&EPA
National Oceangraphic
and Atmospheric Administration
United States
Army Corps of Engineers
Waterways Experiment Station
United States
Environmental Protection
Agency
Office of Water
(WH-556F)
EPA 503/2-89/001
April 1989
Compendium of Methods
For Marine And Estuarine
Environmental Studies
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TABLE OF CONTENTS
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GENERAL INFORMATION
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TABLE OF CONTENTS
GENERAL INFORMATION
1. BACKGROUND
2. FORMAT FOR COMPENDIUM OF METHODS
2.1 Sampling Methods Section
22. Analytical Methods Section
3. FORMAT FOR PRESENTATION OF METHODS
4. REFERENCES
SAMPUNG METHODS METHOD NO.
1.0 WATER SAMPLERS
1.1 Water column samplers
1.1.1 Discrete samplers
1.1.2 Pump samplers
12 Sea-surface microlayer samplers
1.2.1 Plate samplers
1.2.2 Rotating drum samplers
1.3 Precipitation samplers
2.0 SEDIMENT SAMPLERS
2.1 Grab samplers
22 Core samplers
2.2.1 Piston corers
222 Box corers
2.3 Dredges
2.4 Sediment traps
3.0 BIOLOGICAL SAMPLERS
3.1 Nets
3.2 Trawls
3.3 Dredges
3.4 Lines
3.5 Traps
4.0 AIR SAMPLERS
4.1 High-volume ambient air samplers
4.2 Stack samplers
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ANALYTICAL METHODS
METHOD NO.
1.0 WATER
1.1 Marine and Estuarine Seawater
1.1.1 Physical Characteristics
1.1.1.1 Currents and Water Column Structure
1.1.1.2 Water Mass Movements
1.1.2 Water Quality/Biochemical Parameters
1.1.3 Organic Compounds
1.1.3.1 Purgeable Organic Compounds
1.1.3.2 Extractable Organic Compounds
1.1.4 Inorganic Compounds
1.1.4.1 Trace Metals
1.1.4.2 Nutrients
1.1.4.2.1 NITROGEN
Colorimetric Automated Phenate Method for
Ammonia Nitrogen
Automated Phenate Method for the Determination
of Ammonia Nitrogen
Automated Method for the Determination of
Ammonia Nitrogen
Manual Method for the Determination of
Ammonia Nitrogen
Colorimetric, Semi-Automated, Block Digester
Method for the Determination of
Total Kjeldahl Nitrogen
Semi-Automated Method for the Determination of
Total Kjeldahl Nitrogen
Manual Method for the Determination of
Total Kjeldahl Nitrogen
Colorimetric, Automated, Cadmium Reduction
Method for Nitrate-Nitrite Nitrogen
Automated Method for the Determination of
Nitrate Plus Nitrite Nitrogen
Automated Method for the Determination of
Nitrite Nitrogen
Manual Method for the Determination of
Nitrite Nitrogen
Manual Method for the Determination of
Nitrate Nitrogen
Determination of Ammonium Nitrogen
Determination of Nitrite Nitrogen
Determination of Nitrate plus Nitrite Nitrogen
Determination of Kjeldahl Nitrogen
Determination of Ammonia
Determination of Ammonia plus Amino Acids
Determination of Reactive Nitrite
Determination of Soluble Organic Nitrogen,
Kjeldahl Digestion
Determination of Solube) Organic Nitrogen by
Ultraviolet Oxidation
Distillation Method for the Determination of Ammonia Nitrogen
A-NrTROGEN-1
A-NfTROGEN-2
A-NfTROGEN-3
A-NfTROGEN-4
A-NITROGEN-5
A-NITROGEN-6
A-NITROGEN-7
A-NPTROGEN-a
A-NfTROGEN-9
A-NITROGEN-10
A-NITROGEN-11
A-NITROGEN-12
A-NITROGEN-13
A-NITROGEN-14
A-NfTROGEN-15
A-NITROGEN-16
A-NITROGEN-17
A-NITROGEN-18
A-NITROGEN-20
A-NJTROGEN-21
A-NITROGEN-22
A-NJTROGEN-23
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Potentiometric Method for the Determination of
Ammonia Nitrogen A-NITROGEN-24
Colorimetric, Automated Phenate Method for the
Determination of Total KjeWahl Nitrogen A-NITROGEN-25
1.1.4.2.2 PHOSPHORUS
Colorimetric, Automated, Block Digester Method for
the Determination of Total Phosphorus A-PHOS-1
Colorimetric, Automated. Ascorbic Add Method
for the Determination of Phosphorus A-PHOS-2
Automated Method for the Determination of Phosphorus A-PHOS-3
Manual Method for the Determination of Phosphorus A-PHOS-4
Determination of Orthophosphate A-PHOS-5
1.1.4.2.3 CHLOROPHYLL
Ruorometric Determination of Chlorophyll a A-CHLOR-1
Spectrophotometric Determination of Chlorophylls
and Total Carotenoids A-CHLOR-2
Determination of Chlorophyll c A-CHLOR-3
Spectrophotometric Determination of Chlorophyll c A-CHLOR-4
1.1.5 Bacteria, Viruses, and Parasites
1.1.6 Toxicity Tests
1.1.6.1 Acute Toxicity
1.1.6.2 Chronic Toxicity
1.1.6.3 Bioaccumulation
1.1.7 Biological Communities
1.1.7.1 Microbial Populations
1.1.7.2 Phytoplankton
1.1.7.3 Zooplankton
1.1.7.4 Nekton Larvae
1.1.7.5 Marine Mammals, Turtles, and Seabirds
1.1.8 Radioactivity
1.1.9 Floatable Materials
12. Marine and Estuarine Sea-Surface Microlayer
1.2.1 Water Quality/Biochemical Parameters
1.2.2 Organic Compounds
1.2.2.1 Purgeable Organic Compounds
1.2.2.2 Extractable Organic Compounds
1.2.3 Inorganic Compounds
1.2.4 Bacteria, Viruses, and Parasites
1.2.5 Toxicity Tests
1.2.5.1 Acute Toxicity
1.2.5.2 Chronic Toxicity
1.2.6 Biological Communities
1.2.6.1 Microbial Populations
1.2.6.2 Phytoplankton
1.2.6.3 Zooplankton
1.2.6.4 Nekton Larvae
1.2.7 Radioactivity
1.3 Precipitation
1.3.1 Water Quality Parameters
1.3.2 Organic Compounds
1.3.2.1 Purgeable Organic Compounds
1.3.2.2 Extractable Organic Compounds
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1.3.3 Inorganic Compounds
1.3.4 Radioactivity
1.4 Sediment Interstitial Water
1.4.1 Water Quality/Biochemical Parameters
1.4.2 Organic Compounds
1.4.2.1 Purgeable Organic Compounds
1.4.2.2 Extractable Organic Compounds
1.4.3 Inorganic Compounds
1.4.4 Toxicity Tests
1.4.4.1 Acute Toxicity
1.4.4.2 Chronic Toxicity
1.4.4.3 Bioaccumulation
2.0 SEDIMENT
2.1 Marine and Estuarine Sediment
2.1.1 Physical Characteristics
2.1.2 Organic Compounds
2.1.2.1 Purgeable Organic Compounds
2.1.2.1 Extractable Organic Compounds
2.1.3 Inorganic Compounds
2.1.4 Bacteria, Viruses, and Parasites
2.1.5 Toxicity Tests
2.1.5.1 Acute Toxicity
2.1.5.2 Chronic Toxicity
2.1.5.3 Bioaccumulation
2.1.6 Biological Communities
2.1.6.1 Infauna
2.1.6.2 Epifauna
2.1.7 Radioactivity
3.0 TISSUE
3.1 Marine and Estuarine Species (Plankton, Nekton, and Benthos)
3.1.1 Physical Characteristics
3.1.2 Organic Compounds
3.1.2.1 Purgeable Organic Compounds
3.1.2.2 Extractable Organic Compounds
3.1.3 Inorganic Compounds
3.1.4 Pathology
4.0 AIR
4.1 Ambient Air
4.1.1 Meteorological Observations
4.1.2 Total Particulates
4.1.3 Organic Compounds
4.1.3.1 Purgeable Organic Compounds
4.1.3.2 Extractable Organic Compounds
4.1.4 Inorganic Compounds
4.1.5 Radioactivity
4.2 Stack Air
4.2.1 Total Particulates
4.2.2 Organic Compounds
4.2.2.1 Purgeable Organic Compounds
4.2.2.2 Extractable Organic Compounds
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4.2.3 Inorganic Compounds
4.2.4 Toxicity Tests
4.2.4.1 Acute Toxicity
4.2.4.2 Chronic Toxicity
4.2.4.3 Bioaccumulation
4.2.5 Radioactivity
5.0 WASTE
5.1 Municipal Sludges
5.1.1 Physical Characteristics
5.1.2 Organic Compounds
5.1.2.1 Purgeable Organic Compounds
5.1.2.2 Extractable Organic Compounds
5.1.3 Inorganic Compounds
5.1.4 Bacteria, Viruses, and Parasites
5.1.5 Toxicity Tests
5.1.5.1 Acute Toxicity
5.1.5.2 Chronic Toxicity
5.1.5.3 Bioaccumulation
5.1.6 Radioactivity
5.1.7 Floatable Materials
52 Dredged Materials
5.2.1 Physical Characteristics
5.2.2 Organic Compounds
5.2.2.1 Purgeable Organic Compounds
5.2.2.2 Extractable Organic Compounds
5.2.3 Inorganic Compounds
5.2.4 Bacteria, Viruses, and Parasites
5.2.5 Toxicity Tests
5.2.5.1 Acute Toxicity
5.2.5.2 Chronic Toxicity
5.2.5.3 Bioaccumulation
5.2.6 Radioactivity
5.3 Wastewater/Effluents
5.3.1 Water Quality/Biochemical Parameters
5.3.2 Organic Compounds
5.3.2.1 Purgeable Organic Compounds
5.3.2.2 Extractable Organic Compounds
5.3.3 Inorganic Compounds
5.3.4 Bacteria, Viruses, and Parasites
5.3.5 Toxicity Tests
5.3.5.1 Acute Toxicity
5.3.5.2 Chronic Toxicity
5.3.5.3 Bioaccumulation
5.3.6 Radioactivity
5.3.7 Floatable Materials
5.4 Industrial Waste
5.4.1 Physical Characteristics
5.4.2 Organic Compounds
5.4.2.1 Purgeable Organic Compounds
5.4.2.2 Extractable Organic Compounds
5.4.3 Inorganic Compounds
5.4.4 Toxicity Tests
5.4.4.1 Acute Toxicity
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5.4.4.2 Chronic Toxicity
5.4.4.3 Bioaccumulation
5.4.5 Radioactivity
5.4.6 Floatable Materials
5.5 Oil Waste
5.5.1 Physical Characteristics
5.5.2 Organic Compounds
5.5.2.1 Purgeable Organic Compounds
5.5.2.2 Extractable Organic Compounds
5.5.3 Inorganic Compounds
5.5.4 Toxicity Tests
5.5.4.1 Acute Toxicity
5.5.4.2 Chronic Toxicity
5.5.4.3 Bioaccumulation
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SAMPLING METHODS
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COMPENDIUM OF METHODS FOR ESTUARINE AND MARINE ENVIRONMENTAL STUDIES
1. BACKGROUND
This document represents a prototype for a compendium of methods recommended by the U.S. Environmental
Protection Agency (EPA) for use in estuarine and marine environmental studies, and in designing and
implementing marine monitoring programs.
This compendium is intended to be part of a cooperative sharing of methods among federal agencies. The
need for such a compendium has been identified by agencies including EPA's Office of Marine and Estuarine
Protection (OMEP)-both the Technical Support Division (TSD) and the Marine Operations Division (MOD)-
Regional EPA Offices, EPA research laboratories in Narragansett and Cincinnati, the U.S. Army Corps of
Engineers (COE), the National Oceanic and Atmospheric Administration (NOAA), and the National Bureau of
Standards (NBS). In August 1988, representatives from many of these agencies participated in an Interagency
Workgroup meeting to discuss the status and availability of methods and analytical reference materials. The
Workgroup participants agreed that their agencies would be interested in seeing EPA OMEP take the lead in
coordinating efforts to compile validated methods and reference materials. The compendium of methods,
when completed, would be available to investigators in both hardcopy and on-line format.
In order to meet the immediate needs of the Interagency Workgroup, a candidate parameter-nutrients in
seawater-was selected as the focus for the prototype compendium. This parameter was chosen because
nutrients are a major concern in nearly all estuaries, and analysis of nutrients is often a problem due to the
lack of validated methods. Therefore, this initial version of the compendium consists of selected methods for
the analysis of nutrients; in particular, nitrogen, phosphorus, and chlorophyll.
Methods assembled in this compendium were collected from many sources. Two documents prepared by EPA
OMEP, entitled "Handbook of Methods for Estuarine Environmental Monitoring" (EPA, 1986) and "Interim
Guidance on Quality Assurance/Quality Control for Estuarine Field and Laboratory Methods" (EPA, 1985) were
consulted, as were the standard operating procedures of the Nutrient Analytical Services Laboratory at the
Chesapeake Biological Laboratory (D'Elia et al., 1988), Strickland and Parsons' A Practical Handbook of
Seawater Analysis (Strickland and Parsons, 1972), and the American Public Health Association's Standard
Methods for the Examination of Water and Wastewater (APHA, 1985). Technical personnel from various EPA
coastal Regional Offices, the U.S. Army Corps of Engineers (COE) Waterways Experiment Station, and other
agencies were also contacted and requested to supply the methods they currently use for the analysis of
nitrogen, phosphorus, and chlorophyll. In addition to methods, data on precision and accuracy or
1
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comparability studies using the methods in a seawater matrix were also requested. Unfortunately, the
availability of these data was very limited. Once this prototype compendium is distributed, it is hoped that
investigators will realize the importance of this information and the need for a repository of comparability
data, and more data of this type will be generated.
2. FORMAT FOR THE COMPENDIUM OF METHODS
In establishing a format for this compendium of methods, a number of other documents and existing
collections of sampling and analytical procedures were consulted. These include two documents published by
the U.S. Army COE-a procedural guide for designation surveys of ocean dredged material disposal sites
(Pequegnat et al.. 1981) and a manual of procedures for handling and chemical analysis of sediment and
water samples (Plumb, 1981); a pilot phase compendium of information and performance data on routinely
used measurement methods (Research Triangle Institute, 1986); a NOAA technical memorandum (NOAA. 1986);
a collection of protocols used for the Puget Sound Estuary Program (TetraTech, 1986); and the standard
operating procedures used by Battelle Ocean Sciences (Battelle, 1988).
The compendium is divided into two major sections according to the two basic types of methods-sampling
methods and analytical methods. Separating methods into their sampling and analytical components minimizes
the need to rewrite collection procedures when a single sampling method can tie applied to a suite of
analytical methods.
2.1 Sampling Methods Section
The sampling methods section is further divided according to sampler types based on the type of
environmental matrix to be sampled. Under each sampler type general categories of sampling equipment are
identified. Occasionally, these categories are further subdivided into more specific groups of equipment,
followed by individual methods for operation of each piece of equipment The divisions of the sampling
section are illustrated in Rgure 1.
The first level of the outline (e.g.. 1.0,2.0) corresponds to the sampler type. Four basic types of samplers
have been identified: water, sediment, biological, and air samplers. The second level of the outline (e.g..
1.1,2.1) corresponds to the category and identifies the general types of sampling equipment (e.g., water
column samplers as a category of water samplers.) The third level (e.g., 1.1.1,2.1.1) or subcategory does not
apply to all environmental samplers, but would include, for example, piston corers and box corers as two
types or subcategories of core samplers. The fourth level of the outline identifies the specific pieces of
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OimjNE STRUCTURE FOR SAMPLING PROTOCOLS
1.0 SAMPLER TYPES
(e.g.. Water. Sediment, Biological, and Air Samplers)
1.1 CATEGORIES OF SAMPLER TYPES
(e.g.. Water Column Samplers. Core Samplers. Microlayer Samplers)
1.1.1 SUBCATEGORIES OF SAMPLER TYPES
(e.g.. Pump Samplers. Piston Corers)
1.1.1.1 SAMPLING EQUIPMENT
(e.g.. Niskin Bottle. Neuston Net. Van Veen Grab Sampler,
Box Core Sampler)
S-1.1.1.1-1
SAMPLING METHODS
(eg.. Collection of Sediment Samples Using the
Box Core Sampler, Collection and At-Sea Processing
of Neuston Samples)
Rgure 1. Outline of the general organization for the sampling methods section of the compendium.
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sampling equipment. Finally, once all the individual methods for the collection of samples have been
selected, they will be added to the outline as the next level. Sampling methods are assigned method
numbers according to the position they take in the outline, beginning with an S, for sampling, followed by
a key word describing the sampler category, and ending with a sequential number.
2.2 Analytical Methods Section
The analytical section of the compendium is further divided according to environmental matrices. Each
environmental matrix will have several categories that characterize the matrix. Under categories, general
types of analytical parameters will be identified. These parameter groups will be further subdivided into
individual methods for measurement of analytical parameters. The proposed divisions of the analytical
section are illustrated in Figure 2.
The first level of the outline (e.g., 1.0,2.0) corresponds to the environmental matrix. Five matrices have
been identified: water, sediment, tissue, air, and waste. The second level of the outline (e.g., 1.1,2.1)
corresponds to the category and more specifically identifies the matrix (e.g., sea-surface microlayer as a
category of water.) The third level (e.g., 1.1.1,2.1.1) corresponds to the analyses that can be performed in
a particular matrix. Groups of analytical parameters may range from physical and chemical measurements on
the matrix to assessments of biological communities living within the matrix. Some groups of analytical
parameters are further subdivided to more specifically identify analytes (e.g., petroleum hydrocarbons as a
division of organic compounds). Finally, once all of the individual methods for the preparation and analysis
of samples have been selected, they will be added to the outline as the next level. Thus, the handbook may
include several methods for the same analytical parameter. Analytical methods are also assigned method
numbers according to the position they take in the outline, beginning with an A, for analytical, followed by
a key word describing the parameter (e.g., NITROGEN, PHOS, or CHLOR), and ending with a sequential
number.
3. FORMAT FOR PRESENTATION OF METHODS
Both the sampling and analytical methods follow one basic format. Information presented in each method is
organized under seven major headings: method title, background and application, specifications, procedure,
data quality requirements and assessments, recordkeeping and data reporting requirements, and special
precautions. Figures 3 and 4 present the formats for sampling methods and analytical methods, respectively.
Guidelines for the types of information to be included in the written methods appear under each of the
headings. At the top of each method, the environmental matrices, categories, and analytical parameters to
which the method can be applied are noted to aid in indexing the methods. Sample collection methods will
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OUTLINE STRUCTURE TOR ANALYTICAL PROTOCOLS
1.0 ENVIRONMENTAL MATRICES
(e.g., Water, Sediment, Tissue, Air, and Waste)
1.1 CATEGORIES OF ANALYSES
(e.g., Estuarine Seawater, Marine Sediment, Ambient Air)
1.1.1 ANALYTICAL PARAMETERS
(e.g.. Physical Characteristics. Organic Compounds. Toxicity
Tests, Biological Communities)
1.1.1.1 SPECIRC ANALYTES
(e.g., Petroleum Hydrocarbons, Coliform Bacteria)
A-1.1.1.1-1 ANALYTICAL METHODS
(e.g., Determination of Ammonium Nitrogen,
Spectrophotometric Determination of Chlorophylls
and Total Carotinoids)
Figure 2. Outline of the general organization for the analytical protocols section of the methods
handbook.
