349.0-1

Method 349.0

 Determination of Ammonia in Estuarine and Coastal Waters

 by Gas Segmented Continuous Flow Colorimetric Analysis

Jia-Zhong Zhang, Cooperative Institute for Marine and Atmospheric Studies

Rosenstiel School of Marine and Atmospheric Science/AOML, NOAA

University of Miami, Miami, FL 33149

Peter B. Ortner, Charles J. Fischer, and Lloyd D. Moore, Jr., National Oceanic and

Atmospheric Administration, Atlantic Oceanographic and Meteorological Laboratory,

Ocean Chemistry Division, Miami, FL 33149

Project Officer

Elizabeth J. Arar

Version 1.0

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Version 1.0 September 1997 349.0-2

Method 349.0

 Determination of Ammonia in Estuarine and Coastal Waters

 by Gas Segmented Continuous Flow Colorimetric Analysis

1.0 Scope and Application

1.1 This method provides a procedure for the

determination of ammonia in estuarine and coastal

waters. The method is based upon the indophenol

reaction, here adapted to automated gas-segmented

1-5

continuous flow analysis.

The term ammonia as used in this method denotes total

concentration of ammonia, including both chemical

forms, NH3 and NH4. Because ionization of NH4 has a

+ + 

pK value of about 9.3, NH is the dominant chemical 4

+ 

form in natural waters. At pH of 8.2 and 25oC only 8.1%

is present as NH3, the form that can be toxic to fish and

other aquatic organisms.

The concentration of ammonia in estuarine and coastal

water shows considerable temporal and spatial variability.

It rarely exceeds 0.005 mg N/L in oxygenated, unpolluted

estuarine and coastal water, but in anoxic water, the

amount of ammonia can be as high as 0.28 mg N/L.6

Although other forms of nitrogen contribute to primary

productivity and nutrient cycling in marine and estuarine

waters, ammonia is particularly important. Because

ammonia represents the most reduced form of inorganic

nitrogen available, it is preferentially assimilated by

phytoplankton. Whereas nitrate is the source of nitrogen,

it must first be reduced to ammonia before it can be

assimilated and incorporated into amino acids and other

compounds. Ammonia is released during the

decomposition of organic nitrogen compounds by

proteolytic bacteria, but also excreted directly by

invertebrates along with urea and peptides. In regions of 7

coastal upwelling, ammonia released by zooplankton can

play a significant role in supplying the nitrogen that

supports phytoplankton production.8

Chemical Abstracts Service

Analyte Registry Numbers (CASRN)

___________________________________________

Ammonia 7664-41-7

___________________________________________

1.2 A statistically determined method detection limit

(MDL) of 0.3 μg N/L has been determined by one

9

laboratory from seawaters of four different salinities. The

method is linear to 4.0 mg N/L using a Flow Solution

System (Alpkem, Wilsonville, Oregon).

1.3 Approximately 60 samples per hour can be

analyzed.

1.4 This method should be used by analysts both

experienced in the use of automated gas segmented

continuous flow colorimetric analyses, and also familiar

with matrix interferences and the procedures used in their

correction. A minimum of 6-months experience under the

close supervision of a qualified analyst is recommended.

2.0 Summary of Method 

2.1 The automated gas segmented continuous flow

colorimetric method is used for the analysis of ammonia

concentration. Ammonia in solution reacts with alkaline

phenol and NaDTT (Sect. 7.2.5) at 60oC to form

indophenol blue in the presence of sodium

nitroferricyanide as a catalyst. The absorbance of

indophenol blue at 640 nm is linearly proportional to the

concentration of ammonia in the sample. A small

systematic negative error caused by differences in the

refractive index of seawater and reagent water, and a

positive error caused by the matrix effect on the color

formation, may be corrected for during data processing.

3.0 Definitions

3.1 Calibration Standard (CAL) -- A solution

prepared from the primary dilution standard solution or

stock standard solution containing analytes. The CAL

solutions are used to calibrate the instrument response

with respect to analyte concentration.

3.2 Laboratory Fortified Blank (LFB) -- An aliquot of

reagent water to which known quantities of the method

analytes are added in the laboratory. The LFB is analyzed
 349.0-3 Version 1.0 September 1997

exactly like a sample, and its purpose is to determine 3.9 Stock Standard Solution (SSS) -- A concentrated

whether method performance is within acceptable control solution of method analyte prepared in the laboratory

limits, and whether the laboratory is capable of making using assayed reference compounds or purchased from

accurate and precise measurements. This is a standard a reputable commercial source.

prepared in reagent water that is analyzed as a sample.

3.3 Laboratory Fortified Sample Matrix solution prepared in the laboratory from stock standard

(LFM) -- An aliquot of an environmental sample to which solutions and diluted as needed to prepare calibration

known quantities of the method analytes are added in the solutions and other needed analyte solutions.

laboratory. The LFM is analyzed exactly like a sample,

and its purpose is to determine whether the sample matrix 3.11 Quality Control Sample (QCS) -- A solution of

contributes bias to the analytical results. The background method analyte of known concentrations which is used to

concentrations of the analytes in the sample matrix must fortify an aliquot of LRB or sample matrix. The QCS is

be determined in a separate aliquot and the measured obtained from a source external to the laboratory and

values in the LFM corrected for background different from the source of calibration standards. It is

concentrations. used to check laboratory performance with externally

3.4 Laboratory Reagent Blank (LRB) -- An aliquot of

reagent water that is treated exactly as a sample including 3.12 Synchronization Peak Solution -- A

exposure to all labware, equipment, and reagents that are synchronization peak is required by most data acquisition

used with other samples. The LRB is used to determine programs to initialize the peak finding parameters. The

if method analytes or other interferences are present in first cup in every run must always be identified as a SYNC

the laboratory environment, the reagents, or apparatus. sample. The SYNC sample is usually a high

3.5 Linear Dynamic Range (LDR) -- The absolute generates a peak at least 25% of full scale. 

quantity or concentration range over which the instrument

response to an analyte is linear.

