistry, v. 49, no. 13, 

p. 2062–2068. 

________1979, Calibration curves with 

nonuniform variance: Analytical 

Chemistry, v. 51, no. 6, p. 723–727. 

U.S. Environmental Protection Agency, 

1983a, Methods for chemical analysis 

of water and wastes: Cincinnati, Ohio, 

Environmental Monitoring and 

Support Laboratory, USEPA-600/4-

79-020, p. 365.1-1 through 365.4-1. 

________1983b, Methods for chemical 

analysis of water and wastes: 

Cincinnati, Ohio, Environmental 

Monitoring and Support Laboratory, 

USEPA-600/4-79-020, p. 351.2-1 

through 351.2-5. 

________1990, Guidelines establishing test 

procedures for the analysis of 

pollutants (App. B, Part 136, 

Definition of procedures for the 

determination of the method detection 

limit—Revision 1.11): revised as of 

July 1, 1999, p. 537–539. 

________1993, Methods for the 

determination of inorganic substances 

in environmental samples: Cincinnati, 

Ohio, Environmental Monitoring and 

Support Laboratory, EPA/600/R93/100, August 1993, 79 p. 

________1997, Determination of carbon 

and nitrogen in sediments and 

particulates of estuarine/coastal waters 

using elemental analysis: Cincinnati, 

Ohio, National Exposure Research 

Laboratory, Office of Research and 

Development, Method 440.0, 10 p., 

accessed May 18, 2000, at URL 

http://www.epa.gov/nerlcwww/ 

m440_0.pdf 

U.S. Geological Survey, 1994, New 

preservation techniques for nutrient 

samples: Office of Water Quality 

Technical Memorandum 94.16, 

accessed May 18, 2000, at URL 

http://water.usgs.gov/public/admin/ 

memo/QW/qw94.16.txt 

________1998, Reporting level changes for 

volatile organic compounds (schedules 

2020/2021), inductively coupled 

plasma–atomic emission spectrometry 

(ICP–AES), ammonia plus organic 

nitrogen and phosphorus (microKjeldahl) in water methods at the 

National Water Quality Laboratory: 

National Water Quality Laboratory 

Technical Memorandum 98.07, 

accessed February 23, 2000, at URL 

http://wwwnwql.cr.usgs.gov/Public/ 

tech_memos/nwql.98-07.html 

________1999, Changes in field treatment 

protocols and bottle types for wholewater samples collected for total 

ammonium plus organic nitrogen and 

total phosphorus determinations: 

Office of Water Quality Technical 

Memorandum 99.04, accessed 

May 18, 2000, at URL 

http://water.usgs.gov/public/admin/ 

memo/QW/qw99.04.txt