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SAMFUNG METHODS
METHOD NO.
INDEX INFORMATION
Envifui mental Matnx
Category
Analytical Parameter
1. Method Tftte.
2. Background and Appfcation. Includes information such as the source of the method, its
application to regulatory requirements, the application of the method to environmental
matrices and analytical methods, and any irrtra- or irrtertaboratory or performance evaluations
conducted on the method.
3. Specifications. Lists supplies and equipment for sampling, including requirements for sample
containers and guidelines for prevention of sample contamination during collection. Also
presents survey vessel and station positioning requirements.
4. Procedure. A step-by-step description of the sampling method, including sample
acceptability criteria, immediate sample processing or preservation, and sample holding
times.
5. Data QuaBty Requirements and Assessments. Discusses data quality considerations such
as representativeness, comparability, precision, and accuracy.
& Recordkeeping and Data Reporting Requirements. Includes description of sampling data
and documentation, such as tracking forms, sample labels, field logs, and measurements.
7. Special Precautions, Presents health and safety information, and training
recommendations for sampling personnel
References.
Figures. Format for samping methods.
6
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ANALYTICAL METHODS
METHOD NO.
INDEX INFORMATION
Environmental Matrix
Category
Analytical Parameter
1. Method Title.
2. Background and Application. Includes information such as the source of the method.
its application to regulatory requirements, the application of the method to
environmental matrices, and compatible sampling methods. This section also presents
detection limits, optimum concentration ranges, and any intra- or irrtertaboratory or
performance evaluations conducted on the method.
3. Specifications. Lists supplies and equipment for analysis, including instrument
calibration and preventive maintenance.
4. Procedure. A step-by-step description of the sample preparation or analytical procedure.
or both.
5. Data Quality Requirements and Assessments. Discusses data quality considerations such
as precision and accuracy, use of blank, spiked, and replicate samples.
& Recordkeeping and Data Reporting Requirements. Includes description of laboratory
data and documentation, such as tracking forms, sample labels, bench logs, and
instalment/computer printouts.
7. Special Precautions. Presents health and safety information, and training
recommendations for laboratory personnel.
a References.
Figure 4. Format far analytical mollmds.
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be written assuming that the investigators have already established the locations of sampling sites
or stations for a particular project or monitoring program as part of the study design. Each
analytical method will include a reference to compatible sample collection methods. Depending on
their complexity, methods for preparation of a sample matrix for analysis and methods for
instrumental analysis of a sample matrix extract can be written either as one method or
separately.
8
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4. REFERENCES
APHA, AWWA. and WPCF. 1985. Standard Methods for the Examination of Water and
Wastewater. 16th ed. APHA. Washington. DC.
Battelle. 1988. Quality Assurance Manual. Battelle Ocean Sciences. Ouxbury. MA.
D'Elia, C.F.. N.L Kaumeyer, C.W. Keefe, K.V. Wood, C.F. Zimmerman. 1988. Nutrient Analytical
Services Laboratory Standard Operating Procedures. Chesapeake Biological Laboratory (CBL),
University of Maryland.
National Oceanic and Atmospheric Administration (NOAA). 1986. Technical Memorandum NMFS
F/NWC-92 Standard Analytical Procedures of the NOAA National Analytical Facility.
Extractable Organic Compounds, 2nd ed.
Pequegnat, W.E., LH. Pequegnat, B.M. James, E.A. Kennedy, R.R. Fay, and A.D. Fredericks. 1981.
Procedural guide for designation surveys of ocean dredged material disposal sites. Technical
report EL-81-1. Submitted to Office, Chief of Engineers, U.S. Army. Washington, DC by
TerEco Corporation. College Station, TX.
Plumb, R.H. 1981. Procedures for handling and chemical analysis of sediment and water samples.
Prepared for U.S. Environmental Protection Agency/Corps of Engineers Technical Committee
on Criteria for Dredged and Fill Material. Technical Report EPA/CE-81-1. Environmental
Laboratory, U.S. Army Engineer Waterways Experiment Station. Vicksburg, MS.
Research Triangle Institute. 1986. Compendium of information and performance data on routinely
used measurement methods (RUMM)-Pilot phase. Draft document submitted to U.S. EPA.
Research Triangle Park, NC.
Strickland, J.D.H., and T.R. Parsons. 1968. A Practical Handbook of Seawater Analysis. Fisheries
Research Board of Canada. Ottawa, Canada.
TetraTech. 1986. Recommended protocols for measuring selected variables in Puget Sound. Final
report submitted to Puget Sound Estuary Program by Tetra Tech, Inc. Bellevue, WA.
U.S. EPA. 1985. Interim guidance on quality assurance/quality control (QA/QC) for the field and
laboratory methods. U.S. EPA Office of Marine and Estuarine Protection. Washington, DC.
U.S. EPA. 1986. Handbook of methods for estuarine environmental monitoring. U.S. EPA Office
of Marine and Estuarine Protection. Washington, DC.
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Water Samplers
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Sediment Samplers
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Biological Samplers
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Air Samplers
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ANALYTICAL METHODS
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Waste
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NUTRIENTS
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METHOD NO. A-NITROGEN-1
INDEX INFORMATION
Matrices: Seawater, waste
Categories: Estuarine and marine seawater and sea-surface microlayer, wastewater/effluents
Parameter. Ammonia nitrogen
1. METHOD TITLE
Colorimetric Automated Phenate Method for the Determination of Ammonia Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrogen, Ammonia. Method 350.1 (Colorimetric, Automated Phenate) Storet
Nos.-Total 00610, Dissolved 00608. In: Methods for Chemical Analysis of Water and Wastes. U.S.
Environmental Protection Agency, Environmental Monitoring and Support Laboratory (EMSL), Cincinnati,
OH. March 1979. EPA-600/4-79-020.
2.2 Regulatory status. This method is approved for NPDES.
2.3 Principle and application.
2.3.1 Description. This method is approved by EPA for the determination of ammonia in saline
water and domestic and industrial wastes (also in drinking and surface water).
Alkaline phenol and hypochlorite react with ammonia to form indophenol blue that is proportional
to the ammonia concentration. The blue color formed is intensified with sodium nitroprusside.
This method will detect ammonia in the range of 0.01 to 2.0 mg/L NH3 as N. This range is for
photometric measurements made at 630-660 nm in a 15 mm or 50 mm tubular flow cell. Higher
concentrations can be determined by sample dilution. Approximately 20 to 60 samples per hour
can be analyzed.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method. Collect samples for ammonia analysis
in glass or plastic bottles. Each bottle and cap should be rinsed thoroughly with the sample water
prior to collection.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
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METHOD NO. A-NITROGEN-1
2.4.2 Precision and accuracy.
Precision. In a single laboratory (EMSL), using surface water samples at concentrations of 0.77,
0.59, and 0.43 mg NHa-N/1, the standard deviation was ± 0.005.
Accuracy. In a single laboratory (EMSL), using surface water samples at concentrations of 0.16
and 1.44 mg NHs-N/1, recoveries were 107 and 99 percent, respectively.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
Technicon AutoAnalyzer Unit (AAI or AAII). consisting of the following components:
1. Sampler
2. Manifold (AAI) or Analytical Cartridge (AAII)
3. Proportioning pump
4. Heating bath with double delay coil (AAI)
5. Colorimeter equipped with 15 mm tubular flow cell and 630-660 nm filters
6. Recorder
7. Digital printer for AAII (optional)
3.2 This method requires the following reagents:
NOTE: All solutions must be made using ammonia-free water.
1. Distilled water. Special precaution must be taken to insure that distilled water is free of ammonia.
Such water is prepared by passage of distilled water through an ion-exchange column consisting of a
mixture of both strongly acidic cation and strongly basic anion-exchange resins. The regeneration of
the ion-exchange column should be carried out according to the manufacturer's instructions.
2. Sulfuric acid, 5N-air scrubber solution. Carefully add 139 ml of concentrated sulfuric acid to
approximately 500 mL of ammonia-free distilled water. Cool to room temperature and dilute to 1 L
with ammonia-free distilled water.
3. Sodium phenolate. In a 1-L Erlenmeyer flask, dissolve 83 g of phenol in 500 mL of distilled water.
In small increments, cautiously add with agitation, 32 g of NaOH. Periodically cool flask under water
faucet. When cool, dilute to 1 L with distilled water.
4. Sodium hypochlorite solution. Dilute 250 mL of a bleach solution containing 5.25 percent NaOCI
(such as Chlorox) to 500 mL with distilled water. Available chlorine should approximate 2 to 3 percent.
Because Chlorox is a proprietary product, its formulation is subject to change. The analyst must
remain alert to detecting any variation in this product significant to its use in this procedure. Due to
the instability of this product, storage over an extended period should be avoided.
-------
METHOD NO. A-NITROGEN-1
5. Disodium ethylenediamine-tetraacetate (EDTA), 5 percent. Dissolve 50 g of EDTA (disodium salt)
and approximately six pellets of NaOH in 1 L of distilled water. NOTE: In salt water samples, where
EDTA does not prevent precipitation of cations, sodium potassium tartrate solution may be used. It is
prepared as follows:
Sodium potassium tartrate solution (NaKC4H4Ofi6-4H2O). 10 percent. To 900 mL of distilled water,
add 100 g sodium potassium tartrate. Add 2 pellets of NaOH and a few boiling chips, boil gently
for 45 min. Cover, cool, and dilute to 1 L with distilled water. Adjust pH to 5.2 ± 0.5 with
H2SO4. After allowing solution to settle overnight in a cool place, filter to remove precipitate.
Add 0.5 ml of Brij-35 solution (a wetting agent recommended and supplied by Technicon Corp. for
use in AutoAnalyzers) and store in a stoppered bottle.
6. Sodium nitroprusside. 0.05 percent Dissolve 0.5 g of sodium nrtroprusside in 1 L of distilled water.
7. Stock solution. Dissolve 3.819 g of anhydrous ammonium chloride (NH4CI), dried at 105 °C, in
distilled water and dilute to 1000 mL 1.0 ml = 1.0 mg NH3-N.
8. Standard solution A. Dilute 10.0 mL of stock solution to 1000 mL with distilled water.
1.0 mL = 0.01 mgNH3-N.
9. Standard solution B. Dilute 10.0 mL of standard solution A to 100.0 mL with distilled water.
1.0 mL = 0.001 mgNHa-N.
10. Calibration standards. Using standard solutions A and B, prepare the following standards in 100-mL
volumetric flasks. (Prepare standards fresh daily).
NHa-N. mg/L mL Standard Solution/100 mL
Solution B
0.01 1.0
0.02 2.0
0.05 5.0
0.10 10.0
Solution A
0.20 2.0
0.50 5.0
0.80 8.0
1.00 10.0
1.50 15.0
2.00 20.0
NOTE: When saline water samples are analyzed, Substitute Ocean Water (SOW) should be used for
preparing the above standards used for the calibration curve (otherwise, distilled water should be used).
Prepare SOW with the following concentrations of compounds:
-------
METHOD NO. A-NITROGEN-1
Substitute Ocean Water (SOW)
NaCI 4.53 g/L NaHCOa 0.20 g/L
MgCl2 5.20 g/L KBr 0.10 g/L
N32SO4 4.09 g/L HaBOa 0.03 g/L
CaCl2 1.16 g/L SrCl2 0.03 g/L
KCI 0.70 g/L NaF 0.003 g/L
If SOW is used, subtract its blank background response from the standards before preparing the standard
curve.
3.3 Equipment/instrument calibration. A calibration curve should be prepared for each day of sample
analysis using the calibration standards described in Section 3.2, Step 10. Concentrations of the
calibration standards should bracket the sample concentrations. If a sample concentration is outside the
range of calibration, then an additional calibration standard should be analyzed to check if the result is
within the linear range of the method. Alternatively, the sample should be diluted to within the
calibration range and then reanalyzed.
4. PROCEDURE
4.1 Sample handling and preservation. Samples must be preserved by the addition of 2 mL concentrated
H2SO4 per liter, and refrigerated at 4 °C until analysis.
42 Interferences.
1. Calcium and magnesium ions may be present in concentrations sufficient to cause precipitation
problems during analysis. The 5 percent EDTA solution should be used in the procedure to prevent the
precipitation of calcium and magnesium ions from river water and industrial waste. For seawater, the
sodium potassium tartrate solution should be used.
2. Sample turbidity and color may also interfere with this method. Turbidity must be removed by
filtration prior to analysis. Sample color that absorbs in the photometric range used will also interfere.
3. Contamination of ammonia samples can occur easily due to the volatile nature of ammonia. To
prevent potential cross-contamination, reagents used for other analyses that contain ammonia (e.g.,
colorimetric phenol) should be isolated from samples and standards used for ammonia determinations. In
addition, cleaning preparations that contain significant quantities of ammonia (e.g., Pine-Sol or wax
removers) should not be used in the laboratory area where ammonia determinations are performed.
4. Contaminated glassware should be rinsed with 1 +1 HCI, followed by distilled water. To check for
contamination, blanks should be analyzed whenever a new reagent is prepared.
4.3 Sample analysis.
1. Because the intensity of the color used to quantify the concentration is pH dependent, the acid
concentration of the wash water and the standard ammonia solutions should approximate that of the
samples. For example, if the standards have been preserved with 2 mL concentrated H2SC-4/L, the wash
water and standards should also contain 2 mL concentrated H2SO4/L
-------
METHOD NO. A-NFTROGEN-I
2. For a working range of 0.01 to 2.00 NHs-N/1 (AAI), set up the manifold as shown in Figure 1. For
a working range of 0.01 to 1.0 mg NHs-N/1 (Mil), set up the manifold as shown in Figure 2. Higher
concentrations may be accommodated by sample dilution.
3. Allow both colorimeter and recorder to warm up for 30 min. Obtain a stable baseline with all
reagents, feeding distilled water through sample line.
4. For the AAI system, sample at a rate of 20/h, 1:1. For the AAII, use a 60/h 6:1 cam with a common
wash.
5. Arrange ammonia standards in the sampler in order of decreasing concentration of nitrogen.
Complete the loading of the sampler tray with unknown samples.
6. Switch the sample line from distilled water to sampler and begin analysis.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5,1 Precision. The analyst should demonstrate the ability to generate acceptable precision with this
method using replicate sample analyses. Duplicate analyses should be conducted on a minimum of 5
percent of the total number of samples.
Analysts should be able to meet the precision criteria obtained by EMSL-using surface water samples at
concentrations of 0.77,0.59, and 0.43 mg NH3-N/1. the standard deviation was ± 0.005.
5.2 Accuracy. The analyst should demonstrate the ability to generate acceptable accuracy with this
method using laboratory recovery samples and blank samples. A spiked sample should be analysis should
be conducted on a minimum of 5 percent of the total number of samples; a blank should be analyzed
with each batch of samples; a U.S. EPA performance evaluation sample should be analyzed at least once
per quarter.
Analysts should be able to meet the accuracy criteria obtained by EMSL-using surface water samples at
concentrations of 0.16 and 1.44 mg NH3-N/1, recoveries were 107 and 99 percent, respectively.
6. RECORDKEEPINQ AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve derived from processing ammonia standards through the
manifold. Calculate concentration of samples by comparing sample peak heights with the standard curve.
6.2 Reporting units. Concentrations of ammonia in unknown samples are reported in units of mg
NH3-N/L to a maximum of three significant figures.
Results should be reported for all determinations, including QA replicates and spiked samples. Any
factors that may have influenced sample quality should also be reported.
-------
METHOD NO. A-NFTROGEN-1
PROPORTIONING
SM* SMALL MIXING CO
LM« LARGE MIXING C
^
HEATING (
BATH37»CV
1
WASH WATER
TO SAMPLER
IL SM
OIL 0000
LM
OOOQQQQQ
LM
00000000
SM OOOO
' L,
1
•»
n
i
r
WASTE
f~"
^
4
PUMP
P B
G G
R R
G G
W W
W W
R R
P P
ml/mln
2.9 WASH
2.0 SAMPLE
0.8 EDTA
2.0 AIR*
9.6 PHENOLATE
0
SAMPLER
20/ hr.
dl
0.6 HYPOCHLORITE
0.6 NITROPRUSSIDE
2i?
| WASTE
RECORDER
<;r.RiiRRFn THRONG
H
COLORIMETER
15mm FLOW CELL
650-660 nm FILTER
5N HA60
2 4
Figure 1. Manifold for Ammonia Determination (AAI)
6
-------
METHOD NO. A-N1TROGEN-1
HEATING
BATH
50»C
WASH WATER
TO SAMPLER
PROPORTIONING
PUMP
ml/mln.
SAMPLER
60/hr.
6-1
0.42 NITROPRUSSIOE
WASTE
COLORIMETER
50 mm FLOW CELL
650-660 nm FILTER
SCRUBBED THROUGH
5N H2S04
Rgure 2. Manifold for Ammonia Determination (AA1I)
7
-------
METHOD NO. A-N(TROGEN-1
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
72. Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
8. REFERENCES
APHA, AWWA, and WPCF. 1975. Standard Methods for the Examination of Water and Wastewater, 14 ed.
Method 604. p. 616. APHA. Washington, DC.
ASTM. 1966. Manual on Industrial Water and Industrial Waste Water, 2nd ed. p. 418.
Booth, R.L and LB. Lobring. 1973. Evaluation of the AutoAnalyzer II: A progress report. In: Advances
in Automated Analysis: 1972 Technicon International Congress. Vol. 8. pp. 7-10. Mediad Incorporated,
Tarn/town, NY.
Fiore, J. and J. E. O'Brien. 1962. Ammonia determination by automatic analysis. Wastes Engineering 33. p.
352.
Hiller, A. and D. Van Slyke. 1933. Determination of ammonia in blood. J. Biol. Chem.102. p. 499.
O'Connor, B., R. Dobbs, B. Villers, and R. Dean. 1967. Laboratory distillation of municipal waste effluents.
JWPCF 39. R 25.
U.S. EPA. 1979. Methods for chemical analysis of water and wastes. U.S. EPA Environmental Monitoring
and Support Laboratory. Cincinnati, OH.
U.S. EPA. 1985. Interim guidance on quality assurance/quality control (QA/QC) for the field and laboratory
methods. U.S. EPA Office of Marine and Estuarine Protection. Washington, D.C.
U.S. EPA. 1986. Handbook of methods for estuarine environmental monitoring. U.S. EPA Office of Marine
and Estuarine Protection. Washington, D.C.
-------
METHOD NO. A-NfTROGEN-2
INDEX INFORMATION
Matrices: Seawater, waste
Categories: Estuarine and marine seawater and sea-surface microlayer, wastewater/effluents
Parameter Ammonia nitrogen
1. METHOD TITLE
Automated Phenate Method for the Determination of Ammonia Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrogen. Ammonia. Method 417 G. In: APHA, AWWA, and WPCF. 1985.