3.6 Method Detection Limit (MDL) -- The minimum

concentration of an analyte that can be identified,

measured and reported with 99% confidence that the

analyte concentration is greater than zero.9

3.7 Reagent Water (RW) -- Type 1 reagent grade

water equal to or exceeding the standards established by

the American Society for Testing and Materials (ASTM).

Reverse osmosis systems or distilling units followed by

Super-Q Plus Water System that produce water with 18

megohm resistance are examples of acceptable water

sources. To avoid contamination of ammonia from the air,

the reagent water should be stored in a sealed or a

collapsible container and used the day of preparation.

3.8 Refractive Index (RI) -- The ratio of the velocity

of light in a vacuum to that in a given medium. The

relative refractive index is the ratio of the velocity of light

in two different media, such as estuarine or sea water

versus reagent water. The correction for this difference is

referred to as refractive index correction in this method.

3.10 Primary Dilution Standard Solution (PDS) -- A

prepared test materials.

concentration standard, but can be any sample that

3.13 Color SYNC Peak Solution -- A colored solution

used to produce a synchronization peak in the refractive

index measurement in which no color reagent is pumped

through system. 

3.14 Sensitivity Drift -- The change in absorbance for

a given concentration of analyte due to instrumental or

chemical drift during the course of measurement.

3.15 Matrix Effect -- The change of absorbance in

different matrices due to the effect of ionic strength and

composition on the kinetics of color forming reactions.

4.0 Interferences

4.1 Hydrogen sulfide at concentrations greater than 2

mg S/L can negatively interfere with ammonia analysis.

Hydrogen sulfide in samples should be removed by

acidification with sulfuric acid to a pH of about 3, then

stripping with gaseous nitrogen.

4.2 The addition of sodium citrate and EDTA

complexing reagent eliminates the precipitation of

calcium and magnesium hydroxides when calcium and 
Version 1.0 September 1997 349.0-4

magnesium in seawater samples mix with high pH (about 6.1.6 Nitrogen gas ( high-purity grade, 99.99%).

13) reagent solution.4

4.3 Sample turbidity is eliminated by filtration or

centrifugation after sample collection.

4.4 As noted in Section 2.1 refractive index and salt

error interferences occur when sampler wash solution

and calibration standards are not matched with samples

in salinity, but are correctable. For low concentration

samples (< 20 μg N/L), low nutrient seawater (LNSW)

with salinity matched to samples, sampler wash solutions

and calibration standards is recommended to eliminate

matrix interferences.

5.0 Safety

5.1 Water samples collected from the estuarine and

coastal environment are rarely hazardous. However, the

individual who collects samples should use proper

technique.

5.2 Good laboratory technique should be used when

preparing reagents. Laboratory personnel should obtain

material safety data sheets (MSDS) for al chemicals used

in this method. A lab coat, safety goggles, and gloves

should be worn when handling the concentrated acid.

5.3 Chloroform is used as a preservative in this

method. Use in a properly ventilated area, such as a fume

hood.

6.0 Equipment and Supplies

6.1 Gas Segmented Continuous Flow Autoanalyzer

Consisting of:

6.1.1 Automatic sampler.

6.1.2 Analytical cartridge with reaction coils and heater.

6.1.3 Proportioning pump.

6.1.4 Spectrophotometer equipped with a tungsten lamp

(380-800 nm) or photometer with a 640 nm interference

filter (maximum 2 nm bandwidth).

6.1.5 Strip chart recorder or computer based data

acquisition system.

6.2 Glassware and Supplies

6.2.1 Gaseous ammonia concentration in the laboratory

air should be minimal to avoid sample or reagent

contamination. Remove any NH4OH solution stored in the

laboratory. Smoking should be strictly forbidden. An air

filtration unit might also be used to obtain ammonia-free

lab air.

6.2.2 All labware used in the analysis must be free of

residual ammonia to avoid sample or reagent

contamination. Soaking with laboratory grade detergent,

rinsing with tap water, followed by rinsing with 10% HCl

(v/v) and then thoroughly rinsing with reagent water was

found to be sufficient when working at moderate and high

concentration of ammonia. Ammonia is known for its high

surface reactivity. When working at low levels of

10

ammonia (< 20 μg N/L), further cleaning of labware is

mandatory. Plastic bottles and glass volumetric flasks

should be cleaned in an ultrasonic bath with reagent

water for 60 minutes. Bottles and sample tubes made of

glass can be easily cleaned by boiling in reagent water.

Repeat the cleaning process with fresh reagent water

prior to use if necessary.

6.2.3 Automatic pipetters with disposable pipet tips

capable of delivering volumes ranging from 100 μL to

1000 μL and 1 mL to 10 mL.

6.2.4 Analytical balance, with accuracy to 0.1 mg, for

preparing standards.

6.2.5 60-mL glass or high density polyethylene sample

bottles, glass volumetric flasks and glass sample tubes.

6.2.6 Drying oven.

6.2.7 Desiccator.

6.2.8 Membrane filters with 0.45 μm nominal pore size.

Plastic syringes with syringe filters.

6.2.9 Centrifuge.

6.2.10 Ultrasonic water bath cleaner. 

7.0 Reagents and Standards

Note: All reagents must be of analytical reagent grade.
 349.0-5 Version 1.0 September 1997

7.1 Stock Reagent Solutions 7.2.4 Phenol Solution - Dissolve 1.8 g of solid phenol

7.1.1 Complexing Reagent - Dissolve 140 g of sodium

citrate dihydrate(Na C H O .2H O, FW 294.11), 5 g of solution fresh daily. 3 6 5 7 2

sodium hydroxide (NaOH, FW 40) and 10 g of disodium

EDTA (Na2C10H14O8N2.2H2O, FW 372.24), in 7.2.5 NaDTT Solution - Dissolve 0.5 g of sodium

approximately 800 mL of reagent water, mix and dilute to

1 L with reagent water. The pH of this solution is

approximately 13. This solution is stable for 2 months.