Standard Methods for the Examination of Water and Wastewater, 16th ed. APHA. Washington. DC.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This is a method that can be used for the determination of ammonia in saline
water and domestic and industrial wastes (also in drinking and surface water).'
Alkaline phenol and hypochlorite react with ammonia to form indophenol blue that is proportional
to the ammonia concentration. The blue color formed is intensified with sodium nitroprusside.
This method will detect ammonia in the range of 0.02 to 2.0 mg/L NHa as N. This range is for
photometric measurements made at 630-660 nm in a 15-mm or 50-mm tubular flow cell. Higher
concentrations can be determined by sample dilution.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
Six synthetic samples containing ammonia and other constituents dissolved in distilled water were
analyzed by five procedures. For a complete discussion of comparability data obtained by
participating laboratories, refer to the method for Ammonia Nitrogen, Method 417, Section 417.4. In:
APHA (1985).
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NrTROGEN-2
2.4.2 Precision and accuracy. For an automated phenate system, in a single laboratory using
surface water samples at concentrations of 1.41,0.77,0.59, and 0.43 mg NHa-N/L, the standard
deviation was ± 0.005, and at concentrations of 0.16 and 1.44 mg NHa-N/L, recoveries were 107 and
99 percent, respectively (APHA, 1985).
3. SPECIFICATIONS
3.1 Equipment For a list of the equipment and apparatus necessary for this method, refer to the
method for Ammonia Nitrogen, Method 417 G, in APHA (1985).
3.2 Reagents. For a list of the reagents necessary for this method, refer to the method for Ammonia
Nitrogen, Method 417 G, in APHA (1985).
3.3 Equipment/instrument calibration. A calibration curve should be prepared for each day of sample
analysis using the ammonia calibration standards as described in the method.
4. PROCEDURE
4.1 Sample handling and preservation. Most reliable results are obtained on fresh samples. Destroy
residual chlorine immediately after sample collection to prevent its reaction with ammonia. If prompt
analysis is impossible, preserve samples with 0.8 ml_ concentrated H2SO4/L sample and store at 4 °C.
The pH of the acid-preserved samples should be between 1.5 and 2. Some wastewaters may require more
concentrated H2SO4 to achieve this pH. If acid preservation is used, neutralize samples with NaOH or
KOH immediately before making the determination (APHA, 1985).
4.2 Interferences.
1. Seawater contains calcium and magnesium ions in concentrations sufficient to cause precipitation
problems during analysis. Adding EDTA and sodium potassium tartrate reduces the problem.
2. Eliminate any marked variation in acidity or alkalinity among samples because intensity of measured
color is pH dependent. Likewise, insure that the pH of wash water and standard ammonia solutions
approximates that of the sample.
3. Mercuric chloride used as a preservative gives a negative interference by complexing with ammonia.
Overcome this effect by adding a comparable amount of HgCl2 to the ammonia standards.
4. Remove interfering turbidity by filtration.
5. Color in the samples that absorbs in the photometric range used for analysis interferes.
4.3 Sample analysis. The procedure for this method is given in Ammonia Nitrogen, Method 417 G. In:
APHA (1985).
-------
METHOD NO. A-NfTROGEN-2
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. No requirements for the demonstration of precision were presented in this method,
however, the analyst should demonstrate the ability to generate acceptable precision with this method
using replicate sample analyses. Refer to Section 2.4.2 for criteria for acceptable precision.
Jx2 Accuracy. No requirements for the demonstration of accuracy were presented in this method,
however, the analyst should demonstrate the ability to generate acceptable accuracy with this method
using laboratory recovery samples and blank samples. Refer to Section 2.4.2 for criteria for acceptable
accuracy.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve derived from processing ammonia standards through the
manifold. Calculate concentration of samples by comparing sample peak heights with the standard curve.
&2 Reporting units. Concentrations of ammonia in unknown samples are reported in units of mg NHa-
N/L
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
72. Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
8. REFERENCES
APHA, AWWA, and WPCF. 1985. Standard Methods for the Examination ofWater and Wastewater, 16th ed.
APHA. Washington, DC.
-------
METHOD NO. A-NITROGEN-3
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Ammonia nitrogen
1. METHOD TTTLE
Automated Method for the Determination of Ammonia Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Ammonia Nitrogen. Analytical Methods Manual for Bottom Sediment Analysis
(Draft). U.S. Environmental Protection Agency, Office of Research and Development July 1974.
22. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
Description. This is a method that can be used for the determination of ammonia nitrogen in
saline water. The intensity of the "indophenol-f elated" blue color, formed by the reaction of
ammonia with alkaline phenol-hypochlorrte, is measured. Sodium nttroprusside is used to intensify
the blue color and mask any positive interference from iron in the samples.
This method will detect ammonia in the range of 0.005 to 1 .0 mg/kg NH3 as N.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
2.4.2 Precision and accuracy. No data are available for precision and accuracy of this method with
a seawater matrix; this method will be updated to include this information as soon as data are
available.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEhW
3. SPECIRCAT1ONS
3.1 This procedure requires the following equipment
Technicon AutoAnalyzer II. Industrial Model, consisting of the following components:
1. Sampler II or IV
2. Analytical cartridge no. 116-D223-01 or equivalent
3. Proportioning pump III or IV
4. Industrial AA-II single channel colorimeter equipped with 15 mm by 1.5 mm I.D. tubular flow cells
and 630 or 650 nm filters.
5. Recorder
3.2 This method requires the following reagents:
1. Distilled water. Special precaution must be taken to insure that distilled water is free of ammonia.
Such water is prepared by passage of distilled water through an ion-exchange column consisting of a
mixture of both strongly acidic cation and strongly basic anion-exchange resins. Since organic
contamination may interfere with this analysis, use of the resin Dowex XE-75 or equivalent, which tends
to remove organic impurities, is advised. The regeneration of the ion-exchange column should be carried
out according to the manufacturer's instructions.
2. Air. Air used for segmenting the stream should be scrubbed (bubbled) through 5N H2SO4 prior to its
introduction into the system.
3. Sodium phenolate. In a 1-L Erlenmeyer flask, dissolve 83 g of phenol (freshly opened reagent grade)
in 50 mL of distilled water. Cautiously add, while cooling under tap water, and in small increments
with agitation, 180 mL of 20 percent NaOH. Filter through a glass fiber filter before use.
4. Sodium hypochlorite solution. (Chlorox) Dilute 200 mL of any good commercially available liquid
household bleach, having 5.25 percent available chlorine, to 1 L with distilled water. NOTE: Check the
label for the 5.25 percent formulation. Beware of the super bleach containing approximately 6 percent
available chlorine.
5. Complexing reagent. Dissolve 33 g of potassium sodium tartrate and 24 g of sodium citrate in 950
mL of distilled water. Adjust the pH of this solution to 5.0 with concentrated H2SO4. Dilute to 1 L
with distilled water. After allowing to settle overnight in a cool place, filter to remove precipitate.
Then add 0.5 mL of Brij-35, (a registered trademark of Atlas Chemical Ind., supplied by Technicon
Corporation under part no. T 21-0110) and store in a stoppered bottle.
6. Sodium nitroprusslde, 0.05 percent. Dissolve 0.5 g of sodium nitroprusside in 1 L of ammonia-free
distilled water. (This reagent is also known as sodium nitroferricyanide.)
7. Stock standard ammonium sulfate (1000 mg/kg N). Dissolve 4.717 g of anhydrous ammonium sulfate,
(NH4)2SO4, dried at 105 °C, in ammonia-free water and dilute to 1 L 1 mL = 1.0 mg NH3-N.
-------
METHOD NO. A-NITROGEN-3
8. Standard solution A. Dilute 10.0 mL of stock solution to 1 L with ammonia-free distilled water. 1 .0
ml = 0.01 mg
9. Standard solution B. Dilute 1 0.0 mL of standard solution A to 1 L with distilled water. 1 .0 mL • -
0.1 n
10. Calibration standards. Using standard solutions A and B, prepare the following standards in 100 mL
volumetric flasks. (Prepare standards fresh daily).
NHa-N. mg/kq mL Standard Solution/100 mL
Solution B
0.005 5.0
0.01 10.0
0.02 20.0
0.05 50.0
0.10 100.0
Solution A
0.20 2.0
0.40 4.0
0.60 6.0
3.3 Equipment/instrument calibration. A calibration curve should be prepared for each day of sample
analysis using the calibration standards described in Section 3.2, Step 10.
4. PROCEDURE
4.1 Sample handling and preservation. Samples can be preserved by the addition of 40 mg HgCIa per kg
of sample, and stored under refrigeration at 4 °C. (Note the HgCl2 interference under Section 4.2.)
Alternatively, quick freezing and storage at -20 °C may be used. Sample handling should be kept to a
minimum in order to avoid absorption of ammonia from the ambient air.
4.2 Interferences. Calcium and magnesium ions may be present in concentrations sufficient to cause
precipitation problems during analysis. This problem is eliminated by using the complexing agent
containing sodium potassium tartrate and sodium citrate. Any marked variation in acidity or alkalinity
among samples should be eliminated, since the intensity of the color used to quantify the concentration
is pH dependent. Mercuric chloride, used as a preservative, gives a negative interference. This is
overcome by using a comparable amount of HgCl2 to the ammonia standards.
4.3 Sample analysis.
1. For a working range of 0.005 to 0.6 mg/kg NH3-N, set up the manifold as shown in Figure 1. A
lower range of 0.003 to 0.15 mg/kg NHa-N can be obtained by using a 50 mm x 1.5 mm flow cell
(Technicon part no. 199-B023-01).
-------
METHOD NO. A-NrTROGEN~3
20 turns
HEATING OATH
20 turns
Waste
Ammonia Nitrogen in Seawater
Range 0.005-0.60 mg/kg N
20 turns
flfifl.W
r^
ToWaslo 4—
COLORIMITER RECORDER
15xL5.mm Tubular f/c
630 nm Filters
01
(Bj
SAMPIIR llorlV
RataySO per hour
A?r.(0.42)0rn/0rn
:oiTi.olexing Keacient fOT80)
Sample* (0.42) Orn/Qrn
rn ^|
EflOOQ) 2:1 sni/wash
rn/0rnj Red/Red
Alkaline Phenol
Bleach (hvoochlorilc) (0.32) Q'K/Blk
Nltroprusside (a 42) Orn/Orn
From flow cell.(1.40)Yci/IJ!u
Proportioning
. Pump
• concentration range may be changed
to 0.003 to 0.15 mg/kg N by changing
(low cell size to 50 mm x 1.5 mm
Figure 1. Manifold for Ammonia Analysis
4
-------
METHOD NO. A-NITROGEN-3
2. Allow both the colorimeter (with 650 nm filters and 15 mm flow cell) and the recorder to warm up
for 30 min. Run a baseline with all reagents, feeding ammonia-free water through the sample line.
Adjust the baseline knob and aperture opening on the colorimeter to obtain a proper baseline.
3. Use a 50 sample/h, 2:1 sample/wash sampler cam and operate the colorimeter in the Damp 1 mode.
All sample cups should be washed with 1 N HCI and rinsed thoroughly with distilled water in order to
remove any traces of ammonia. A final rinse should be made with the sample when filling the sampling
trays. The sample tray should be covered during the run and the tray should be filled with samples just
prior to loading the tray on the sampler.
4. A blank reading for the particular seawater of interest should be determined by sampling the
seawater while the nitroprusside and hypochlorite reagent lines are in distilled water. The blank
obtained should then be subtracted from the readings of the samples with the reagent lines in their
proper solutions.
5. Arrange ammonia standards in the sampler in order of decreasing concentration of nitrogen.
Complete loading of the sampler tray with unknown samples.
6. Switch the sample line from distilled water to sampler and begin analysis.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
Precision and accuracy. No requirements for measurement of precision and accuracy are cited in this
method, however, analysts should demonstrate the ability to generate acceptable precision with this method
using replicate sample analyses, and demonstrate acceptable accuracy using laboratory recovery sample's and
blank samples.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare an appropriate standard curve derived from processing ammonia standards
through the manifold. Calculate concentration of samples by comparing sample peak heights with the
standard curve.
6.2 Reporting units. Concentrations of ammonia in unknown samples are reported in units of
mg/kg
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
-------
METHOD NO. A-NrTROGEN-3
Methods for chemical analysis of water and wastes. 1971. U.S. Environmental Protection Agency, NERC.
AQCU Cincinnati, OH. pp. 141-147.
Harwood. J.E. and D.J. Huyser. 1970. Automated analysis of ammonia in water. Water Research. Pergamon
Press. Vol. 4. pp. 659-704.
-------
METHOD NO. A-NITROGEN-4
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Ammonia nitrogen
1. METHOD TITLE
Manual Method for the Determination of Ammonia Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Ammonia Nitrogen. Analytical Methods Manual for Bottom Sediment Analysis
(Draft). U.S. Environmental Protection Agency, Office of Research and Development July 1974. This
EPA method is based on the procedure developed by Lucia Solorzano:
Solorzano, L Determination of ammonia in natural waters by the phenol-hypochlorite method.
Limnology and Oceanography, Vol. 14,99.799-801.
22. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This is a method that can be used for the direct determination of ammonia in
saline water, without distillation. The combination of high pH and citrate will complex magnesium
and calcium, and permit the reaction of ammonia with phenol-hypochlorite to proceed with a
minimum of interference problems. Nitroprusside is used as a catalyst in the color formation which
is measured at 640 nm.
This method will detect ammonia in the range of 0.005 to 1.0 mg/kg NHa as N.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Statistics on the use of this method are available in the following
article:
Degobbis, D. 1973. On the storage of seawater samples for ammonia determination. Limnology and
Oceanography. Vol. 18, pp. 145-150.
This method will be updated to include any other comparability studies using this method with a
seawater matrix as soon as data are available.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEN-4
2.4.2 Precision and accuracy. Solorzano reported that at 4 /tg/kg the standard deviation was
1
3. SPECIFICATIONS
3.1 Equipment
1 . Spectrophotometer or filter photometer for use at 640 nm and providing a light path of 10 cm. A
shorter light path can be used when values approach 100 /tg/kg.
2. All glassware used in sample preparation must be thoroughly cleaned, washed with warm dilute HCI,
and thoroughly rinsed in ammonia-free distilled water.
3.2 Reagents.
1 . The distilled water used in the preparation of reagents, blanks, and standards must be free of
ammonia. It may be necessary to percolate distilled water through an ion-exchange resin specifically
formulated to remove ammonia.
2. All reagents used in the preparation of solutions must be of the highest quality obtainable and free
of ammonia.
3. The ammonium chloride used to prepare standards must be of reagent grade or better quality.
4. PROCEDURE
4.1 Sample handling and preservation. Samples can be preserved by freezing samples in polyethylene
containers. Conditions may require filtration through 0.45 p filters before samples are frozen. Mercuric
chloride has also been recommended as an inhibitor of biological activity.
42 Interferences. Negligible interference was reported from animo acids and urea. The cleaning of
glassware and analysis of samples must be performed in a well-ventilated smoke-free laboratory. (Smoke
from tobacco has been shown to provide a positive interference.)
4.3 Sample analysis. The procedure for this method is found in the following article:
Solorzano, L Determination of ammonia in natural waters by the phenol-hypochlorite method.
Limnology and Oceanography, Vol. 14, 99. 799-601.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
Precision and accuracy. The analyst should demonstrate the ability to generate acceptable precision and
accuracy with this method using a laboratory control standard. Solorzano reported that at 4 /*g/kg the
standard deviation was 1 /*g/kg.
-------
METHOD NO. A-NITROGEN-4
6. RECORDKEEPINQ AND DATA REPORTING REQUIREMENTS
Concentrations of ammonia in unknown samples are reported in units of pg/kg NHs-N.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should initially work under the guidance of
an experienced supervisor until he/she can demonstrate proficiency in the laboratory techniques
described in this method.
a REFERENCES
Degobbis, D. 1973. On the storage of seawater samples for ammonia determination. Limnology and
Oceanography. Vol. 18, pp. 145-150.
Head, P.C. 1971. An automated phenol-hypochlorite method for the determination of ammonia in seawater.
Deep Sea Research, Vol. 18. pp. 531-532.
Jenkins, D. 1965. A study of methods suitable for the analysis and preservation of nitrogen forms in an
estuarine environment. Report to the USPHS, Region IX, WSPC Division, SERL No. 65-13, College of
Engineering and School of Public Health, University of California.
Solorzano, L 1969. Determination of ammonia in natural waters by the phenol-hypochlorite method.
Limnology and Oceanography, Vol. 14,99.799-801.
Strickland, J.D.H. and T.R. Parsons. 1968. Determination of ammonia. In: A Practical Handbook of
Seawater Analysis. Fisheries Research Board of Canada, Ottawa, Canada, pp. 87-92.
Weber, C.I. 1967. The preservation of plankton grab samples. Water Pollution Surveillance System
Applications and Development Report No. 26, FWPCA, Dept. of the Interior.
-------
METHOD NO. A-NJTROGEN-5
INDEX INFORMATION
Matrices: Seawater. waste
Categories: Estuarine and marine seawater and sea-suriace microlayer, wastewater/effluents
Parameter Total Kjeldahl nitrogen
1. METHOD TITLE
Colorimetric, Semi-Automated, Block Digester Method for the Determination of Total Kjeldahl Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrogen, Kjeldahl. Total. Method 3512. (Colorimetric, Semi-Automated Block
Digester) Storet No. 00625. In: Methods for Chemical Analysis of Water and Wastes. U.S.
Environmental Protection Agency, Environmental Monitoring and Support Laboratory (EMSL), Cincinnati,
OH. March 1979. EPA-600/4-79-020.
22. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This is a method that can be used for the determination of total Kjeldahl
nitrogen (TKN) in saline waters and in domestic and industrial wastes. (It can also be used for
analysis of drinking and surface waters.)
The procedure converts nitrogen components of biological origin such as amino acids, proteins, and
peptides to ammonia, by may not convert nitrogenous compounds of some industrial wastes such as
amines, nrtro compounds, hydrazones, oximes, semicarbazones, and some refractory tertiary amines.
The sample is heated in the presence of H2SO4, K2SO4. and HgSO4 for 2.5 h. The residue is
cooled, diluted to 25 mL, and placed on the Technicon AutoAnalyzer for ammonia determination.
This digested sample may also be used for phosphorus determination.
The applicable range of this method is 0.1 to 20 mg/L TKN. The range may be extended with
sample dilution.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2^3 Definitions.
1. Total Kjeldahl Nitrogen is defined as the sum of free-ammonia and organic nitrogen compounds
which are converted to ammonium sulfate. (NH4)2SO4, under the conditions of digestion described
by this method.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NrTROGEN-5
2. Organic Kjeldahl Nitrogen is defined as the difference obtained by subtracting the free-ammonia
value (Method 350.2, Nitrogen, Ammonia, In: Methods for Chemical Analysis of Water and Wastes.
U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory (EMSL),
Cincinnati, OH. March 1979. EPA-600/4-79-020) from the total Kjeldahl nitrogen value.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
2.4.2 Precision and accuracy.
1. Precision. In a single laboratory (EMSL), using sewage samples with concentrations of 1.2,2.6,
and 1.7 mg N/L, the precision was ± 0.07, ± 0.03, and ± 0.15, respectively.
2. Accuracy. In a single laboratory (EMSL), using sewage samples with concentrations of 4.7 and
8.74 mg N/L, the recoveries were 99 and 99 percent, respectively.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. Block Digestor-40
2. Technicon Manifold for Ammonia (Rgure 1)
3. Teflon boiling chips
3.2 This method requires the following reagents:
NOTE: All solutions must be made using ammonia-free water.
1. Distilled water. Special precaution must be taken to ensure that distilled water is free of ammonia.
Such water is prepared by passage of distilled water through an ion-exchange column consisting of a
mixture of both strongly acidic cation and strongly basic anion-exchange resins. The regeneration of the
ion-exchange column should be carried out according to the manufacturer's instructions.
2. Mercuric sulfate. Dissolve 8 g red mercuric oxide (HgO) in 50 mL of 1:4 H2SO4 (10 mL concentrated
H2SO4 to 40 mL distilled water) and dilute to 100 mL with distilled water.
3. Digestion solution-sulfuric acid/mercuric sulfate/potassium sulfate solution. Dissolve 133 g of K2SO4
in 700 mL of distilled water and 200 mL of concentrated H2SO4. Add 25 mL of mercuric sulfate
solution (item no. 1) and dilute to 1 L
4. Sulfuric acid solution, 4 percent. Add 40 mL of concentrated H2SO4 to 800 mL of ammonia-free
distilled water, cool, and dilute to 1 L
5. Stock sodium hydroxide. 20 percent. Dissolve 200 g of NaOH in 900 mL of ammonia-free distilled
water and dilute to 1 L
-------
METHOD NO. A-NITROGEN-5
ml/min
CRY CRY 1.0 4* H]SO4
10 TURNS
ftOOO
116-0489-01
37«C
S TURNS I57-B773-03 10 TURNS
tOOO I 0000 I 0000
"G" COIL
TO PUMP TUBE
COLORIMETER
660nm
50mm F/C « 1.5mm ID
157-8089
20 TURNS
QOOQ
BIK BIK
RED RED
ORN YEl
BIK BIK
RED RED
ORN YEl
BIK BIK
ORN Yfl
CRY CRY
0.32 AIR
O.BO DILUENT WATER
•SAMPLE
TO PHOSPHORUS
SAMPLE LINE
116-BOOO
0.3? AIR
0.80 WORKING BUFFER
•RESAMPLE
0.32 SAUCYIATE-NITROPRUSSIDE
0.16 HYPOCHLORITE
WASTE
1.0 WASTE
•SEE CHART FOR RANGE SELECTION (Tobl. J3)
AMMONIA MANIFOLD AAII
Figure 1. Manifold for Total KjeWahl Nitrogen Determination
3
-------
METHOD NO. A-NITROGEN-5
6. Stock sodium-potassium tartrate solution. 20 percent Dissolve 200 g potassium tartrate in 800 mL of
ammonia-free distilled water and dilute to 1 L
7. Stock buffer solution. Dissolve 134.0 g of sodium phosphate, dibasic (N32HPO4) in 800 ml of
ammonia-free distilled water and dilute to 1 L
8. Working buffer solution. Combine the following reagents in this order: Add 250 mL of stock
sodium-potassium tartrate solution (item no. 6) to 200 mL of stock buffer solution (item no. 7) and mix
Add an appropriate amount of sodium hydroxide solution (refer to Table 1 for concentration ranges) and
dilute to 1 L
9. Sodium salicylate/sodium nitroprusside solution. Dissolve 150 g of sodium salicylate and 0.3 g of
sodium nitroprusside in 600 mL of ammonia-free distilled water and dilute to 1 L
10. Sodium hypochlorite solution. Dilute 6.0 mL sodium hypochlorite solution (ChloroxR) to 100 mL
with ammonia-free distilled water.
1 1 . Ammonium chloride, stock solution. Dissolve 3.819 g NhUCI in distilled water and dilute to 1 L in a
volumetric flask. 1 mL = 1.
4. PROCEDURE
4.1 Sample handling and preservation. Analysis should be conducted as soon as possible after sample
collection. If samples cannot be analyzed immediately, samples can be preserved by the addition of 2 mL
of concentrated H£SO4 per liter of sample and stored under refrigeration at 4 °C. However, even when
preserved in this manner, conversion of organic nitrogen to ammonia may occur.
4.2 Sample analysis.
4.2.1 Digestion.
1. To 20 or 25 mL of sample, add 5 mL of digestion solution and mix, using a vortex mixer.
2. Add several (4-8) Teflon boiling chips. (Too many boiling chips may cause the sample to boil
over.)
3. With the Block Digester in its manual mode, set both the low and high temperatures at 160 °C
and preheat the unit to 160 °C. Place tube in the digester and switch to automatic mode. Set the
low temperature timer for 1 h. Reset the high temperature to 380 °C and set the timer for 2.5 h.
4. After digestion, cool the sample and dilute to 25 mL with ammonia-free distilled water.
4.2.2 Cokximetric analysis.
1. Check the levels of all reagent containers to ensure an adequate supply.
2. Excluding the salicylate line, place all reagent lines in their respective containers, connect the
sample probe to the Sampler IV, and start the proportioning pump.
-------
METHOD NO. A-NITROGEN-5
Table 1. Concentration Ranges (Nitrogen)
ml slock NaOH
Dilution loops Appro*. Range per liter
Initial sample Resample std. cal. PPM N working buffer
No. Sample line Diluent line Resample line Diluent line setting (£10%) solution
I .80 (RED/RED) .80 (RED/RED) .32 (BLK/BLK) .80 (RED/RED) 700 0-0.5 250
2 80 (RED/RED) .80 (RED/RED) .32 (BLK/BLK) .80 (RED/RED) 100 0-1.5 250
3 .16 (ORN/YEL) .80 (RED/RED) .32 (BLK/BLK) .80 (RED/RED) 700 0-1 120
.16 (ORN/YEL) .80 (RED/RED) .32 (BLK/BLK) .80 (RED/RED) 100 0-5 120
5 .16 (ORN/YEL) 80 (RED/RED) .16 (ORN/YEL) .80 (RED/RED) 700 0-2 80
6 .16 (ORN/YEL) .80 (RED/RED) .16 (ORN/YEL) .80 (RED/RED) 100 0-10 80
-------
METHOD NO. A-NFTROGEN-S
3. Flush the Sampler IV wash receptacle with about 25 mL of 4.0 percent H2SO4.
4. When reagents have been pumping for at least 5 min, place the salicylate line in its respective
container and allow the system to equilibrate. If a precipitate forms after the addition of
salicylate, the pH is too low. Immediately stop the proportioning pump and flush the coils with
water using a syringe. Before restarting the system, check the concentration of the sulfuric acid
solutions and/or the working buffer solution.
5. To prevent precipitation of sodium salicylate in the waste tray, which can clog the tray outlet,
keep the nitrogen flowcell pump tube and the nitrogen colorimeter waste outlet tube separate from
all other lines or keep tap water flowing in the waste tray.
6. Once a stable baseline has been obtained, start the sampler.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. The analyst should demonstrate the ability to generate acceptable precision with this
method using a laboratory control standard. Analysts should be able to meet the precision criteria
obtained by EMSL-using sewage samples with concentrations of 1.2,2.6, and 1.7 mg N/L, the precision
was ± 0.07, ± 0.03, and ± 0.15, respectively.
5.2 Accuracy. The analyst should demonstrate the ability to generate acceptable accuracy with, this
method using a laboratory control standard. Analysts should be able to meet the accuracy criteria
obtained by EMSL-using sewage samples with concentrations of 4.7 and 8.74 mg N/L, the recoveries
were 99 and 99 percent, respectively.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare an appropriate standard curve by plotting peak heights of processed standards
against concentration values. Calculate concentrations by comparing sample peak heights with the
standard curve.
6.2 Reporting units. Concentrations of total Kjeldahl nitrogen in unknown samples are reported in units
of mg N/L
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
6
-------
METHOD NO. A-NITROGEN-5
a REFERENCES
Gales, M.E. and R.L Booth. 1972. Evaluation of organic nitrogen methods. EPA Office of Research and
Monitoring.
Gales, M.E. and R.L Booth. 1974. Simultaneous and automated determination of total phosphorus and total
Kjeldahl nitrogen. Methods Development and Quality Assurance Research Laboratory.
Gales, M.E. and R.L Booth. 1978. Evaluation of the block digestion system for the measurement ot total
Kjeldahl nitrogen and total phosphorus. EPA-600/4-78-015, Environmental Monitoring and Support
Laboratory, Cincinnati, OH.
McDaniel, W.H., R.N. Hemphill, and W.T. Donaldson. 1967. Automated determination of total Kjeldahl
nitrogen in estuarine water. Technicon Symposia. Vol.1, pp. 362-367.
Technicon Corp. 1974. Total Kjeldahl nitrogen and total phosphorus BD-40 digestion procedure for water.
-------
METHOD NO. A-NITROGEN-6
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Total Kjeldahl nitrogen
1. METHOD TTTLE
Semi-Automated Method for the Determination of Total Kjeldahl Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrogen, Kjeldahl. Total. Method 4.1.1.1. Analytical Methods Manual for
Bottom Sediment Analysis (Draft). U.S. Environmental Protection Agency, Office of Research and
Development July 1974.
2.2. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
Description. This is a method that can be used for the determination of total Kjeldahl
nitrogen (TKN) in saline waters. The procedure converts nitrogen components of biological origin
such as amino acids, proteins, and peptides to ammonia, but may not convert the nitrogenous
compounds such as amines, nitro compounds, hydrazones, oximes, semi-carbazones, and some
refractory tertiary amines.
The applicable range of this method is 0.05 to 5.0 rag/kg TKN.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
233 Definitions.
1 . Total Kjeldahl Nitrogen is defined as the sum of free-ammonia and organic nitrogen compounds
which are converted to ammonium sulfate, (NH4)2SO4, under the conditions of digestion described
by this method.
2. Organic Kjeldahl Nitrogen is defined as the difference obtained by subtracting the free-ammonia
value from the total Kjeldahl nitrogen value.
2.4 Standanjization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NrTROGEN-6
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
Technicon AutoAnalyzer II, Industrial Model, consisting of the following components:
1. Sampler II or IV
2. Analytical cartridge no. 116-D223-01 or equivalent
3. Proportioning pump III or IV
4. Industrial AA-II single channel colorimeter equipped with 15 mm by 1.5 mm I.D. tubular flow cells
and 630 or 650 nm filters
5. Recorder
3.2 This method requires the following reagents:
1. Distilled water. Special precaution must be taken to insure that distilled water is free of ammonia.
Such water is prepared by passage of distilled water through an ion-exchange column consisting of a
mixture of both strongly acidic cation and strongly basic anion-exchange resins. Since organic
contamination may interfere with this analysis, use of the resin Dowex XE-75 or equivalent, which tends
to remove organic impurities, is advised. The regeneration of the ion-exchange column should be carried
out according to the manufacturer's instructions.
2. Digestion reagent. Dissolve 134 g of potassium sulfate, K2S04, in 650 mL ammonia-free distilled
water and 200 mL concentrated H2SO4. Add; while stirring, a solution prepared by dissolving 2 g of red
mercuric oxide, HgO, in 25 mL 6N H2SO4. Dilute the combined solution to 1 L Store above 20 °C to
prevent crystallization.
3. Boiling chips. Hengar, non-selenized
4. Air. Air used for segmenting the stream should be scrubbed (bubbled) through 5N H2SO4 prior to its
introduction into the system.
5. Sodium phenolate. In a 1-L Erlenmeyer flask, dissolve 83 g of phenol (freshly opened reagent grade)
in 50 mL of distilled water. Cautiously add, while cooling under tap water, and in small increments
with agitation, 180 mL of 20 percent NaOH. Filter through a glass fiber filter before use.
6. Sodium hypochlorite solution. (Chlorox) Dilute 200 mL of any good commercially available liquid
household bleach, having 5.25 percent available chlorine, to 1 L with distilled water. NOTE: Check the
label for the 5.25 percent formulation. Beware of the super bleach containing approximately 6 percent
available chlorine.
7. Complexing reagent. Dissolve 33 g of potassium sodium tartrate and 24 g of sodium citrate in
950 mL of distilled water. Adjust the pH of this solution to 5.0 with concentrated H2SO4. Dilute to
1 L with distilled water. After allowing to settle overnight in a cool place, filter to remove precipitate.
Then add 0.5 mL of Brij-35, (a registered trademark of Atlas Chemical Ind., supplied by Technicon
Corporation under part no. T 21-0110) and store in a stoppered bottle.
-------
METHOD NO. A-NITROGEN-6
8. Sodium nitroprusside. 0.05 percent. Dissolve 0.5 g of sodium nitroprusside in 1 L of ammonia-free
distilled water. (This reagent is also known as sodium nitroferricyanide.)
9. Stock standard ammonium sulfate (1000 mg/kg N). Dissolve 4.717 g of anhydrous ammonium sulfate,
(NH4)2SO4, dried at 105 °C, in ammonia-free water and dilute to 1 L 1 mL = 1.0 mg NH3-N.
10. Standard solution A. Dilute 10.0 mL of stock solution to 1 L with ammonia-free distilled water.
1.0 mL = 0.01 mgNH3-N.
11. Calibration standards. Using standard solution A, prepare the following standards in 100 mL
volumetric flasks. (Prepare standards fresh daily).
NHa-N. mg/ko mL Standard Solution A/100 mL
0.10 1.0
0.30 3.0
0.50 5.0
1.0 10.0
3.0 30.0
5.0 50.0
3.3 Equipment/instrument calibration. A calibration curve should be prepared for each day of sample
analysis using the calibration standards described in Section 3.2, Step 10.
4. PROCEDURE
4.1 Sample handling and preservation. Samples can be preserved by the addition of 40 rr~ HgCl2 per kg
of sample, and stored under refrigeration at 4 °C. (Note the HgCfc interference under Section 4.2.)
Alternatively, quick freezing and storage at -20 °C may be used. Sample handling should be kept to a
minimum in order to avoid absorption of ammonia from the ambient air.
4.2 Interferences. Calcium and magnesium ions may be present in concentrations sufficient to cause
precipitation problems during analysis. This problem is eliminated by using the complexing agent
containing sodium potassium tartrate and sodium citrate. Any marked variation in acidity or alkalinity
among samples should be eliminated, since the intensity of the color used to quantify the concentration
is pH dependent. Mercuric chloride, used as a preservative, gives a negative interference. This is
overcome by using a comparable amount of HgCl2 to the ammonia standards.
4.3 Sample digestion. To 50 mL of sample or standard, add 10 mL of digestion reagent and a few
Hengar boiling chips. Heat the flasks sufficiently to bring the samples to a brisk boil, and continue
until S03 fumes are obtained. Heat the solution further until it becomes colorless or pale yellow, then
continue digestion for an additional 30 min. Cool the residue, then dilute with 50 mL of distilled water.
Standards, as well as samples, should be digested in this manner.
4.4 Sample analysis.
1. For a working range of 0.05 to 5.0 mg/kg NHs-N, set up the manifold as shown in Figure 1.
3
-------
METHOD NO A-NITROGEN 5
20 turns 22 turns
Total KjglcfaM Nitrogen
after Manual Digestion
Range 0-5 mg/kg
20 turns j-
T
To Sampler
;.V:iCi ' -=• '.-:.•
To Waste
To Wast!
Air to range (.42)
iiiiflle lOrn-Blu (.051
iluK*1 Wafer Orn-WW
.4, SAMPLER IV
Rate: 30 per
hour
-Q
l art-rate
•(£>;
leach
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Pump
23)
Red (. 80}
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COLefUMKItR RICOBDEB
ISjnrn Tubular f/c
fo30 nrn RUers
,,)fA'' ..M.D.V1 HIVROGEH AFTER HAHU.AL DIGESTION MANIFOLD
Rgure 1. Manifold for Total KjekJahl Nitrogen Determination
4
-------
METHOD NO. A-NITROGEN-6
2. Allow both the colorimeter (with 650 nm filters and 15 mm flow cell) and the recorder to warm up
for 30 min. Run a baseline with all reagents, feeding ammonia-free water through the sample line.
Adjust the baseline knob and aperture opening on the colorimeter to obtain a proper baseline.
3. Use a 50 sample/h, 2:1 sample/wash sampler cam and operate the colorimeter in the Damp 1 mode.
All sample cups should be washed with 1 N HCI and rinsed thoroughly with distilled water in order to
remove any traces of ammonia. A final rinse should be made with the sample when filling the sampling
trays. The sample tray should be covered during the run and the tray should be filled with samples just
prior to loading the tray on the sampler.
4. A blank reading for the particular seawater of interest should be determined by sampling the
seawater while the nitroprusside and hypochlorite reagent lines are in distilled water. The blank
obtained should then be subtracted from the readings of the samples with the reagent lines in their
proper solutions.
5. Arrange ammonia standards in the sampler in order of decreasing concentration of nitrogen.
Complete loading of the sampler tray with unknown samples.
6. Switch the sample line from distilled water to sampler and begin analysis.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
Precision and Accuracy. No requirements for measurement of precision and accuracy are cited by this
method, however, analysts should demonstrate the ability to generate acceptable precision with this methos
using replicate sample analyses, and demonstrate acceptable accuracy using laboratory recovery samples and
blank samples.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare an appropriate standard curve derived from processing ammonia standards
through the manifold. Calculate concentration of samples by comparing sample peak heights with the
standard curve.
6.2 Reporting units. Concentrations of Kjeldah! nitrogen in unknown samples are reported in units of
mg/kg NHa-N.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
72. Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
-------
METHOD NO. A-NHROGEN-6
8. REFERENCES
APHA, AAWA, WPCF. 1971. Standard Methods for the Examination of Water and Wastewater, 13th ed. pp.
244-248.
U.S. Environmental Protection Agency. 1971. Methods for Chemical Analysis of Water and Wastes. U.S.
EPA, NERC, AQCL, Cincinnati, OH. pp. 141-147.
6
-------
METHOD NO. A-NFTROGEN-7
INDEX INFORMATION
Ma.rix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Total Kjeldahl nitrogen
1. METHOD TITLE
Manual Method for the Determination of Total Kjeldahl Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrogen, Kjeldahl, Total. Method 4.1.1.2. Analytical Methods Manual for
Bottom Sediment Analysis (Draft). U.S. Environmental Protection Agency, Office of Research and
Development. July 1974.
22. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This is a method that can be used for the determination of total Kjeldahl
nitrogen (TKN) in marine waters. The Kjeldahl nitrogen is defined as the summation of ammonia
nitrogen and those forms of nitrogen which, under the conditions of a vigorous catalyzed sulfuric
acid step, are converted to ammonia. The digestion step is usually followed by distillation into an
acidified aqueous solution.
The use of macro, semi-micro, and micro systems which do not change the chemistry of the
procedure is considered to be equivalent. The sample is heated in the presence of sulfuric acid,
potassium sulfate, and catalyst (HgSO4, SeO2, CuSo4, or combinations of all) to drive off water.