7.1.2 Stock Ammonium Sulfate Solution (100 mg N/L) -

Quantitatively transfer 0.4721 g of pre-dried (105 C for 2 o

hours) ammonium sulfate ((NH4) 2SO4, FW 132.15) to a

1000 mL glass volumetric flask containing approximately

800 mL of reagent water and dissolve the salt. Add a few

drops of chloroform as a preservative. Dilute the solution

to the mark with reagent water. Store in a glass bottle in

the refrigerator at 4 C. It is stable for 2 months. o 11

7.1.3 Low Nutrient Sea Water (LNSW) - Obtain natural

low nutrient seawater from surface water of the Gulf

Stream or Sargasso Sea (salinity 36 %o, < 7 μg N/L) and

filter it through 0.3 micron pore size glass fiber filters. If

this is not available, commercial low nutrient sea water

( < 7 μg N/L) with salinity of 35 %o (Ocean Scientific

International, Wormley, U.K. ) can be substituted. NOTE:

Don’t use artificial seawater in this method.

7.2 Working Reagents

7.2.1 Brij-35 Start-up Solution - Add 2 mL of Brij-35

surfactant (ICI Americas, Inc.) to 1000 mL reagent water

and mix gently.

Note: Brij-35 is a trade name for polyoxyethylene(23)

lauryl ether (C12H25(OCH2CH2)23OH, FW=1199.57, CASRN

9002-92-0).

7.2.2 Working Complexing Reagent - Add 1 mL Brij-35

to 200 mL of stock complexing reagent, mix gently.

Prepare this solution daily. This volume of solution is

sufficient for an 8-hour run.

7.2.3 Sodium Nitroferricyanide Solution - Dissolve 0.25

g of sodium nitroferricyanide (Na2Fe(CN)5NO.2H2O, FW

297.97) in 400 mL of reagent water, dilute to 500 mL with

reagent water. Store in an amber bottle at room

temperature.

(C6H5OH, FW 94.11) and 1.5 g of sodium hydroxide

(NaOH, FW 40) in 100 mL of reagent water. Prepare this

hydroxide (NaOH, FW 40) and 0.2 g dichloroisocyanuric

acid sodium salt (NaDTT, NaC3Cl2N3O3 , FW 219.95) in

100 mL of reagent water. Prepare this solution fresh daily.

7.2.6 Colored SYNC Peak Solution - Add 50 μL of blue

food coloring solution to 1000 mL reagent water and mix

thoroughly. Further dilute this solution to obtain a peak of

between 25 to 100 percent full scale according to the

AUFS setting used for refractive index measurement.

7.2.7 Primary Dilution Standard Solution - Prepare a

primary dilution standard solution ( 5 mg N/L) by diluting

5.0 mL of stock standard solution to 100 mL with reagent

water. Prepare this solution daily.

Note: This solution should be prepared to give an

intermediate concentration appropriate for further dilution

in preparing the calibration solutions. Therefore, the

concentration of a primary dilution standard solution must

be adjusted according to the desired concentration range

of calibration solutions. 

7.2.8 Calibration Standards - Prepare a series of

calibration standards (CAL) by diluting suitable volumes

of a primary dilution standard solution (Section 7.2.7) to

100 mL with reagent water or low nutrient seawater.

Prepare these standards daily. The concentration range

of calibration standards should bracket the expected

concentrations of samples and not span more than two

orders of magnitude. At least five calibration standards

with equal increments in concentration should be used to

construct the calibration curve.

When working with samples of a narrow range of

salinities (± 2 %o) or samples containing low ammonia

concentration (< 20 μg N/L), it is recommended that the

CAL solutions be prepared in Low Nutrient Seawater

(Section 7.1.4) diluted to the salinity of samples, and the

Sampler Wash Solution also be Low Nutrient Seawater

(Section 7.1.4) diluted to the same salinity. NOTE: If this

procedure is employed, it is not necessary to perform the

matrix effect and refractive index corrections outlined in

Sections 12.2 and 12.3. 
Version 1.0 September 1997 349.0-6

When analyzing samples of moderate and high ammonia 8.1.4 Turbid samples must be filtered through a 0.45

concentration (> 20 μg N/L) with varying salinities,

calibration standard solutions and sampler wash

solutions can be prepared in reagent water. The

corrections for matrix effect and refractive index should be

subsequently applied (Sections 12.2 and 12.3).

7.2.9 Saline Ammonia Standards - If CAL solutions are

not prepared to match sample salinity, then saline centrifugation.

ammonia standards must be prepared in a series of

salinities in order to quantify the matrix effect (the change

in the colorimetric response of ammonia due to the are used for sample storage. Sample bottles should be

change in the composition of the solution). The following rinsed 3 times with about 20 mL of sample, shaking with

dilution of Primary Dilution Standard Solution (Section the cap in place after each rinse. Pour the rinse water into

7.2.7) and LNSW with reagent water to 100 mL in the cap to dissolve and rinse away salt crusts trapped in

volumetric flasks, are suggested. the threads of the cap. Finally, fill the sample bottle about

Salinity Volume of Volume of Conc.

(%o) LNSW(mL) PDS(mL) mg N/L

_______________________________________

0 0 2 .10

9 25 2 .10

18 50 2 .10

27 75 2 .10

35 98 2 .10

_______________________________________

8.0 Sample Collection, Preservation and

Storage

8.1 Sample Collection - Samples collected for

nutrient analyses from estuarine and coastal waters are

normally collected using one of two methods: hydrocast

or submersible pump systems.