After the water has been evaporated, the digestion is continued until all organic matter is
converted to carbon dioxide and expelled. When the reflux mixture becomes colorless or pale
yellow, and sulfur dioxide fumes are visible, the reflux action is continued for another 30 min to
assure complete digestion.
The digested sulfuric acid mixture is cooled. The neutralization step will also require cooling
unless this is accomplished in a closed system with release of vapors into the acidified distillation
receiver.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEN-7
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. A digestion apparatus with adequate venting systems to assist in evaporation of water and to trap
chlorine and sulfur dioxide fumes.
2. A distillation system that will permit neutralization of the acid solution with the distillate which is
received submerged in a weak acid receiver to quantitatively entrap all ammonia vapor.
3. Spectrophotometer or filter photometer for use at 640 nm and providing a light path of 10 cm. A
shorter light path can be used as values approach 100 jig/kg.
4. All glassware used in sample preparation must be thoroughly cleaned, washed with warm dilute
hydrochloric acid, and thorougly rinsed in ammonia-free distilled water.
3.2 This procedure requires the following reagents:
1. The H2SO4 must be nitrogen-free. Each batch of H2SO4 must be compared for ammonia blanks.
Those batches that provide high ammonia blanks are to be discarded.
2. The distilled water used in preparation of reagents, blanks, and standards must be free of ammonia.
It may be necessary to percolate distilled water through an ion-exchange resin specifically formulated to
remove ammonia.
3. All reagents used in preparation of solutions must be of the highest quality obtainable and free of
ammonia.
4. The ammonium chloride used to prepare standards must be of reagent or better quality.
4. PROCEDURE
4.1 Sample handling and preservation. Samples should be analyzed as soon as possible after collection,
however, samples can be stored frozen in polyethylene containers. Mercuric chloride has also been
recommended as an inhibitor of biological activity.
4.2 Interferences. The cleaning of glassware and analysis of samples must be performed in a well-
ventilated smoke-free laboratory. (Smoke from tobacco has been shown to provide a positive
interference.) No other information is available on interferences with this method.
4.3 Sample analysis. Follow the procedure in U.S. Environmental Protection Agency. 1971. Methods
for Chemical Analysis of Water and Wastes. U.S. EPA, NERC, AQCL, Cincinnati, OH.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
Precision and accuracy. No data are available for precision and accuracy of this method with a seawater
matrix; this method will be updated to include this information as soon as data are available.
-------
METHOD NO. A-NITROGEN-7
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
Concentrations of Kjeldahl nitrogen in unknown samples are reported in units of mg/kg N.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should initially work under the guidance of
an experienced supervisor until he/she can demonstrate proficiency in the laboratory techniques
described in this method.
8. REFERENCES
Jenkins, D. 1965. A study of methods suitable for the analysis and preservation of nitrogen forms in an
estuarine environment. Report to the USPHS, Region IX, WSPC Division, SERL No. 65-13, College of
Engineering and School of Public Health, University of California.
Solorzano, L 1969. Determination of ammonia in natural waters by the phenol-hypochlorite method.
Limnology and Oceanography, Vol. 14, 99. 799-801.
U.S. Environmental Protection Agency. 1971. Methods for Chemical Analysis of Water and Wastes. U.S.
EPA, NERC, AQCL, Cincinnati, OH.
Weber, C.I. 1967. The preservation of plankton grab samples. Water Pollution Surveillance System
Applications and Development Report No. 26, FWPCA, Dept. of the Interior.
-------
METHOD NO. A-NITROGEN-a
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter: Nitrate-nitrite nitrogen
1. METHOD TITLE
Colorimetric Automated Cadmium Reduction Method for the Determination of Nitrate-Nitrite Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrogen, Nitrate-Nitrite. Method 353.2 (Colorimetric, Automated, Cadmium
Reduction) Storet No. 00630. In: Methods for Chemical Analysis of Water and Wastes. U.S.
Environmental Protection Agency, Environmental Monitoring and Support Laboratory (EMSL), Cincinnati,
OH. March 1979. EPA-600/4-79-020.
2.2 Regulatory status. This method is approved for NPDES and SDWA.
2.3 Principle and application.
2.3.1 Description. This method is approved by EPA for the determination of nitrite singly, or
nitrite and nitrate combined, in marine and estuarine waters, and domestic and industrial wastes.
The applicable range of this method is 0.05 to 10.0 mg/L nitrate-nitrite nitrogen. The range can be
extended with sample dilution.
A filtered sample is passed through a granulated copper-cadmium column to reduce nitrate to
nitrite. The nitrite (that which was originally present plus the reduced nitrate) is determined by
diazotizing with sulfanilamide and coupling with N-(1-naphthyl)-ethylenediamine dihydrochloride to
form a highly colored azo dye which is then measured colorimetrically. Separate, rater than
combined nitrate-nitrite values are readily obtained by carrying out the procedure first with, then
without the Cu-Cd reduction step.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method. Nitrate-nitrite samples can be collected
in glass or plastic containers. Prior to sample collection, each container and cup should be
thoroughly rinsed with sample water.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NrTROGEN-8
2.4.2 Precision and accuracy. Three laboratories participating in an EPA method study analyzed
four natural water samples containing exact increments of inorganic nitrate with the following
results:
Increment as
Nitrate N
(mgN/L)
0.29
0.035
2.31
2.48
Precision as
Standard Deviation
(mgN/L)
0.012
0.092
0.318
0.176
Accuracy as
Bias, percent
+ 5.75
+ 18.10
+ 4.47
- 2.69
Bias, mgN/L
+ 0.017
+ 0.063
+ 0.103
- 0.067
3. SPECIFICATIONS
3.1 This procedure requires the following equipment:
Technicon AutoAnalyzer Unit (AAI or AAII), consisting of the following components:
1. Sampler
2. Manifold (AAI) or Analytical Cartridge (AAII)
3. Proportioning pump
4. Heating bath with double delay coil (AAI)
5. Colorimeter equipped with 15 mm tubular flow cell and 630-660 nm filters
6. Recorder
7. Digital printer for AAII (optional)
3.2 This method requires the following reagents and supplies:
1. Distilled water. Because of possible contamination, this should be prepared by passage through an
ion-exchange column consisting of a mixture of both strongly acidic cation- and strongly basic anion-
exchange resins. The regeneration of the ion-exchange column should be carried out according to
manufacturer's instructions.
2. Dilute hydrochloric acid, 6N. Dilute 50 mL of concentrated HCI to 100 ml with distilled water.
3. Copper sulfate solution, 2 percent. Dissolve 20 g of CuSO4-5H2O in 500 mL of distilled water and
dilute to 1 L
4. Granulated cadmium. 40-6 mesh.
-------
METHOD NO. A-NfTROGEN-8
5. Copper-cadmium. The cadmium granules (new or used) are cleaned with dilute HCI and copperized
with a 2 percent solution of copper sulfate in the following manner:
• Wash the cadmium with HCI and rinse with distilled water. The color of the cadmium after this
treatment should be silver.
• SwirM 0 g of cadmium in 100 mL portions of 2 percent copper sulfate for 5 min or until blue color
partially fades. Decant and repeat with fresh copper sulfate until a brown colloidal precipitate
forms.
• Wash the copper-cadmium with distilled water for at least 10 min to remove all the precipitated
copper. The color of the cadmium after this treatment should be black.
6. Preparation of reduction column AAI. The reduction column is an 8 by 50 mm glass tube with the
ends reduced in diameter to permit insertion into the system. Copper-cadmium granules are placed in
the column between glass wool plugs. The packed reduction column is positioned in an up-flow 20°
incline to minimize channelling.
7. Preparation of reduction column AAII. The reduction column is a U-shaped, 35-cm long, 2-mm I.D.
glass tube. (In place of the 2-mm glass tube, a 0.081 I.D. pump tube-purple-can be used.) Fill the
reduction column with distilled water to prevent entrapment of air bubbles during the filling operation.
Transfer the copper-cadmium granules to the reduction column and place a glass wool plug in each end.
To prevent entrapment of air bubbles in the reduction column, ensure that all pump tubes are filled with
reagents before putting the column into the analytical system.
8. Color reagent. To approximately BOO mL of distilled water, add, while stirring, 100 mL concentrated
phosphoric acid, 40 g sulfanilamide, and 3 g N-(1-naphthyl)-ethylenediamine dihydrochloride. Stir until
dissolved and dilute to 1 L Store in an amber bottle and protect form light when not in use. This
solution is stable for several months.
9. Wash solution. Use distilled water for unpreserved samples. For samples preserved with H2SO4, use
2 mL H2SO4 per liter of wash water.
10. Ammonium chloride-EDTA solution. Dissolve 85 g of reagent grade ammonium chloride and 0.1 g of
disodium ethylenediamine tetraacetate in 900 mL of distilled water. Adjust the pH to 8.5 with
concentrated ammonium hydroxide and dilute to 1 L with distilled water. Add 0.5 mL Brij-35 solution (a
wetting agent recommended and supplied by Technicon Corp. for use in AutoAnalyzers).
11. Stock nitrate solution. Dissolve 7.216 g KNO3 in 500 mL of distilled water and dilute to 1 L in a
volumetric flask. Preserve with 2 mL of chloroform per liter; solution is stable for 6 mos.
1 mL = 1.0 mg NOa-N.
12. Stock nitrite solution. Dissolve 6.072 g KNO2 in 500 mL of distilled water and dilute to 1 L in a
volumetric flask. Preserve with 2 mL of chloroform and store under refrigeration.
1 mL = 1.0mgNO2-N.
13. Standard nitrate solution. Dilute 10.0 mL of stock nitrate solution to 1 L with distilled water.
Preserve with 2 mL of chloroform; solution is stable for 6 mos.
1 mL = 0.01 mg NO3-N.
-------
METHOD NO. A-NITRO -EN-8
14. Standard nitrite solution. Dilute 0.0 mL of stock nitrite solution to 1 L with distilled water. This
solution is unstable, so prepare as needed.
1 ml = 0.01 mg NO2-N.
15. Calibration standards. Using the standard nitrate solution, prepare trie following standards in 100.0
mL volumetric flasks. At least one nitrite standard should be compared :o a nitrate standard at the
same concentration to verify the efficiency of the column.
Concentration, - il_ Standard Solution/
mg NO2-N or NO3-N iOOmL
0.0
0.05
0.10
0.20
0.50
1.00
2.00
4.00
6.00
0.0
0.5
1.0
2.0
5.0
1.0
20.0
40.0
60.0
NOTE: When saline water samples are analyzed, Substitute Ocean Water (SOW) should be used for
preparing the above standards used for the calibration curve (otherwise, distilled water should be
used). Prepare SOW with the following concentrations of compounds:
Substitute Ocean Water (SOW)
NaCI
MgCl2
4.53 g/L
5.20 g/L
4.09 g/L
1.1 6 g/L
0.70 g/L
NaHCOa 0.20 g/L
KBr 0.10 g/L
H3BO3 0.03 g/L
SrCl2 0.03 g/L
NaF 0.003 g/L
CaCl2
KCI
If SOW is used, subtract its blank background response from the standards before preparing the
standard curve.
3.3 Equipment/instrument calibration. The efficiency of each reduction column should be checked by
comparing a nitrite standard to a reduced nitrate standard at the same concentration. This efficiency
check should be made at the beginning and the end of each sample run and at a minimum frequency of
every 10 samples. Reactivate the copper-cadmium granules when reduction falls below 75 percent.
A calibration curve should be prepared for each day of sample analysis using the calibration standards
described in Section 3.2. Step 15. Concentrations of the calibration standards should bracket the sarrole
concentrations. If a sarrple concentration is outside the range of calibration, then an additional
calibration standard she jld be analyzed to check if the result is within the linear range of the method.
Alternatively, the sample should be diluted to within the calibration range and then reanalyzed.
-------
METHOD NO. A-NITROGEN-8
4. PROCEDURE
4.1 Sample handling and preservation. Analysis should be conducted as soon as possible after sample
collection. Samples can be stored under refrigeration at 4 °C for up to 24 h. If samples must be stored
for more than 24 h, they should be preserved with 2 mL H2SO4 per liter of sample before refrigeration.
NOTE: Samples for reduction column must not be preserved with mercuric chloride.
42 Interferences. Various components of effluent can interfere with the analysis. Possible
interferences include suspended solids, residual chlorine, oil and grease, and high concentrations of iron,
copper, and other metals.
Build up of suspended matter in the reduction column will restrict sample flow. Because nitrate nitrogen
is found in a soluble state, the sample may be pre-filtered. Low results could be obtained for samples
containing high concentrations of iron, copper, or other metals. EDTA is added to the samples to
eliminate this interference. Samples containing high concentrations of oil and grease will coat the
surface of the cadmium. This interference is eliminated by pre-extracting the sample with an organic
solvent.
The area where nitrate-nitrite analyses are performed should also be isolated from exposure to nitric
acid or nitric acid fumes.
4.3 Sample analysis.
1. If the pH of the sample is less than 5 or greater than 9, adjust the pH to between 5 and 9 with
either concentrated HCI or NH/tOH.
2. Set up the manifold as shown in Figure 1 (AAI) or Figure 2 (AAII). Note that the copper-cadmium
reduction column should be in a 20° inclined position (AAII). Care should be taken not to introduce air
into the reduction column on the AAII.
3. Allow both the colorimeter and recorder to warm up for 30 min. Obtain a stable baseline with all
reagents while feeding distilled water through the sample line. NOTE: If a new reduction column is
being used, condition the column by running the 1 mg/L standard for 10 min. Subsequently, wash the
column with reagents for 20 min.
4. Place the appropriate nitrate and/or nitrite standards in the sampler in order of decreasing
concentration of nitrogen. Complete the loading of the sampler tray with unknown samples.
5. For the AAI system, sample at a rate of 30/h, 1:1 cam. For the AAII, use a 40/h, 4:1 cam and a
common wash.
6. Switch the sample line from distilled water to sampler and start analysis.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. The analyst should demonstrate the ability to generate acceptable precision with this
method using replicate sample analyses. Duplicate analyses should be conducted on a minimum of 5
percent of the total number of sarnples.
-------
METHOD NO. A-NITROGEN-S
10 SAMPLE WASH
WAJI£
ml/nil
iPS-3
0000
C-3* MIXER
W»SIE
"-c"
DOUIU MIXER
HO
COLUMN
HUE
0
BLUE
BLUE
BLUE
SAMPLER 2
RATE: 30 PER HR.
0.42
1.60
0.10 AIR
2.00
H}0
0.42
REAGENT
2.00
t.EO SAMPLE
1.20 8.5% NHjCL
1.20 AIR
WAStl
COLORIMETER
50» TURUIAR l/c
SipH FILTERS
PROPORTIONING PUMP
RECORDER
FROM C-3 TO SAMPLE LINE USE
.030 « .041 POLTETHTLENE TUBING.
SEE FIGURE 1. FOR DETAIL. COLUMN
SHOULD BE IN 20° INCLINE POSITION
RANGE EXPANDER
NITRATE • NITRITE MANIFOLD AA-I
Figure 1. Manifold for Nitrate-Nitrite Analysis (AAI)
6
-------
METHOD NO. A-NITROGEN-8
DIGITAL
PRINTER
PUMP TUBE "*""
RECORDER
RE
CO
WASTE
t
*
^
r
COLORIMETE
520 nm FILT
15mm FLOW
W
T
DUCTION^
LUMN
OOOOCX
^U/ACTI^ T
PUMP Tl
R
ER
CELL
ASH WATE
0 SAMPLE
•^^^^M
A2
0000
3
QUO
RP
1
J
R ^
R
ml/mln
BLACK m 0.32 AIR
(7
Y Y
BLACK
BLACK
BLACK
W W
GREY
G G
1.2 AMMONIUM
CHLORIDE
0.32 SAMPLE
0.32 AIR
032 COLOR
REAGENT
06 ui-
L0 WA«
2.0 WASH
0
SAMPLER
40/hr
»TE
5TE
PROPORTIONING
PUMP
NITRATE-NITRITE MANIFOLD AAII
Figure 2. Manifold for Nitrate-Nitrite Analysis (AAII)
7
-------
METHOD NO. A-NITROGEN-8
5.2 Accuracy. The analyst should demonstrate the ability to generate acceptable accuracy with this
method using laboratory recovery samples and blank samples. A spiked sample should be analysis should
be conducted on a minimum of 5 percent of the total number of samples; a blank should be analyzed
with each batch of samples; a U.S. EPA performance evaluation sample should be analyzed at least once
per quarter.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve derived from processing nitrate and/or nitrite standards
through the manifold. Calculate concentration of samples by comparing sample peak heights with the
standard curve.
6.2 Concentrations of nitrate in unknown samples are reported in units of mg NOs-N/L to a maximum
of three significant figures.
Results should be reported for all determinations, including QA replicates and spiked samples. Any
factors that may have influenced sample quality should also be reported.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
8. REFERENCES
Armstrong, F.A., C.R. Stearns, and J.D. Strickland. 1967. The measurement of upwelling and subsequent
biological processes by means of the Technicon AutoAnalyzer and associated equipment. Deep Sea
Research t4. pp. 381-389.
ASTM. 1967. Annual Book of ASTM Standards. Part 31. Water. Standard D1254. p. 366.
ASTM. 1976. Annual Book of ASTM Standards, Part 31. Water. Standard D 1141-75, Substitute Ocean
Water, p. 48.
Department of the Interior. 1966. Chemical Analyses for Water Quality Manual, Department of the Interior,
FWPCA, R.A. Taft Sanitary Engineering Center Training Program, Cincinnati, OH.
Fiore, J. and J.E. O'Brien. 1962. Automation in sanitary chemistry - parts 1 and 2 - determination of
nitrates and nitrites. Wastes Engineering 33,128 and 238.
-------
METHOD NO. A-NITROGEN-9
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter: Nitrate plus nitrite nitrogen
1. METHOD TITLE
Automated Method for the Determination of Nitrate Plus Nitrite Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrogen, Nitrate plus Nitrite. Method 4.1.4. In: Analytical Methods Manual for
Bottom Sediment Analysis (Draft). U.S. Environmental Protection Agency, Office of Research and
Development. July 1974.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This is a method for the determination of nitrates and nitrites, single or
combined, present in saline waters. The initial step is to reduce the nitrates to nitrites by using a
cadmium reductant. The nitrites (those originally present plus reduced nitrates) are then reacted
with sulfanilamide to form the diazo compound, which is then coupled in an acid solution (pH 2.0 to
2.5) with N-(l-naphthyl)-ethylenediamine to form the azo dye. The azo dye intensity, which is
proportional to the nitrite concentrations, is then measured.
The prescribed specifications permit analyses of samples in the range of 0.010 to 2.00 mg/kg N
present as NO2-N, NOs-N, or both.
2.3.3 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment:
Technicon AutoAnalyzer II (Industrial system), consisting of the following components:
1. Sampler
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEN-r
2. Analytical cartridge, Technicon part no. 116-D-I33-01 or equivalent
3. Industrial colorimeter, single channel, equipped with 15 mm x 1.5 mm tubular flow cells and 520 nm
filters
4. Recorder
5. Cadmium reduction column.
• Preparation. Shake the 16-28 mesh coarse cadmium powder (supplied unscreened by Technicon
Corporation under part no. T11-5063 or by E.M. Merck Co.) with a solution of 2 percent (w/v)
copper sulfate pentahydrate. A weight of solution equal to five times the weight of the cadmium is
used. Wash the excess blue copper solution from the cadmium granules as well as the brown
colloidal copper material. The remaining cadmium should be coated with a dark gray material wh ch
is the active reductant. Be careful to minimize washing this material off of the cadmium metal
surface. Fill the reductor tube with water to prevent the entrapment of air bubbles during the
filling operation. Transfer the prepared cadmium granules to the column using a small glass funnel
with a shortened stem connected to the U-tube with a piece of rubber or Tygon tubing. The
entire tube should also be filled with water. The U-tube should be filled from both ends. When
the entire column is filled with granules, insert a small piece of glass wool loosely in both ends.
• Regeneration. When the column has ;ost its reduction efficiency, the cadmium granules should e
removed and discarded. The column should then be repacked with new treated cadmium gran: as.
It is practical to have several prepared columns in storage under distilled water.
3.2 This method requires the following reagents:
1. Distilled water: Because of possible contamination, this should be prepared by passage through an
ion-exchange column consisting of a mixture of both strongly acidic cation- and strongly basic anion-
exchange resins. The regeneration of the ion-exchange column should be carried out according to
manufacturer's instructions.
2. Sulfanilamide. To about 800 mL of distilled water add 100 mL concentrated HCI and mix. Add 1 0 g
of sulfanilamide. Dissolve completely and dilute to 1 L This reagent is stable for at least one monvr
3. N-(l-Naphthyl)-ethylenediamine dihydrochloride (NEDA). To about 800 mL of distilled water, a
-------
METHOD NO. A-NfTROGEN-9
7. Stock nitrite solution. Dissolve 6.072 g KNO2 in distilled water and dilute to 1 L Add 40 mg HgCl2
to the solution. Store in a dark bottle. 1 mL = 1.0 mg NO2-N.
8. Standard nitrate solution. Dilute 10.0 ml of the stock nitrate solution to 1 L Add 40 mg HgCIa to
the solution. Store in a dark bottle. 1 mL = 0.01 mg NOs-N.
9. Standard nitrite solution. Dilute 10.0 mL of the stock nitrite solution to 1 L Add 40 mg HgCl2 to
the solution. Store in a dark bottle. 1 mL = 0.01 mg NO2-N.
10. Working standards. Using either the standard nitrate or standard nitrite solutions and distilled
water, prepare working standards in the range of 0.001 to 0.200 mg/kg. Add 40 mg HgCl2 to each liter
of working standard. Store in amber plastic bottles.
4. PROCEDURE
4.1 Sample handling and preservation. Samples may be preserved by the addition of 40 mg HgCl2 per
kg and stored at 4 °C. Samples may also be preserved by quick freezing in plastic containers at -
20 °C.
4.2 Interferences. There are very few known interferences at concentrations less that 1000 times that
of the nitrite; however, a recent addition of strong oxidants or reductants to the samples will readily
affect the nitrite concentrations. High alkalinity (greater than 600 mg/kg) will give low results due to a
shift in pH of the color reaction.
4.3 Sample analysis.
1. When the cadmium column is not in use on the cartridge, it should be disconnected from the inlet
debubbler at the point between the debubbler and the column. The inlet end of the column should then
be sealed using a knotted piece of Tygon tubing to prevent its draining and to keep air from entering
the column. Before turning on the pump during startup, the side-arm leg of the debubbler should also
be disconnected to allow extra bubbles to escape. When the bubble pattern is near normal, both of the
disconnected legs of the debubbler are reconnected. The debubbler is connected to the cadmium column
after removing the piece of Tygon tubing seal from it. The connection should be made quickly to
prevent an air bubble from entering the column. Air in the column will prevent surface contact by the
sample with the active cadmium surface. If a small amount of air accidentally enters the column, usually
running the system for several minutes will result the air being absorbed by the water being pumped
through the column.
2. Allow both the colorimeter and recorder to warm up for 30 min. Run a baseline with all reagents,
feeding distilled water through the sample line. Adjust the baseline knob to obtain a proper baseline.
Operate in Damp 1 mode at 50 samples per hour with a 2:1 sample to wash ratio.
2. Arrange the nitrate and nitrite standards in the sampler in order of decreasing concentration of
nitrogen. Complete loading of the sampler tray with unknown samples.
3. Switch the sample line from distilled water to sampler and begin analysis.
-------
METHOD NO. A-NITROGEN-9
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision and accuracy. No data are available for precision and accuracy of this method with a
seawater matrix; this method will be updated to include this information as soon as data are available.
5.2 Blank analyses. A blank reading for the particular seawater of interest should be determined by
sampling the seawater while running distilled water through the NEDA reagent line. The blank reading
should then be subtracted from the readings ot the unknowns.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare two standard curves from processing nitrite and nitrate standards through the
system. Calculate the concentration of samples by comparing sample peak heights with the standard
curve. Any sample having a computed concentration less than 10 percent of its immediate predecessor
must be rerun.
6.2 Reporting units. Concentrations of nitrite in unknown samples are reported in units of
mg/kg NQ-2-N; concentrations of nitrate in unknown samples are reported in units of mg/kg N03-N.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
8. REFERENCES
U.S. Environmental Protection Agency. 1971. Methods for Chemical Analysis of Water and Wastes. U.S. EPA,
NERC, AQCL, Cincinnati, OH. pp. 175-183.
-------
METHOD NO. A-NfTROGEN-10
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter. Nitrite nitrogen
1. METHOD TITLE
Automated Method for the Determination of Nitrite Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrite Nitrogen. Method 4.1.3.1 Analytical Methods Manual for Bottom
Sediment Analysis (Draft). U.S. Environmental Protection Agency, Office of Research and Development.
July 1974.
22 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This is a method that can be used for the determination of nitrites present in
saline water. The prescribed specifications permit analysis in the range of 0.001 to 0.2 mg/kg N
present as NO2-
The diazonium compound formed by diazotization of sulfanilamide by nitrite in water under acid
conditions is coupled with N-(1-naphthyl)-ethylenediamine to produce a reddish-purple color which
is measured at 520 nm.
Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as
soon as data are available.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
Technicon AutoAnalyzer Unit II (Industrial), consisting of the following components:
1 . Sampler II or IV and pump IV
2. Analytical cartridge, Technicon part no. 1 16-D241-01 or equivalent
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEN-10
3. Industrial colorimeter, single channel, equipped with 15 mm x 1.5 mm tubular -low cells and 520 nm
filters
4. Recorder
3.2 This procedure requires the following reagents:
1. Distilled water. Because of possible contamination, this should be preparec oy passage through an
ion-exchange column comprised of a mixture of bcth strongly acidic-cation ar strongly basic-cation
exchange resins. The regeneration of the ion-exchange column should be earned out according to
manufacturer's instructions.
2. Sulfanilamide. To about 800 mL of distilled water, add 100 mL concentrated HCI and mix. Add 1.0 g
of sulfanilamide. Dissolve completely and dilute to 1 L This reagent is stable for at least 1 month.
3. N-(1-Naphthyl)-ethylenediamine dihydrochloride (NEDA). To about 800 mL of distilled water, add 1
g NEDA and 1 mL Brij-35. (Brij-35 is a registered trademark of Atlas Chemical Ind., and is supplied by
Technicon Corporation under part no. T 21-0110.) Dissolve completely and dilute to 1 L This reagent
is stable for at least 1 month.
4. Wash solution. Use distilled water.
5. Stock nitrite solution. Dissolve 6.072 g KNO2 in distilled water and dilute to 1 L Add 40 mg HgCl2
to the solution. Store in a dark bottle. 1 mL = 1.0 mg NO2-N.
6. Standard nitrite solution. Dilute 10.0 mL of the stock nitrite solution to 1 L Add 40 mg HgCl2 to
the solution. Store in a dark bottle. 1 mL = 0.01 mg NC-2-N.
7. Working standards. Using the standard nitrite solution and distilled water, prepare working standards
in the range of 0.001 to 0.200 mg/kg. Add 40 mg HgCl2 to each liter of working standard. Store in
amber plastic bottles.
4. PROCEDURE
4.1 Sample handling and preservation. Samples may be preserved by the addition of 40 mg HgC > per
kilogram and stored at 4 °C. Samples may also be preserved by quick freezing in plastic containers at
-20 °C.
4.2 Interferences. There are very few known interferences at concentrations less that 1000 times that
of the nitrite; however, a recent addition of strong oxidants or reductants to the samples will read y
affect the nitrite concentrations. High alkalinity (greater than 600 mg/kg) will give low results due to a
shift in pH of the color reaction.
4.3 Sample analysis.
1. Allow both the colorimeter and recorder to warm up for 30 mir. Run a baseline with all reagr its,
feeding distilled water through the sa nple line. Adjust the baseli a knob to obtain a proper baseline.
Operate in Damp 1 mode at 50 samr. es per hour with a 2:1 sarr ;le to wash ratio.
-------
METHOD NO. A-NITROGEN-10
2. Arrange nitrite working standards in the sampler in order of decreasing concentration of nitrogen.
Complete loading of the sampler tray with unknown samples.
3. Switch the sample line from distilled water to sampler and begin analysis.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision and accuracy. No requirements for measurement of precision and accuracy are cited by
this method, however, analysts should demonstrate the ability to generate acceptable precision with this
method using replicate sample analyses, and demonstrate acceptable accuracy using laboratory recovery
samples and blank samples.
5.2 Blank analyses. A blank reading for the particular seawater of interest should be determined by
sampling the seawater while running distilled water through the NEDA reagent line. The blank reading
should then be subtracted from the readings of the unknowns.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve from processing nitrite standards through the system.
Calculate the concentration of samples by comparing sample peak heights with the standard curve. Any
sample having a computed concentration less than 10 percent of its immediate predecessor must be rerun.
6.2 Reporting units. Concentrations of nitrite in unknown samples are reported in units of mg/kg
N02-N.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
72 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
8. REFERENCES
U.S. Environmental Protection Agency. 1974. Analytical Methods Manual for Bottom Sediment Analysis
(Draft). U.S. Environmental Protection Agency, Office of Research and Development.
-------
METHOD NO. A-NfTROGEN-11
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Nitrite nitrogen
1. METHOD TITLE
Manual Method for the Determination of Nitrite Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrite Nitrogen. Method 4.1.3.2. Analytical Methods Manual for Bottom
Sediment Analysis (Draft). U.S. Environmental Protection Agency, Office of Research and Development.
July 1974.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This is a method that can be used for the determination of nitrite nitrogen in
seawater. Nitrite forms are transient in oxidation of ammonia or the reduction of nitrate. High
concentrations of nitrite ions are indicative of unstable conditions in natural seawaters.
The nitrite ion reacts with a primary aromatic amine to form a diazonium compound. The diazo
compound is then reacted with an aromatic amine, with the ability to couple, to produce a colored
azo dye.
2.3.3 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as
soon as data are available.
3. SPECIFICATIONS
3.1 Equipment A spectrophotometer or filter photometer capable of measuring absorbance at a
wavelength of 543 nm using 10 cm cells is required.
3.2 Reagents. For a list of reagents, refer to Method II.7, Determination of Reactive Nitrite, in
Strickland etal. (1968).
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NfTROGEN-1 1
4. PROCEDURE
4.1 Sample handling and preservation. Samples should be analyzed immediately after collection. If
analysis is delayed, preserve samples with mercuric chloride, such that the final concentration of
mercuric chloride is 40 mg/kg. The use of mercuric chloride is recommended because of its effectiveness
as an inhibitor of biological activity. Samples can also be preserved by freezing samples in polyethylene
containers. (Do not use mineral acid as a preservative.)
4.2 Sample analysis. For the procedure for this method, refer to Method II.7, Determination of
Reactive Nitrite, in Strickland and Parsons (1968).
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
Precision and accuracy. No requirements for demonstration of precision and accuracy were provided in this
method. However, the analyst should demonstrate the ability to generate acceptable precision and accuracy
with this method using replicate sample analyses and analysis of a laboratory control standard. Precision
levels are reported in Method II.7, Determination of Reactive Nitrite, in Strickland et al. (1968).
6. RECORDKEEPINQ AND DATA REPORTING REQUIREMENTS
Concentrations of nitrite in unknown samples are reported in units of /*g/kg NO2-N.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of all equipment. Personnel conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency in the laboratory
techniques described in this method.
a REFERENCES
Jenkins, D. 1965. A study of methods suitable for the analysis and preservation of nitrogen forms in an
estuarine environment. Report to the USPHS Region IX, WSPC Division, SERL NO. 65-13, College of
Engineering and School of Public Health, University of California.
Strickland, J.D.H., and T.R. Parsons. 1968. Determination of Reactive Nitrite. In: A Practical Handbook of
Seawater Analysis. Fisheries Research Board of Canada, Ottawa, Canada.
U.S. Environmental Protection Agency. 1971. Methods for Chemical Analysis of Water and Wastes. U.S.
EPA, NERC, AQCL, Cincinnati, OH. pp. 195-197.
-------
METHOD NO. A-NITROGEN-11
Weber, C.I. 1967. The preservation of plankton grab samples. Water Pollution Surveillance System
Applications and Development Report No. 26. FWPCA. Dept. of the Interior.
-------
METHOD NO. A-NFTROGEN-12
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter: Nitrate nitrogen
1. METHOD TITLE
Manual Method for the Determination of Nitrate Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrate Nitrogen. Method 4.3.5 Analytical Methods Manual for Bottom Sediment
Analysis (Draft). U.S. Environmental Protection Agency, Office of Research and Development. July
1974.
22. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This is a method that can be used for the determination of nitrates present in
saline water. The method is based on the reduction of nitrate to nitrite and the measurement of
nitrite. Nitrite analysis must be accomplished at the same time to differentiate between the nitrate
nitrogen and the nitrite nitrogen. The method of choice is that found in A Practical Handbook of
Seawater Analysis by J.D.H. Strickland and T.R. Parsons.
2.3.3 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available.
3. SPECIFICATIONS
3.1 Equipment The details on the reducing column are presented in Method II.6, Determination of
Reactive Nitrate, in Strickland et al. (1968).
32 Reagents. Reagent grade chemicals, free of nitrates are to be used in preparation. Follow the
details for preparation of reagents presented in Method II.6, Determination of Reactive Nitrate, in
Strickland and Parsons (1968).
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEN-12
4. PROCEDURE
4.1 Sample handling and preservation. Samples should be analyzed immediately after collection. If
analysis is delayed, preserve samples with mercuric chloride, such that the final concentration of
mercuric chloride is 40 mg/kg. The use of mercuric chloride is recommended because of its effectiveness
as an inhibitor of biological activity. Samples can also be preserved by freezing samples in polyethylene
containers.
42 Interferences.
1. Waters with high sulfide concentration will interfere because of the reaction of the sulfide with
cadmium.
2. Suspended material will plug up-the reductant column. Filter waters that appear turbid.
4.2 Sample analysis. Follow the details of the procedure presented in Method II.6, Determination of
Reactive Nitrate, in Strickland and Parsons (1968).
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
Precision and accuracy. No requirements for demonstration of precision and accuracy were provided in this
method. However, the analyst should demonstrate the ability to generate acceptable precision and accuracy
with this method using replicate sample analyses and analysis of a laboratory control standard. Precision
levels are reported in Method II.6, Determination of Reactive Nitrate, in Strickland, et al. (1968).
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
Reporting units. Concentrations of nitrate in unknown samples are reported in units of ^g/kg NOs-N.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of all equipment. Personnel conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency in the laboratory
techniques described in this method.
8. REFERENCES
Jenkins, D. 1965. A study of methods suitable for the analysis and preservation of nitrogen forms in an
estuarine environment. Report to the USPHS Region IX, WSPC Division, SERL NO. 65-13, College of
Engineering and School of Public Health, University of California.
-------
METHOD NO. A-NITROGEN-12
Strickland. J.D.H., and T.R. Parsons. 1968. Determination of Reactive Nitrite. In: A Practical Handbook of
Seawater Analysis. Fisheries Research Board of Canada, Ottawa. Canada.
Weber, C.I. 1967. The preservation of plankton grab samples. Water Pollution Surveillance System
Applications and Development Report No. ~6. FWPCA. Dept. of the Interior.
-------
METHOD NO. A-NITROGEN-13
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Ammonium nitrogen
1. METHOD TITLE
Determination of Ammonium Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. D'Elia. C. F.. N. L Kaumeyer. C.W. Keefe, K. V. Wood. C.F. Zimmerman. 1988.
Nutrient Analytical Services Laboratory Standard Operating Procedures. Ammonium. Chesapeake
Biological Laboratory (CBL), University of Maryland. Box 38, Solomons, Maryland 20688. Tel. (301)
326-4281.
This CBL method is based on the following procedure: Technicon Industrial Method No. 154-71 W/B
EPA. 1979. Chemical Analysis of Water and Wastes. USEPA-600/4-79-20. Method no. 365.1.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This method for the determination of ammonium in seawater is used by the
Nutrient Analytical Services Laboratory at the Chesapeake Biological Laboratory for analyses
conducted as part of the Chesapeake Bay Program.
Ammonium in a filtered sample reacts with alkaline phenol and hypochlorite to form indophenol blue
that is proportional to the ammonia concentration. The color is intensified by sodium
nitroprusside and is measured colorimetrically.
The limit of detection (the lowest concentration of an analyte that the analytical procedure can
reliably detect) is defined as three times the standard deviation of the mean of a minimum of seven
replicate analyses of one sample. At concentrations less than 0.25 mg/L the detection limit for
ammonium is 0.0051 mg/L At concentrations greater than 0.25 mg/U the detection limit for
ammonium is 0.0100 mg/L
This method requires the use of a segmented continuous flow analyzer, such as the AutoAnalyzer II,
where samples and reagents are continuously added in a specific sequence along a path of glass
tubing and mixing coils.Air bubbles are injected at precise intervals to sweep the walls of the
tubing and to help prevent diffusion between successive samples. The reactions in the
AutoAnalyzer do not develop to completion as in manual methods, by reach identical stages of
development in each sample, because every sample follows the same path, timing, and exposure to
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEN-13
specific reagents. The basic function of each component of the segmented continuous flow analyzer
is briefly discussed in Section 3.1. The explanation is similar to that of Sanborn and Larrance
(1972).
2.3.2 Reference to compatible sampling procedures. Surface, bottom, and water samples from above
and below the pycnocline are collected via a submersible pump system.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
Technicon AutoAnalyzer II system, consisting of the following components:
1. Sampler. A sampler probe alternately draws fluid from a tray of discrete samples and then from a
wash-fluid receptacle. The probe dips into the sample to be extracted, and at a timed interval, moves to
a wash solution while a tray of samples advances one position. A bubble of air, which acts as a
diffusion barrier, is aspirated into the sample stream between sample and wash. The ratio of sample to
wash time, as well as the number of samples analyzed per hour, are controlled by a cam located in the
top well of the sampler assembly. Cams are easily changed and are available for a varied range of
sampling rates.
The wash solution separates successive samples by alternating minima (wash) and maxima (sample). The
sample probe is connected to a stream divider that delivers identical samples simultaneously to each
manifold via the pump.