8.1.1 A hydrocast uses a series of sampling bottles

(Niskin, Go-Flo or equivalent) attached at fixed intervals to

a hydro wire. These bottles are sent through the water

column open and are closed either electronically or via a

mechanical messenger when the bottles have reached

the desired depth.

8.1.2 In a submersible pump system, a weighted hose

is sent to the desired depth in the water column and water

is pumped from that depth to the deck of the ship for

sample processing.

8.1.3 For collecting surface samples, an acid - cleaned

plastic bucket or a large plastic bottle can be used as 

convenient samplers. Wash the sampler three times with

sample water before collecting samples.

μm membrane filter as soon as possible after collection.

Wash the filter with reagent water before use. Pass at

least 100 mL of sample through the filter and discard

before taking the final sample. Care must be taken to

avoid the contamination of ammonia especially handling

low concentrations of ammonia ( < 20 μg N/L ) samples.10

An alternative technique to remove particulate is

8.1.5 60-mL glass or high density polyethylene bottles

3/4 full, and screw the cap on firmly.

8.2 Sample Preservation - After collection and

filtration or centrifugation, samples should be analyzed as

soon as possible. If samples will be analyzed within 3

hours then keep refrigerated in tightly sealed, glass or

high density polyethylene bottles in the dark at 4 C until

o

the analysis can be performed.

8.3 Sample Storage - At low concentrations of

ammonia (< 20 μg N/L), no preservation technique is

satisfactory. Samples must be analyzed within 3 hours of

collection. At moderate and high concentrations of

ammonia (> 20 μg N/L) samples can be preserved by the

addition of 2 mL of chloroform per liter of sample and

refrigerated in the dark at 4 C. Samples can be stored in o

either glass or high density polyethylene bottles. A

maximum holding time for preserved estuarine and

coastal water samples with moderate to high

concentrations of ammonia is two weeks.12

9.0 Quality Control

9.1 Each laboratory using this method is required to

implement a formal quality control (QC) program. The

minimum requirements of this program consists of an

initial demonstration of performance, continued analysis

of Laboratory Reagent Blanks (LRB), laboratory

duplicates and Laboratory Fortified Blanks (LFB) with

each set of samples as a continuing check on

performance.
 349.0-7 Version 1.0 September 1997

9.2 Initial Demonstration of Performance then these samples must be diluted and reanalyzed. 

(Mandatory)

9.2.1 The initial demonstration of performance is used (Mandatory)

to characterize instrument performance by determining

the MDL and LDR and laboratory performance by

analyzing quality control samples prior to analysis of should analyze at least one LRB with each set of

samples using this method. samples. LRB data are used to assess contamination

9.2.2 A method detection limit (MDL) should be in the LRB exceed the MDL, then laboratory or reagent

established for the method analyte, using a low level

seawater sample containing, or fortified at, approximately

5 times the estimated detection limit. To determine MDL

values, analyze at least seven replicate aliquots of water

which have been processed through the entire analytical

method. Perform all calculations defined in the method

and report concentration in appropriate units. Calculate

the MDL as follows:

MDL = (t)(S) samples. The LFB must be at a concentration within the

where, S = the standard deviation of the calculate accuracy as percent recovery. If the recovery of

replicate analyses the analyte falls outside the required control limits of 90

-110%, the source of the problem should be identified

 t = Student's t value for n-1 degrees of and resolved before continuing the analyses.

freedom at the 99% confidence 

limit; t = 3.143 for six degrees 9.3.3 The laboratory must use LFB data to assess

of freedom. 

MDLs should be determined every 6 months or whenever

a significant change in background or instrument

response occurs or a new matrix is encountered.

 

9.2.3 The LDR should be determined by analyzing a

minimum of eight calibration standards ranging from

0.002 to 2.00 mg N/L across all sensitivity settings Upper Control Limit = x + 3S

(Absorbance Units Full Scale output range setting) of the Lower Control Limit = x - 3S

detector. Standards and sampler wash solutions should

be prepared in low nutrient seawater with salinities similar The optional control limits must be equal to or better than

to that of samples to avoid the necessity to correct for salt the required control limits of 90-110%. After each 5 to 10

error, or refractive index. Normalize responses by new recovery measurements, new control limits can be

multiplying the response by the Absorbance Units Full calculated using only the most recent 20 to 30 data

Scale output range setting. Perform the linear regression points. Also the standard deviation (S) data should be

of normalized response vs. concentration and obtain the used to establish an ongoing precision statement for the

constants m and b, where m is the slope and b is the y- level of concentrations included in the LFB. These data

intercept. Incrementally analyze standards of higher must be kept on file and available for review.

concentration until the measured absorbance response,

R, of a standard no longer yields a calculated

concentration C , that is within 100 ± 10% of known c

concentration, C, where C = (R-b)/m. That concentration c 

defines the upper limit of the LDR for the instrument.

Should samples be encountered that have a

concentration that is $ 90% of the upper limit of LDR,

9.3 Assessing Laboratory Performance

9.3.1 Laboratory Reagent Blank (LRB) - A laboratory

from the laboratory environment. Should an analyte value

contamination should be suspected. When the LRB value

constitutes 10% or more of the analyte concentration

determined for a sample, duplicates of the sample must

be prepared and analyzed again after the source of

contamination has been corrected and acceptable LRB

values have been obtained.

9.3.2 Laboratory Fortified Blank (LFB) - A laboratory

should analyze at least one LFB with each set of

daily calibration range. The LFB data are used to

laboratory performance against the required control limits

of 90 -110%. When sufficient internal performance data

become available (usually a minimum of 20 to 30

analyses), optional control limits can be developed from

the percent mean recovery (x) and standard deviation (S)

of the mean recovery. These data can be used to

establish the upper and lower control limits as follows:
Version 1.0 September 1997 349.0-8

9.4 Assessing Analyte Recovery - Laboratory 10.3 Analyze the calibration standards, in duplicate,

Fortified Sample Matrix (LFM) before the actual samples.