2. Proportioning pump. The proportioning pump is a peristaltic-type pump that continuously delivers
air, reagents, and samples to the manifold. Plastic pump tubes of various diameters are pressed between
a series of moving rollers and a platen. The motion of the rollers along the tubes delivers a continuous
flow. The delivery rate is determined by the inside diameter of the tube, because the rollers move at a
constant rate. These pump tubes are available in a large assortment of delivery rates. The pump will
hold a maximum of 28 tubes and has an air bar that mechanically measures and injects identical air
bubbles into the analytical stream. The pump tubes, which deliver reagents, air, and samples, are
connected to appropriate manifolds.
3. Manifold. Each analysis requires a manifold specifically designed for the chemical method being
used. The manifolds are composed of a series of horizontal glass coils, injection fittings, and heating
baths arranged for the proper sequence of reactions leading to color development. The samples and the
reagents mix within the glass coils. As two solutions with different densities travel around each turn of
the mixing coil, the denser solution falls through the less dense one, causing mixing and resulting in a
homogenous mixture of the two solutions. The length of the coil determines the amount of time allowed
for chemical reaction between the addition of successive reagents. Injection fittings for each of the
reagents are placed between mixing coils. Thus, a sample enters one end of the manifold, a reagent is
added, the solution is mixed and given time to react, and then another reagent is added and mixed.
After all reagents have been added and an adequate reaction time has passed, the solution flows into a
colorimeter.
-------
METHOD NO. A-NITOOGEN-13
4. Colorimeter. The colorimeter measures the absorption of monochromatic light by the solution in the
flow cell. Light from a single source passes through two separate but identical interference filters that
emit light within a narrow spectral band, then through the appropriate flow cell, and finally projects
onto a phototube which generates an electrical signal in response to the intensity of the impinging light.
The output from each phototube is a measure of transmittance and is converted electronically by the
colorimeter to a signal proportional to absorbance. The relationship between transmittance and
absorbance is given by the equation A = log I/T, where A = absorbance and T = transmittance. The
resulting signal is linear in absorbance and is directly proportional to concentration. As each sample
passes through the cell, the signals are sent to a recorder.
5. Recorder. Results of the analyses are continuously recorded by strip chart recorders. Each recorder
simultaneously monitors two separate analyses. The output of the colorimeter is proportional to
absorbance, and standards of known concentrations must be analyzed to relate absorbance to
concentration on the chart The analog signals can be converted to absorbance values by referring to
the Technicon reference curve and the standard calibration control.
&2 This method requires the following reagents:
1. Deionized water. Throughout this method, deionized water is defined as 18.3 megohm water. NOTE:
CBL uses a Barnstead Nanopure II System that produces Type 1 reagent grade water equal to or
exceeding the standards established by ASTM. Water is first filtered through a string prefilter and then
goes through a reverse-osmosis membrane. Final product water then passes through a series of five
filters (organic colloid, two mixed-bed cartridges, organic-free cartridge, and a 0.3 pm final filter).
2. Complexing reagent.
33.0 g Potassium sodium tartrate (KNaC4H4Oe 2H2O)
24.0 g Sodium citrate [HOC(COONa)(CH2OONa)2-2H2O]
lOOOmL Deionized water
0.5 mL Brij-35 (a wetting agent recommended and supplied by Technicon Corp. for use in
AutoAnalyzers).
Dissolve 33.0 g of potassium sodium tartrate and 24.0 g of sodium citrate in 950 ml deionized water.
Dilute to 1 L with deionized water and add 0.5 mL Brij-35. NOTE: Technicon states that the pH of
this reagent is adjusted to 5.0 with concentrated H2SO4. This step has been deleted by using less NaOH
than Technicon recommends in the alkaline phenol reagent.
3. Alkaline phenol reagent
22 mL Phenol (liquefied, 80 percent)
40 mL Sodium hydroxice (20 percent w/v NaOH)
188 mL Deionized water
-------
METHOD NO. A-NfTROGEN-13
To approximately 100 mL deionized water, add 22 mL liquid phenol. Cautiously add, in small increments,
40 mL of 20 percent NaOH and dilute to a volume of 250 mL with deionized water. WARNING: Phenol
is an extremely dangerous chemical and should be handled accordingly. Prepare fresh weekly under a
fume hood, and wear plastic gloves.
4. Sodium hypochlorite solution. Any commercially available household bleach containing 5.25 percent
available chlorine may be used. Dilute 200 mL of bleach to 1 L with deionized water. Prepare every
second day.
5. Sodium nitroprusside (Sodium nitroferricyanide).
0.5 g Sodium nitroprusside [Na2Fe(CN)sNO-2H2O]
lOOOmL Deionized water
Dissolve 0.5 g sodium nitroprusside in 900 mL deionized water and dilute to 1 L
6. Stock standard. Dissolve 0.100 g ammonium sulfate (NH4)2SO4 in approximately 900 mL deionized
water, then dilute to a volume of 1000 mL with deionized water. Add 1.0 mL chloroform to act as a
preservative. As a general rule, stock standards should be prepared every 6 mos, and the preparation
date logged.
7. Working standards. Prepare working standards daily. Concentrations of standards should encompass
the range of the samples.
0.10 mL of stock standard diluted to 100 mL with deionized water will yield 1.5 j*g at-N/L
(0.021 mg-N/L).
0.25 mL of stock standard diluted to 100 mL with deionized water will yield 3.75 jig at-N/L
(0.0525 mg-N/L).
0.5 mL of stock standard diluted to 100 mL with deionized water will yield 7.5 ?g at-N/L (0.105 mg-N/L).
1.0 mL of stock standard diluted to 100 mL with deionized water will yield 15 /ig at-N/L (0.21 mg-N/L).
2.0 mL of stock standard diluted to 100 mL with deionized water will yield 30 /*g at-N/L (0.42 mg-N/L).
3.0 mL of stock standard diluted to 100 mL with deionized water will yield 45 /«g at-N/L (0.63 mg-N/L).
3.3 Equipment/instrument calibration. The AutoAnalyzer is calibrated with each run. Refer to Section
4.2.3 for calibration procedures and Section 6.1 for calculations.
4. PROCEDURE
4.1 Sample handling and preservation. After collection, water samples are filtered through GF/F filters
(nominal pore size 0.7 /*m) and are placed in either polypropylene bottles or directly into 4 mL
Autoanalyzer cups. The samples are then stored frozen at > 20 °C until analysis (up to 28 days).
-------
METHOD NO. A-NrTROGEN-13
4.2 Sample analysis.
4.2.1 Glassware. Prior to use, wash all glassware with 1N HCI followed by numerous rinses with
deionized water.
4.Z2 Instrument specifications.
1. Manifold assembly. Refer to Figure 1 for manifold diagram
2. Standard calibration settings. 6.0.3.0, and 1.0
3. Damp. Normal
4. Sampling rate. 60 samples/h; 6:1 sample/wash ratio
5. Filter. 630 nm
6. Phototube. 199-B021-04
7. Flowcell. 50mm
4.Z3 Operating procedures.
1. Colorimeter. Turn the power on and allow 10 min for warm-up. Check standard calibration
setting for the desired determination.
2. Recorder. Turn the power on and allow 10 min for warm-up; check recorder paper supply.
3. Water reservoirs. Check and fill the deionized water reservoirs.
4. Pump tubes. Connect pump tubes and attach platen to pump.
5. Pump. Start the pump with deionized water flowing through the system. Check for leaks in
tubes at the connections and for a regular bubble pattern in the manifold.
7. Recorder. Turn the recorders on (chart paper should start moving).
8. Colorimeter. Check ZERO and FULL SCALE on the recorder. ZERO simulates a zero output so
that ZERO adjustment of the recorder can be made with a screwdriver.
9. Baseline control. With deionized water pumping through the system, establish a zero baseline
using the BASELINE CONTROL adjustment at a STD. CAL of 1.0.
10. Reagent blank. Allow reagents to pump through; note any rise in the baseline and readjust to
zero. Refer to this rise as the REAGENT BLANK (at a STD. CAL of 1.0).
11. Standard calibration control. An extremely wide range of nutrient concentrations, both
temporally and spatially, are found in estuarine and marine waters. The standard calibration
control setting (STD. CAL), located on the colorimeter, allows the operator to adjust the electrical
output to the concentration range of the standards or samples. Extremely low concentrations
-------
METHOD NO. A-NITROGEN-13
To Sarpler Wast Receptacle-
50°C
Heating
Coil
20 Turns
QQOQO
• 20 Turns
OOOC£»
Waste
COLORIMETER
630 nra filters
50 mm x 1.5 mm Flow/cell
199-B021-01 Phototube
or
199-B021-04 Phototube
HANIF3D OC»FIGURATION FX» \MMONim
GRN/ORN (Water)
BLK/BLK (Air)
RED/RED (Con^)lexing)
ORN/C1N (Sanple)
ORN/ORN (Phenol)
BLK/BLK (Sodium Hypochlor ite)
ORN/ORN (Sodium Nitroprusside)
YEL/BLU (From F/C)
.ampler
60/hr
6:1
Figure 1. ManHbM for Ammonium Nitrogen Analysis
6
-------
METHOD NO. A-NfTROGEN-13
(/*g/L) require high STD. CAL settings, or high sensitivity, whereas high concentrations (mg/L)
require lower STD. CAL settings, or lower sensitivity.
If a calibration curve encompassing a wide range of concentrations is necessary to analyze samples
that would otherwise go off scale, run all calibration standards at STD. CAL settings 1.0,3.0, and
6.0. (There should be no deflection of the pen at zero baseline if the STD. CAL setting is
switched back to 1.0.) Record the peak heights of standards at the various STD. CAL settings,
along with the STD. CAL settings.
12. Sample analysis. After the initial run of calibration standards, intersperse standards in the run
after approximately every 20 samples. Include at least one standard analyzed at each STD. CAL
setting employed during analysis of the preceding 40 samples. A visual comparison with the day's
initial standard curve should indicate no greater variance than 5 percent of the peak height (e.g., if
the initial standard peak height is 60.0, subsequent standards may vary from 57.0 to 63.0). If the
variance is greater than 5 percent, the source of the problem must be identified and corrected, and
the affected samples must be reanalyzed. The baseline should be adjusted after approximately every
20 samples. If an adjustment of more than 1 unit is required, the source of the problem must be
identified and corrected, and the affected samples must be reanalyzed.
4.2.4 Shutdown procedure.
1. At the end of the run, disconnect the reagents and place the tubes in distilled water.
2. Turn off recorder.
3. Wash the system with 1N HCI for 15 min; place the pump tubes in deionized water and wash
with deionized water for an additional 15 min.
4. Turn off the pump, release the proportioning platen, and loosen the pump tubes.
5. Turn off the colorimeter.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. Precision of this method for ammonium analysis is demonstrated by analysis of laboratory
duplicates. A total of four duplicates are analyzed at random per batch of samples collected (or per
cruise). Duplicate analyses are performed during the course of a run. After a sample is analyzed, the
same sample cup is removed from its position in the tray, and placed further along the sample tray to
be reanalyzed. The mean of the two values is reported as the concentration of that sample.
Results of the duplicate analyses are placed in a separate QA/QC data file along with the sample
number, sample date, and analysis date.
5.2 Accuracy. Accuracy of this method for ammonium analysis is demonstrated by analysis laboratory
spiked samples. A total of four spikes are analyzed at random per batch of samples collected (or per
cruise)-one each for the first and third sample collection days, and two for the second day. A spike is
prepared by adding a known volume of standard to a known volume of sample. This sample is then
analyzed and calculated as if it were a normal sample. A comparison is then made of the determined
-------
METHOD NO. A-NfTROGEN-13
value of the spiked sample and its expected value (calculated as the original sample concentration plus
the concentration of the spike).
These three concentrations (original, determined, and expected) are placed in a separate QA/QC data file
along with the sample number, sample date, and analysis data.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
ai Calculations. Prepare a standard curve in which the concentrations of the standards are entered
as the independent variables, and their corresponding peak heights are the dependent variable.
Concentrations of ammonium in samples are calculated from the linear regression obtained from the
standard curve.
When a broad range of sample concentrations requires that several standard calibration settings be
employed during a run, a separate regression must be determined for calculating concentrations from
peak heights read at each standard calibration setting. All standards analyzed during the run at a
particular STD. CAL setting are included in the calculations for that regression; and only samples whose
peak heights were measured at the same STD. CAL setting are calculated using that regression. For
example, peak heights taken from standards analyzed at STD. CAL 1.0 are used to determine the linear
regression at STD. CAL 1.0, and only concentrations of samples analyzed at STD. CAL 1.0 are
calculated using the regression at STD. CAL 1.0.
6.2 Data handling procedures.
1. The data are input to a predetermined format onto floppy disks via LOTUS 1-2-3 and a Compaq 386
microcomputer.
2. Printouts of the data are then verified by laboratory personnel, corrections are made, and all files
are sorted by date and sample number in ascending order.
3. The nitrite analysis data, along with other nutrient data, are then checked via a series of computer
programs to ascertain information such as NOa, NO2, and NH4 < Total dissolved nitrogen.
4. Print files are created from the LOTUS files.
5. If more than one data set is collected per month, the data are combined and sorted by date.
6.3 Data reporting. All analysis documents are kept in bound notebooks with a carbon copy given to
the investigator or granting agency. Information includes the name of analysis, collection date, source
of samples, analyst, analysis date, sample number, peak height, STD. CAL setting, sample concentration,
standard concentrations, standard peak heights, standard peak heights interspersed through the run,
regression statistics, results of duplicate analyses, results of spike analyses, and reagent blank readings.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
8
-------
METHOD NO. A-NFTROGEN-13
72 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
8. REFERENCES
Sanborn, H. and J. Larrance. 1972. An operations manual of the AutoAnalyzer for seawater nutrient
analysis. NOAA/NMFS, Seattle, Washington. 44 pp.
-------
METHOD NO. A-NfTROGEN-14
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter: Nitrite nitrogen
1. METHOD TIRE
Determination of Nitrite Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method D'Elia. C. F.. N. L Kaumeyer. C.W. Keefe, K. V. Wood. C.F. Zimmerman. 1988.
Nutrient Analytical Services Laboratory Standard Operating Procedures. Nitrite. Chesapeake Biological
Laboratory (CBL), University of Maryland. Box 38, Solomons, Maryland 20688. Tel. (301) 326-4281.
This CBL method is based on Technicon Industrial Method No. 161-71 W.
22. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This method for the determination of nitrite in seawater is used by the
Nutrient Analytical Services Laboratory at the Chesapeake Biological Laboratory for analyses
conducted as part of the Chesapeake Bay Program.
Nitrite in a filtered sample is determined by diazotizing with sulfanilamide and coupling with N-1-
naphthylethylenediamine dihydrochloride to form a colored azo dye, which is then measured
colorimetrically.
The limit of detection (the lowest concentration of an analyte that the analytical procedure can
reliably detect) is defined as three times the standard deviation of the mean of a minimum of seven
replicate analyses of one sample. At concentrations less than 0.05 mg/L, the detection limit for
nitrite is 0.0008 mg/L At concentrations greater than 0.05 mg/L, the detection limit for nitrite is
0.0011 mg/L
This method requires the use of a segmented continuous flow analyzer, such as the AutoAnalyzer II,
where samples and reagents are continuously added in a specific sequence along a path of glass
tubing and mixing coils. Air bubbles are injected at precise intervals to sweep the walls of the
tubing and to help prevent diffusion between successive samples. The reactions in the
AutoAnalyzer do not develop to completion as in manual methods, by reach identical stages of
development in each sample, because every sample follows the same path, timing, and exposure to
specific reagents.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEN-14
The basic function of each component of the segmented continuous flow analyzer is briefly
discussed in Section 3.1. The explanation is similar to that of Sanbom and Larrance (1972).
2J32 Reference to compatible samping procedures. Surface, bottom, and water samples from above
and below ihepycnodine are collected via a submersible pump system.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available
3. SPECIFICATIONS
3.1 This procedure requires the foBowing equipment
Technicon AutoAnalyzer II system, consisting of the following components:
1. Sampler. A sampler probe alternately draws fluid from a tray of discrete samples and then from a
wash-fluid receptacle. The probe dios into the sample to be extracted, and at a timed interval, moves to
a wash solution while a tray of samples advances one position. A bubble of air. which acts as a
diffusion barrier, is aspirated into the sample stream between sample and wash. The ratio of sample to
wash time, as well as the number of samples analyzed per hour, are controlled by a cam located in the
top well of the sampler assembly. Cams are easily changed and are available for a varied range of
sampling rates.
The wash solution separates successive samples by alternating minima (wash) and maxima (sample). The
sample probe is connected to a stream divider that delivers identical samples simultaneously to each
manifold via the pump.
*
2. Proportioning pump. The proportioning pump is a peristaltic-type pump that continuously delivers
air, reagents, and samples to the manifold. Plastic pump tubes of various diameters are pressed between
a series of moving rollers and a platen. The motion of the rollers along the tubes delivers a continuous
flow. The delivery rate is determined by the inside diameter of the tube, because the rollers move at a
constant rate. These pump tubes are available in a large assortment of delivery rates. The pump will
hold a maximum of 28 tubes and has an air bar that mechanically measures and injects identical air
bubbles into the analytical stream. The pump tubes, which deliver reagents, air, and samples, are
connected to appropriate manifolds.
3. Manifold. Each analysis requires a manifold specifically designed for the chemical method being
used. The manifolds are composed of a series of horizontal glass coils, injection fittings, and heating
baths arranged for the proper sequence of reactions leading to color development The samples and the
reagents mix within the glass coils. As two solutions with different densities travel around each turn of
the mixing coil, the denser solution falls through the less dense one, causing mixing and resulting in a
homogenous mixture of the two solutions. The length of the coil determines the amount of time allowed
for chemical reaction between the addition of successive reagents. Injection fittings for each of the
reagents are placed between mixing coils. Thus, a sample enters one end of the manifold, a reagent is
added, the solution is mixed and given time to react, and then another reagent is added and mixed.
After all reagents have been added and an adequate reaction time has passed, the solution flows into a
colorimeter.
-------
METHOD NO. A-NITROGEN-14
4. Colorimeter. -The colorimeter measures the absorption of monochromatic light by the solution in the
flow cell. Light from a single source passes through two separate but identical interference filters that
emit light within a narrow spectral band, then through the appropriate flow cell, and finally projects
onto a phototube which generates an electrical signal in response to the intensity of the impinging light.
The output from-each phototube is a measure of transmittance and is converted electronically by the
colorimeter to a signal proportional to absorbance. The relationship between transmittance and
absorbance is given by the equation A = log I/T, where A = absorbance and T = transmittance. The
resulting signal is linear in absorbance and is directly proportional to concentration. As each sample
passes through the cell, the signals are sent to a recorder.
5. Recorder. Results of the analyses are continuously recorded by strip chart recorders. Each recorder
simultaneously monitors two separate analyses. The output of the colorimeter is proportional to
absorbance, and standards of known concentrations must be analyzed to relate absorbance to
concentration on the chart The analog signals can be converted to absorbance values by referring to
the Technicon reference curve and the standard calibration control.