9.4.1 A laboratory should add a known amount of 10.4 The calibration curve containing five data points

analyte to a minimum of 5% of the total number of

samples or one LFM per sample set, whichever is

greater. The analyte added should be 2-4 times the

ambient concentration and should be at least four times

greater than the MDL. 

9.4.2 Calculate percent recovery of analyte, corrected

for background concentration measured in a separate

unfortified sample. These values should be compared

with the values obtained from the LFBs. Percent

recoveries may be calculated using the following

equation:

 (C - C) s

R = --------------- x 100

S

where, R = percent recovery

C = measured fortified sample s 

 addition in mg N/L

C = sample background 

 concentration (mg N/L)

S = concentration in mg N/L added 

to the environmental sample.

9.4.3 If the recovery of the analyte falls outside the

required control limits of 90-110%, but the laboratory

performance for that analyte is within the control limits,

the fortified sample should be prepared again and

analyzed. If the result is the same after reanalysis, the

recovery problem encountered with the fortified sample is

judged to be matrix related and the sample data should

be flagged accordingly.

10.0 Calibration and Standardization

10.1 At least five calibration standards should be

prepared fresh daily for system calibration. 

10.2 A calibration curve should be constructed for

each sample set by analyzing a series of calibration

standard solutions. A sample set should contain no more

than 60 samples. For a large number of samples make

several sample sets with individual calibration curves. 

or more that bracket the conentrations of samples should

have a correlation coefficient, r, of 0.995 or better and the

range should not be greater than two orders of

magnitude. 

10.5 Use a high CAL solution followed by two blank

cups to quantify system carryover. The difference in peak

heights between two blank cups is due to the carryover

from the high CAL solution. The carryover coefficient, k,

is calculated as follows:

 Pb1 - Pb2

k = -----------------

Phigh 

where, P = the peak height of the high high

ammonia standard

P = the peak height of the b1 

first blank sample

P = the peak height of the b2 

second blank sample

The carryover coefficient, k, should be measured in seven

replicates to obtain a statistically significant number. The

carryover coefficient should be remeasured with any

change in manifold plumbing or upon replacement of

pump tubes.

The carryover correction (CO) of a given peak, i, is

proportional to the peak height of the preceding sample,

P .

i-1

CO = (k)x(P ) i-1

To correct a given peak height reading, Pi, subtract the

carryover correction.13,14

Pi,c = Pi - CO

where P is corrected peak height. The correction for i,c

carryover should be applied to all the peak heights

throughout a run. The carryover coefficient should be less

than 5% in this method.
 349.0-9 Version 1.0 September 1997

10.6 Place a high standard solution at the end of each 11.8 Begin pumping the Brij-35 start-up solution

sample run to check for sensitivity drift. Apply sensitivity (Section 7.2.1) through the system and obtain a steady

drift correction to all the samples. The sensitivity drift baseline. Place the reagents on-line. The reagent

during a run should be less than 5%. baseline will be higher than the start-up solution baseline.

After the reagent baseline has stabilized, reset the

Note: Sensitivity drift correction is available in most data baseline.

acquisition software supplied with autoanalyzers. It is

assumed that the sensitivity drift is linear with time. An Note: To minimize the noise in the reagent baseline,

interpolated drift correction factor is calculated for each clean the flow system by sequentially pumping the

sample according to the sample position during a run. sample line with reagent water, 1 N HCl solution, reagent

Multiply the sample peak height by the corresponding water, 1 N NaOH solution for few minutes each at tahe

sensitivity drift correction factor to obtain the corrected end of the daily analysis. Make sure to rinse the system

peak height for each sample. well with reagent water after pumping NaOH solution to

prevent precipitation of Mg(OH) when seawater is

11.0 Procedure

11.1 If samples are stored in a refrigerator, remove

samples and equilibrate to room temperature prior to

analysis.

11.2 Turn on the continuous flow analyzer and data

acquisition components and warm up at least 30 minutes.

11.3 Set up cartridge and pump tubes as shown in

Figure 1.

11.4 Set spectrophotometer wavelength to 640 nm,

and turn on lamp.

11.5 Set the Absorbance Unit Full Scale (AUFS) range

on the spectrophotometer at an appropriate setting

according to the highest concentration of ammonia in the

samples. The highest setting appropriate for this method

is 0.2 AUFS for 6 mg N/L.

11.6 Prepare all reagents and standards.

11.7 Choose an appropriate wash solution for sampler

wash. For analysis of samples with a narrow range of

salinities (± 2 %o) or for samples containing low ammonia

concentrations (< 20 μg N/L), it is recommended that the

CAL solutions be prepared in Low Nutrient Seawater

(Section 7.1.4) diluted to the salinity of samples, and that

the Sampler Wash Solution also be Low Nutrient

Seawater diluted to the same salinity. For samples with

varying salinities and higher ammonia concentrations (> 12.1 Concentrations of ammonia in samples are

20 μg N/L), it is suggested that the reagent water used for calculated from the linear regression, obtained from the

the sampler wash solution and for preparing calibration standard curve in which the concentrations of the 

standards and procedures in Section 12.2 and 12.3 be

employed.

2

introduced into the system. Keep the reagents and

samples free of particulate. Filter the reagents and

samples if necessary.

If the baseline drifts upward, pinch the waste line for a few

seconds to increase back pressure. If absorbance drops

down rapidly when back pressure increases, this indicates

that there are air bubbles trapped in the flow cell. Attach

a syringe at the waste outlet of the flowcell. Air bubbles

in the flowcell can often be eliminated by simply attaching

a syringe for a few minutes or, if not, dislodged by

pumping the syringe piston. Alternatively, flushing the

flowcell with alcohhol was found to be effective in

removing air bubbles from the flowcell.

11.9 The sampling rate is approximately 60 samples

per hour with 30 seconds of sample time and 30 seconds

of wash time.

11.10 Use cleaned sample cups or tubes (follow the

procedures outlined in Section 6.2.2). Place CAL

solutions and saline standards (optional) in sampler.

Complete filling the sampler tray with samples, laboratory

reagent blanks, laboratory fortified blanks, laboratory

fortified sample matrices, and QC samples. Place a blank

after every ten samples.

11.11 Commence analysis.

12.0 Data Analysis and Calculations
Version 1.0 September 1997 349.0-10

calibration standards are entered as the independent baseline), subtract the absorbances of samples of various

variable, and their corresponding peak heights are the salinities from that of reagent water. The results are the

dependent variable. apparent absorbance due to the change in refractive

12.2 Refractive Index Correction for Estuarine and reagent water baseline.

Coastal Samples

12.2.1 If reagent water is used as the wash solution, the

operator has to quantify the refractive index correction from its peak height corrected to reagent water baseline

due to the difference in salinity between sample and wash (Section 12.2.5) and the regression equation of ammonia

solution. The following procedures are used to measure standards obtained with color reagent being pumped

the relationship between the sample salinity and refractive through the system (Section 12.2.2). Salinity is entered as

index on a particular detector. the independent variable and the apparent ammonia

12.2.2 First, analyze a set of ammonia standards in dependent variable. The resulting regression allows the

reagent water with color reagent using reagent water as

the wash and obtain a linear regression of peak height

versus concentration.

12.2.3 Second, replace reagent water wash solution with

Low Nutrient Seawater wash solution.

Note: In ammonia analysis absorbance of the reagent

water is higher than that of the LNSW. When using

reagent water as a wash solution, the change in refractive

index causes the absorbance of seawater to become

negative. To measure the absorbance due to refractive

index change in different salinity samples, Low Nutrient

Seawater must be used as the wash solution to bring the

baseline down.

12.2.4 Third, replace the phenol solution (Section 7.2.4)

and NaDTT solution (Section 7.2.5) with reagent water.

All other reagents remain the same. Replace the

synchronization sample with the colored SYNC peak

solution (Section 7.2.6).

12.2.5 Prepare a series of different salinity samples by

diluting the LNSW. Commence analysis and obtain peak

heights for different salinity samples. The peak heights for

the refractive index correction must be obtained at the

same AUFS range setting and on the same

spectrophotometer as the corresponding standards

(Section 12.2.2).

12.2.6 Using LNSW as the wash water, a maximum

absorbance will be observed for reagent water. No

change in refractive index will be observed in the

seawater sample. Assuming the absolute absorbance for

reagent water ( relative to the seawater baseline ) is equal

to the absorbance for seawater ( relative to reagent water

index between samples of various salinities relative to the

12.2.7 For each sample of varying salinity, calculate the

apparent ammonia concentration due to refractive index

concentration due to refractive index is entered as the

operator to calculate apparent ammonia concentration

due to refractive index when the sample salinity is known.

Thus, the operator would not be required to obtain

refractive index peak heights for all samples.

12.2.8 The magnitude of refractive index correction can

be minimized by using a low refractive index flowcell. An

example of a typical result using a low refractive index

flowcell follows:

_______________________________________

Salinity Apparent ammonia conc. due 

(% ) to refractive index (μg N/L) o

_______________________________________ 

0.0 0.00

4.5 0.18

9.1 0.45

13.9 0.66

17.9 0.86

27.6 1.30

36.2 1.63

_______________________________________

Note: You must calculate the refractive index correction

for your particular detector. The refractive index must be

redetermined whenever a significant change in the design

of the flowcell or a new matrix is encountered.

12.2.9 An example of a typical equation is:

Apparent ammonia (μg N/L) = 0.0134 + 0.0457S

where S is sample salinity in parts per thousand. The

apparent ammonia concentration due to refractive index

so obtained should then be added to samples of 
 349.0-11 Version 1.0 September 1997

corresponding salinity when reagent water was used as Corrected concentration (mg N/L) 

the wash solution for samples analysis. = Uncorrected concentration /1.17(mg N/L)

If a low refractive index flowcell is used and ammonia 12.3.4 Results of sample analyses should be reported

concentration is greater than 200 μg N/L, the correction

for refractive index becomes negligible.

12.3 Correction for Matrix Effect in Estuarine and

Coastal Samples 

12.3.1 When calculating concentrations of samples of

varying salinities from standards and wash solution

prepared in reagent water, it is necessary to first correct

for refractive index errors, then correct for the change in

color development due to the differences in composition

between samples and standards (matrix effect). Even

where the refractive index correction may be small, the

correction for matrix effect can be appreciable.

12.3.2 Plot the salinity of the saline standards (Section

7.2.9) as the independent variable, and the apparent

concentration of ammonia (mg N/L) from the peak height

(corrected for refractive index) calculated from the

regression of standards in reagent water, as the

dependent variable for all saline standards. The resulting

regression equation allows the operator to correct the

concentrations of samples of known salinity for the color

enhancement due to matrix effect. An example of a

typical result follows:

_______________________________________

Peak height of UncorrectedNH3

Salinity 0.140 mg N/L conc. calculated

 (% ) from standards o

in reagent water

 (mg N/L)

_______________________________________

0 2420 0.1400

4.5 2856 0.1649

9.1 2852 0.1649

13.9 2823 0.1635

17.9 2887 0.1673

27.6 2861 0.1663

36.2 2801 0.1633

_______________________________________

12.3.3 Using the reagent described in Section 7.0, as

shown above, matrix effect becomes a single factor

independent of sample salinity. An example of a typical

equation to correct for matrix effect is:

in mg N/L or in μg N/L.

mg N/L = ppm (parts per million)

μg N/L = ppb (part per billion)

13.0 Method Performance

13.1 Single Laboratory Validation

13.1.1 Method Detection Limit- A method detection limit

(MDL) of 0.3 μg N/L has been determined by one

laboratory from spiked LNSW of three different salinities

as follows:

_____________________________________

Salinity [NH3] SD Recovery MDL

(% ) (μg N/L) (μg N/L) (%) (μg N/L) o

_____________________________________

36.2 0.7 0.0252 95.4 0.0792

36.2 0.7 0.0784 100.8 0.2463

36.2 1.4 0.0826 104.7 0.2595

36.2 1.4 0.0966 105.6 0.3035

17.9 0.7 0.0322 106.5 0.1012

17.9 0.7 0.0182 92.2 0.0572

17.9 1.4 0.0938 109.1 0.2947

17.9 1.4 0.0882 100 0.2771

4.5 0.7 0.0672 95.1 0.2111

4.5 1.4 0.1008 94.1 0.3167

4.5 1.4 0.126 106.7 0.3959

0.0 0.7 0.077 98.2 0.2419

0.0 0.7 0.0784 100.8 0.2463

0.0 1.4 0.0854 101.9 0.2683

_____________________________________

13.1.2 Single Analyst Precision - A single laboratory

analyzed three samples collected from the Miami River

and Biscayne Bay, Florida. Seven replicates of each

sample were processed and analyzed with salinity

ranging from 4.8 to 35.0. The results were as follows:
Version 1.0 September 1997 349.0-12

______________________________________ acidification is not suitable preservation technique.

Sample Salinity Concentration RSD Addition of phenol increases the absorbance of samples.

 (%o) (μg N/L) (%) Phenol is not recommended as a suitable preservative

______________________________________ although samples preserved with phenol were stable as

1 35.5 6.3 7.19 those preserved by chloroform.

2 20.0 72.1 1.57

3 4.8 517.6 0.64 For moderate and high concentrations of ammonia (> 20

______________________________________ μg N/L) samples, it is suggested samples be preserved

13.1.3 Laboratory Fortified Sample Matrix - Laboratory and refrigerated in the dark at 4 C. A maximum holding

fortified sample matrices were processed in three

different salinities ranging from 4.8 to 35.0 and ambient

ammonia concentrations from 0.0 to 72.1 μg N/L. Seven

replicates of each sample were analyzed and the results

were as follows:

_______________________________________

Salinity Concentration RSD Recovery

 ambient fortified

(% ) (μg N/L) (%) (%) o

______________________________________

35.5 6.3 70 5.01 98.3

20.0 72.1 140 1.71 98.3

4.8 0.0 280 1.81 98.1

______________________________________

13.1.4 Linear Dynamic Range - A linear dynamic range

(LDR) of 4.0 mg N/L has been determined by one

laboratory from spiked LNSW using a Flow Solution

System (Alpkem, Wilsonville, Oregon).

13.1.5 Sample Preservation Study - Natural samples

have been preserved by freezing, acidification and

addition of chloroform and phenol as preservatives to the

samples stored in glass and high density polyethylene

bottles. Table 1 summarized the results of preservation

study.

There is no significant difference in recovery of ammonia

from samples stored in glass and high density

polyethylene bottles, suggesting either glass or high

density polyethylene bottles can be used for storage of

ammonia samples.

For low concentration of ammonia samples (< 20 μg N/L,

sample 1 in table 1), no preservation technique is

satisfactory. Samples must be analyzed within 3 hours of

collection. 

Freezing cannot preserve ammonia in samples for more

than one week. Acidified samples must be neutralized

with NaOH solution prior to analysis. Addition of NaOH to

acidified samples induces the precipitation of Mg(OH)2

and Ca(OH) . Centrifuging the samples cannot 2

completely eliminate the interference, therefore,

12

by the addition of 2 mL of chloroform per liter of sample

o

time for preserved estuarine and coastal water samples

with moderate to high concentrations of ammonia is two

weeks.10

13.2 Multi-Laboratory Validation

Multi-laboratory data is unavailable at this time.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

USEPA has established a preferred hierarchy of

environmental management techniques that places

pollution prevention as the management option of first

choice. Whenever feasible, laboratory personnel should

use pollution prevention techniques to address their waste

generation. When wastes cannot be feasibly reduced at

the source, the agency recommends recycling as the next

best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research

institutions, consult Less is Better: Laboratory Chemical

Management for Waste Reduction, available from the

American Chemical Society, Department of Government

Relations and Science Policy, 1155 16th Street N.W.,

Washington D.C. 20036, (202) 872-4477.

15.0 Waste Management

15.1 The U.S. Environmental Protection Agency

requires that laboratory waste management practices be

conducted consistent with all applicable rules and

regulations. The Agency urges laboratories to protect the

air, water, and land by minimizing and controlling all

releases from hoods and bench operations, complying

with the letter and spirit of any sewer discharge permits

and regulations, and by complying with all solid and

hazardous waste regulations, particularly the hazardous

waste identification rules and land disposal restrictions. 
 349.0-13 Version 1.0 September 1997

For further information on waste management consult 11. Aminot A. and R. Kerouel, 1996. Stability and

The Waste Management Manual for Laboratory

Personnel, available from the American Chemical Society nutrients. Mar. Chem. 52:173-181.

at the address listed in Section 14.2.

16.0 References

1. Solorzano, L. 1969. Determination of ammonia in

natural waters by the phenylhypochlorite method.

Limnol. Oceanogr., 14:799-801.

2. Head, P.C., 1971. An automated

phenolhypochlorite method for the determination of

ammonia in sea water. Deep-Sea Research,

18:531-532.

3. Slawyk, G., and MacIsaac, J.J., 1972. Comparison

of two automated ammonia methods in a region of

coastal upwelling. Deep-Sea Research, 19:521-

524.

4. Hansen, H.P. and Grasshoff , K. 1983, Automated

Chemical Analysis, In Methods of Seawater

Analysis (Grasshoff, K., M. Ehrhardt and K.

Kremling, Eds) Weinheim, Verlag Chemie,

Germany. pp363-365. 

5. Mautoura, R.F.C. and E.M.S. Woodward, 1983.

Optimization of the indophenol blue method for the

automated determination of ammonia in estuarine

waters. Estuarine, Coastal and Shelf Science,

17:219-224.

6. Zhang J-Z, and F. J. Millero 1993. The chemistry of

anoxic waters in the Cariaco Trench. Deep-Sea

Res., 40:1023-1041.

7. Raymont, J.E.G. 1980. Plankton and productivity in

the oceans. Pergamon Press, Oxford, England. 

8. Smith, S.L. and T.E. Whitledge. 1977. The role of

zooplankton in the regeneration of nitrogen in a

coastal upwelling off northwest Africa. Deep-Sea

Res. 24: 49-56. 

9. Code of Federal Regulations 40, Ch. 1, Pt. 136

Appendix B. Definition and Procedure for the

Determination of Method Detection Limit. Revision

1.11.

10. Eaton, A.D. and V. Grant, 1979. Sorption of

ammonium by glass frits and filters: implications for

analyses of blakish and freshwater. Limnol.

Oceanogr. 24:397-399.

preservation of primary calibration solutions of

12. Degobbis, D. 1973. On the storage of seawater

samples for ammonia determination. Limnol.

Oceanogr., 18:146-150.

13. Angelova, S, and H.W.Holy. 1983. Optimal speed

as a function of system performance for continuous

flow analyzers. Analytica Chimica Acta, 145:51-58.

14. Zhang, J.-Z. 1997. Distinction and quantification of

carry-over and sample interaction in gas

segmented continuous flow analysis. Journal of

Automatic Chemistry, 19(6):205-212.
1

2

3

4

5

6

7

9

10

0.41

0.25

1.57

Pump

mL/min

Debubbler

Detector

Manifold

Sample

Reagent Water 

or Low Nutrient Seawater

Wash To Sampler

To Waste

To Waste

Sample:Wash = 30":30"

640nm

0.32 Complexing Reagent

Nitrogen

1.01

0.10 Phenol

0.10 NaDTT

0.10 Nitroferricyanide

 C 8

0.41

Coil

o

o

60 C

60

Heater

Version 1.0 September 1997 349.0-14

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Figure 1. Manifold Configuration for Ammonia Analysis.
 349.0-15 Version 1.0 September 1997

Table 1 . Percentage Recovery of Ammonia From Natural Water Samples Preserved by Freezing, Acidification,

A 

Addition of Chloroform and Phenol.

______________________________________________________________________________

sample method bottle time(day) B C D

0 7 14 21 28

_______________________________________________________________________________

1 none HDPE 100 349 345 18 91

freezing glass 100 100 0 0 0

HDPE 100 102 0 0 0

H2SO4 glass 200 564 285 73 55 E

HDPE 200 113 64 45 36

CHCl3 glass 193 135 29 47 36

HDPE 193 193 18 44 36

phenol glass 153 36 44 0 0

 F

HDPE 153 36 0 0 0

1+ freezing glass 100 101 82 77 102

HDPE 100 97 76 61 81

H2SO4 glass 95 105 69 54 37 E

HDPE 95 91 91 88 116

CHCl3 glass 96 105 85 78 89

HDPE 96 102 85 78 92

phenol glass 130 133 110 148 123 F

HDPE 130 128 102 103 118

2 none HDPE 100 32 0 0 0

freezing glass 100 109 93 77 88

HDPE 100 107 82 67 91

H2SO4 glass 252 162 66 62 50 E

HDPE 252 193 45 41 27

CHCl3 glass 99 114 83 75 96

HDPE 99 98 80 70 83

phenol glass 108 107 88 74 93 F

HDPE 108 101 83 74 86

2+ freezing glass 99 108 109 111 106

HDPE 99 106 95 78 91

H2SO4 glass 100 107 51 86 88 E

HDPE 100 102 39 98 107

CHCl3 glass 99 106 116 94 105

HDPE 99 107 98 95 103

phenol glass 117 121 106 105 116 F

HDPE 117 124 107 106 117

3 none HDPE 100 104 14 1 0

freezing glass 100 - 116 64 106

HDPE 100 108 105 65 75

H2SO4 glass 101 106 44 74 61 E

HDPE 101 108 111 106 109

CHCl3 glass 100 96 98 96 94

HDPE 100 93 97 95 95

phenol glass 112 106 107 112 125 F

HDPE 112 112 108 110 112

Recovery is calculated based on the ammonia concentration in non-preserved sample at day 0. Samples with A

recoveries higher than 100% are subject to interference either from precipitation or phenol.

For salinity and concentration of ammonia in samples 1, 2, 3 see Section 13.1.2. B
Version 1.0 September 1997 349.0-16

Sample 1+ and 2+ are the fortified samples 1 and 2 at ammonia concentrations 76.3 and 202.1 μg N/L,

respectively.

Methods of preservation: C

None: stored the samples in high density polyethylene carboys at room temperature without any

preservative added.

Freezing: Frozen and stored at -20 C. o

H2SO4 : Acidified to pH 1.8 with H2SO4, and stored at 4 C. Neutralized the acid with o

NaOH solution before analysis.

CHCl : Added 2 mL chloroform per 1000 mL sample, and stored at 4 C. 3

o

Phenol: Added 8 g phenol per 1000 mL sample, and stored at 4 C. o

Glass and high density polyethylene bottles were compared to determine the effect of sample C

bottle type on the preservation.

Adding NaOH to neutralize acidified samples induced the precipitation of Mg(OH) and Ca(OH) .

E

2 2

Centrifuging the samples can not completely eliminate the interference, therefore, acidification is

not suitable preservation technique. 

Although samples preserved with phenol were stable as those preserved by chloroform, F

an absorbance increase was observed, therefore, this is not recommended as a suitable 

preservation technique.