3.2 This method requires the following reagents:
1. Deionized water. Throughout this method, deionized water is defined as 18.3 megohm water. NOTE:
CBL uses a Barnstead Nanopure II System that produces Type 1 reagent grade water equal to or
exceeding the standards established by ASTM. Water is first filtered through a string prefilter and then
goes through a reverse-osmosis membrane. Final product water then passes through a series of five
filters (organic colloid, two mixed-bed cartridges, organic-free cartridge, and a 0.3 /*m final fitter).
2. Color reagent.
20.0 g Sulfanilamide
200.0 mL Concentrated phosphoric acid (80 percent)
1.0 g N-(1 -naphthy)lethylenediamine dihydrochloride
2000 mL Deionized water
1.0 mL Brij-35 (a wetting agent recommended and supplied by Technicon Corp. for use in
AutoAnalyzers).
To approximately 1500 mL of distilled deionized water, add 200 mL concentrated phosphoric acid (80
percent) and 20 g of sulfanilamide. Dissolve completely. Add 1.0 g of N-(l-naphthy)lethylenediamine
dihydrochloride and dissolve. Dilute to 2 L with deionized water and add 1.0 mL of Brij-35. Store in a
refrigerator; prepare fresh reagent every 6 weeks.
3. Stock standard. Dissolve 0.345 g sodium nitrite in approximately 900 mL deionized water, then dilute
to a volume of 1000 mL with deionized water (1 mL = 5 /*g at-N). Add 1.0 mL chloroform to act as a
preservative. As a general rule, stock standards should be prepared every 6 mos, and the preparation
date logged.
4. Working standards. Prepare working standards daily. Concentrations of standards should encompass
the range of the samples.
-------
METHOD NO. A-NfTROGEN-14
1.25 mL of stock standard diluted to 100 mL with deionized water will yield 0.5 /*g at N/L
(0.007 mg N/L).
2.5 mL of stock standard diluted to 100 mL with deionized water will yield 1 /*g at N/L (0.014 mg N/L).
5 mL of stock standard diluted to 100 mL with deionized water will yield 2 pg at N/L (0.028 mg N/L).
10 mL of stock standard diluted to 100 mL with deionized water will yield 4 jig at N/L (0.056 mg N/L).
15 mL of stock standard diluted to 100 mL with deionized water will yield 6 pg at N/L (0.084 mg N/L).
3.3 Equipment/instrument calibration. The AutoAnalyzer is calibrated with each run. Refer to Section
4.2.3 for calibration procedures, and Section 6.2 for calculations.
4. PROCEDURE
4.1 Sample handling and preservation. After collection, water samples are filtered through GF/F filters
(nominal pore size 0.7 /im) and are placed in either polypropylene bottles or directly into 4-mL
Autoanalyzer cups. The samples are then stored frozen at >20 °C until analysis (up to 28 days).
4.2 Sample analysis.
4.2.1 Glassware. Prior to use, wash all glassware with 1 N HCI followed by numerous rinses with
deionized water.
4.Z2 Instrument specifications.
1. Manifold assembly. Refer to Figure 1 for manifold diagram
2. Standard calibration settings. 9.0,6.0. and 0.5
3. Damp. Normal
4. Sampling rate. 60 samples/h; 6:1 sample/wash ratio
5. Filter. 550 nm
6. Phototuba 199-8021-01
7. Flowcell. 50mm
4.23 Operating procedures.
1. Colorimeter. Turn the power on and allow 10 min for warm-up. Check standard calibration
setting for the desired determination.
2. Recorder. Turn the power on and allow 10 min for warm-up; check recorder paper supply.
3. Water reservoirs. Check and fill the deionized water reservoirs.
4
-------
A2
METHOD NO. A-NITROGEN-14
MANIFOLD CONFIGURATION FOR NITRITE
To Sampler Wash Receptacle-
5 Turns
Coooo
22 Turns
oooooo
Debubbler
Waste _
Waste
COLORIMETER
550 nm
50 nm F/C x 1.5 nm H)
199-B021-01 Phototube
GRN/GRN (Water)
BLK/BLK (Air)
YEL/YEL (Water)
BLK/BLK (Sample)..
BLK/BLK (Air)
BLK/BLK (Color Reagent)
WHT/WHT
Sampler
60/hr.
6:1
GRY/GRY (From F/C)
Figure 1. Manifold for Nitrite Nitrogen Analysis
5
-------
METHOD NO. A-NITROGEN-14
4. Pump tubes. Connect pump tubes and attach platen to pump.
5. Pump. Start the pump with deionized water flowing through the system. Check for leaks in
tubes at the connections and for a regular bubble pattern in the manifold.
7. Recorder. Turn the recorders on (chart paper should start moving).
8. Colorimeter. Check ZERO and FULL SCALE on the recorder. ZERO simulates a zero output so
that ZERO adjustment of the recorder can be made with a screwdriver.
9. Baseline control. With deionized water pumping through the system, establish a zero baseline
using the BASELINE CONTROL adjustment at a STD. CAL of 1.0.
10. Reagent blank. Allow reagents to pump through; note any rise in the baseline and readjust to
zero. Refer to this rise as the REAGENT BLANK (at a STD. CAL of 1.0).
11.. Standard calibration control. An extremely wide range of nutrient concentrations, both
temporally and spatially, are found in estuarine and marine waters. The standard calibration
control setting (STD. CAL), located on the colorimeter, allows the operator to adjust the electrical
output to the concentration range of the standards or samples. Extremely low concentrations
(/ig/L) require high STD. CAL settings, or high sensitivity, whereas high concentrations (mg/L)
require lower STD. CAL settings, or lower sensitivity.
If a calibration curve encompassing a wide range of concentrations is necessary to analyze samples
that would otherwise go off scale, run all calibration standards at STD. CAL settings 9.0,6.0, and
0.5. (There should be no deflection of the pen at zero baseline if the STD. CAL setting is
switched back to 1.0.) Record the peak heights of standards at the various STD. CAL settings,
along with the STD. CAL settings.
13. Sample analysis. After the initial run of calibration standards, intersperse standards in the run
after approximately every 20 samples. Include at least one standard analyzed at each STD. CAL.
setting employed during analysis of the preceding 40 samples. A visual comparison with the day's
initial standard curve should indicate no greater variance than 5 percent of the peak height (e.g., if
the initial standard peak height is 60.0, subsequent standards may vary from 57.0 to 63.0). If the
variance is greater than 5 percent, the source of the problem must be identified and corrected, and
the affected samples must be reanalyzed. The baseline should be adjusted after approximately
every 20 samples. If an adjustment of more than 1 unit is required, the source of the problem
must be identified and corrected, and the affected samples must be reanalyzed.
42.4 Shutdown procedure.
1. At the end of the run, disconnect the reagents and place the tubes in distilled water.
2. Turn off recorder.
3. Wash the system with 1N HCI for 15 min; place the pump tubes in deionized water and wash
with deionized water for an additional 15 min.
4. Turn off the pump, release the proportioning platen, and loosen the pump tubes.
6
-------
METHOD NO. A-NITROGEN-14
4. Turn off the colorimeter.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. Precision of this method for nitrite analysis is demonstrated by analysis of laboratory
duplicates. A total of four duplicates are analyzed at random per batch of samples collected (or per
cruise). Duplicate analyses are performed during the course of a run. After a sample is analyzed, the
same sample cup is removed from its position in the tray, and placed further along the sample tray to
be reanalyzed. The mean of the two values is reported as the concentration of that sample.
Results of the duplicate analyses are placed in a separate QA/QC data file along with the sample
number, sample date, and analysis date.
5.2 Accuracy. Accuracy of this method for nitrite analysis is demonstrated by analysis laboratory
spiked samples. A total of four spikes are analyzed at random per batch of samples collected (or per
cruise)-one each for the first and third sample collection days, and two for the second day. A spike is
prepared by adding a known volume of standard to a known volume of sample. This sample is then
analyzed and calculated as if it were a normal sample. A comparison is then made of the determined
value of the spiked sample and its expected value (calculated as the original sample concentration plus
the concentration of the spike).
These three concentrations (original, determined, and expected) are placed in a separate QA/QC data file
along with the sample number, sample date, and analysis data.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve in which the concentrations of the standards are entered
as the independent variables, and their corresponding peak heights are the dependent variable.
Concentrations of nitrite in samples are calculated from the linear regression obtained from the standard
curve. Concentrations of nitrite are reported in units of mg N/L
When a broad range of sample concentrations requires that several standard calibration settings be
employed during a run, a separate regression must be determined for calculating concentrations from
peak heights read at each standard calibration setting. All standards analyzed during the run at a
particular STD. CAL setting are included in the calculations for that regression; and only samples whose
peak heights were measured at the same STD. CAL. setting are calculated using that regression. For
example, peak heights taken from standards analyzed at STD. CAL 1.0 are used to determine the linear
regression at STD. CAL 1.0, and only concentrations of samples analyzed at STD. CAL. 1.0 are
calculated using the regression at STD. CAL 1.0.
6.2 Data handling procedures.
1. The data are input to a predetermined format onto floppy disks via LOTUS 1-2-3 and a Compaq 386
microcomputer.
2. Printouts of the data are then verified by laboratory personnel, corrections are made, and all files
are sorted by date and sample number in ascending order.
-------
METHOD NO. A-NFTROGEN-14
3. The nitrite analysis data, along with other nutrient data, are then checked via a series of computer
programs to ascertain information such as:
NO3, NO2, and NH4 < Total dissolved nitrogen
NO2 < NO3 + NO2
4. Print files are created from the LOTUS files.
5. If more than one data set is collected per month, the data are combined and sorted by date.
6.3 Data reporting. All analysis documents are kept in bound notebooks with a carbon copy given to
the investigator or granting agency. Information includes the name of analysis, collection date, source
of samples, analyst, analysis date, sample number, peak height STD. CAL setting, sample concentration,
standard concentrations, standard peak heights, standard peak heights interspersed through the run,
regression statistics, results of duplicate analyses, results of spike analyses, and reagent blank readings.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
a REFERENCES
Sanborn, H. and J. Larrance. 1972. An operations manual of the AutoAnalyzer for seawater nutrient
analysis. NOAA/NMFS, Seattle, Washington. 44pp.
8
-------
METHOD NO. A-NITROGEN-15
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Nitrate plus nitrite nitrogen
1. METHOD TITLE
Determination of Nitrate plus Nitrite Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. D'Elia, C. F.. N. L Kaumeyer. C.W. Keefe. K. V. Wood, C.F. Zimmerman. 1988.
Nutrient Analytical Services Laboratory Standard Operating Procedures. Nitrate plus Nitrite. Chesapeake
Biological Laboratory (CBL), University of Maryland. Box 38, Solomons, Maryland 20688. Tel.
(301) 326-4281.
This CBL method is based on Technicon Industrial Method No. 158-71 W/A EPA. 1979. Chemical
Analysis of Water and Wastes, USEPA-600/4-79-020. Method No. 353.2.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This method for the determination of nitrate plus nitrite in seawater is used by
the Nutrient Analytical Services Laboratory at the Chesapeake Biological Laboratory for analyses
conducted as part of the Chesapeake Bay Program.
Filtered samples are passed through a granulated copper-cadmium column to reduce nitrate to
nitrite. The nitrite (that which was originally present plus the reduced nitrate) is then determined
by diazotizing with sulfanilamide and coupling with N-(1-naphthy)lethylenediamine dihydrochloride to
form a colored azo dye. Nitrate is obtained by subtracting NO2 values from NO2 +
The limit of detection (the lowest concentration of an analyte that the analytical procedure can
reliably detect) is defined as three times the standard deviation of the mean of a minimum of seven
replicate analyses of one sample. At concentrations less than 0.05 mg/L, the detection limit for
nitrate plus nitrite is 0.001 1 mg/L; at concentrations ranging from 0.05 to 0.12 mg/L, the detection
limit for nitrate plus nitrite is 0.0037 mg/L; at concentrations ranging from 0.12 to 0.30 mg/L, the
detection limit for nitrate plus nitrite is 0.0092 mg/L; at concentrations greater than 0.30 mg/L, the
detection limit for nitrate plus nitrite is 0.0121 mg/L
This method requires the use of a segmented continuous flow analyzer, such as the AutoAnalyzer II
where samples and reagents are continuously added in a specific sequence along a path of glass
tubing and mixing coils. Air bubbles are injected at precise intervals to sweep the walls
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by Jhe U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEN-15
of the tubing and to help prevent diffusion between successive samples. The reactions in the
AutoAnalyzer do not develop to completion as in manual methods, by reach identical stages of
development in each sample, because every sample follows the same path, timing, and exposure to
specific reagents.
The basic function of each component of the segmented continuous flow analyzer is briefly
discussed in Section 3.1. The explanation is similar to that of Sanborn and Larrance (1972).
2.3.2 Reference to compatible sampling procedures. Surface, bottom, and water samples from above
and below the pycnocline are collected via a submersible pump system.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
Technicon AutoAnalyzer II system, consisting of the following components:
1. Sampler. A sampler probe alternately draws fluid from a tray of discrete samples and then from a
wash-fluid receptacle. The probe dips into the sample to be extracted, and at a timed interval, moves to
a wash solution while a tray of samples advances one position. A bubble of air, which acts as a
diffusion barrier, is aspirated into the sample stream between sample and wash. The ratio of sample to
wash time, as well as the number of samples analyzed per hour, are controlled by a cam located in the
top well of the sampler assembly. Cams are easily changed and are available for a varied range of
sampling rates.
The wash solution separates successive samples by alternating minima (wash) and maxima (sample). The
sample probe is connected to a stream divider that delivers identical samples simultaneously to each
manifold via the pump.
2. Proportioning pump. The proportioning pump is a peristaltic-type pump that continuously delivers
air, reagents, and samples to the manifold. Plastic pump tubes of various diameters are pressed between
a series of moving rollers and a platen. The motion of the rollers along the tubes delivers a continuous
flow. The delivery rate is determined by the inside diameter of the tube, because the rollers move at a
constant rate. These pump tubes are available in a large assortment of delivery rates. The pump will
hold a maximum of 28 tubes and has an air bar that mechanically measures and injects identical air
bubbles into the analytical stream. The pump tubes, which deliver reagents, air, and samples, are
connected to appropriate manifolds.
3. Manifold. Each analysis requires a manifold specifically designed for the chemical method being
used. The manifolds are composed of a series of horizontal glass coils, injection fittings, and heating
baths arranged for the proper sequence of reactions leading to color development. The samples and the
reagents mix within the glass coils. As two solutions with different densities travel around each turn of
the mixing coil, the denser solution falls through the less dense one, causing mixing and resulting in a
homogenous mixture of the two solutions. The length of the coil determines the amount of time allowed
-------
METHOD NO. A-NITROGEN-15
for chemical reaction between the addition of successive reagents. Injection fittings for each of the
reagents are placed between mixing coils. Thus, a sample enters one end of the manifold, a reagent is
added, the solution is mixed and given time to react, and then another reagent is added and mixed.
After all reagents-have been added and an adequate reaction time has passed, the solution flows into a
colorimeter.
4. Colorimeter. The colorimeter measures the absorption of monochromatic light by the solution in the
flow cell. Light from a single source passes through two separate but identical interference filters that
emit light within a narrow spectral band, then through the appropriate flow cell, and finally projects
onto a phototube which generates an electrical signal in response to the intensity of the impinging light.
The output from each phototube is a measure of transmittance and is converted electronically by the
colorimeter to a signal proportional to absorbance. The relationship between transmittance and
absorbance is given by the equation.A = log I/T, where A = absorbance and T = transmittance. The
resulting signal is linear in absorbance and is directly proportional to concentration. As each sample
passes through the cell, the signals are sent to a recorder.
5. Recorder. Results of the analyses are continuously recorded by strip chart recorders. Each recorder
simultaneously monitors two separate analyses. The output of the colorimeter is proportional to
absorbance, and standards of known concentrations must be analyzed to relate absorbance to
concentration on the chart. The analog signals can be converted to absorbance values by referring to
the Technicon reference curve and the standard calibration control.
3.2 This method requires the following reagents:
1. Deionized water. Throughout this method, deionized water is defined as 18.3 megohm water. NOTE:
CBL uses a Barnstead Nanopure II System that produces Type 1 reagent grade water equal to or
exceeding the standards established by ASTM. Water is first filtered through a string prefilter and then
goes through a reverse-osmosis membrane. Final product water then passes through a series of five
filters (organic colloid, two mixed-bed cartridges, organic-free cartridge, and a 0.3 pm final filter).
2. Ammonium chloride reagent.
10.0g Ammonium chloride (NH4CI)
1000 ml Alkaline water. Prepare alkaline water by adding approximately 2 mL concentrated ammonium
hydroxide to 1 L of deionized water. This solution should attain a pH of 8.5.
Dissolve 10.0 g ammonium chloride in alkaline water and dilute to 1 L
3. Color reagent.
20.0 g Sulfanilamide
200.0 ml Concentrated phosphoric acid (80 percent)
1.0 g N-(l-naphthyl)-ethylenediamine dihydrochloride
2000 mL Deionized water
-------
METHOD NO. A-NfTROGEN-15
1.0 mL Brij-35 (a wetting agent rei ommended and supplied by Technicon Corp. for use in
AutoAnalyzers).
To approximately 1500 mL of distilled deionized water, add 200 mL concentrated phosphoric acid (80
percent) and 20 g of sulfanilamide. Dissolve completely. Add 1.0 g of N-(1-naphthyl)-ethylenediamine
dihydrochloride and dissolve. Dilute to 2 L with deionized water and add 1.0 mL of Brij-35. Store in a
refrigerator; prepare fresh reagent every 6 weeks.
4. Stock standard. Dissolve 0.5055 g potassium nitrate in approximately 900 mL deionized water, then
dilute to a volume of 1000 mL with deionized water (1 mL = 5 /ig-at N). As a general rule, stock
standards should be prepared every 6 mos, and the preparation date logged.
5. Working standard A. 0.8 mL of stock standard diluted to 1UO mL with deionized water will yield
40 /«g-at N/L (0.56 mg N/L).
6. Working standards.
0.8 mL of stock standard diluted to 200 mL with deionized water will yield 20 pg-at N/L (0.28 mg N/L).
1.0 mL of stock standard diluted to 100 mL with deionized water will yield 50 pg-at N/L (0.70 mg N/L).
1.5 mL of stock standard diluted to 100 mL with deionized water will yield 75 pg-at N/L (1.05 mg N/L).
2.5 mL of Working Standard A diluted to 100 mL with deionized water will yield 1.0 /ig-at N/L
(0.014 mg N/L).
5.0 mL of Working Standard A diluted to 100 mL with deionized water will yield 2.0 /*g-at N/L
(0.028 mg N/L).
10.0 mL of Working Standard A diluted to 100 mL with deionized water will yield 4.0 /*g-at N/L
(0.056 mg N/L).
15.0 mL of Working Standard A diluted to 100 mL with deionized water will yield 6.0 /*g-at N/L
(0.0.84 mg N/L).
25.0 mL of Working Standard A diluted to 100 mL with deionized water will yield 10.0 /*g-at N/L
(0.14 mgN/L).
NOTE: When analyzing samples at concentrations greater than 0.56 mg N/L (NOy + NO2~), substitute
the yellow/blue tube for ammonium chloride and the orange/yellow tube for the sample.)
3.3 Preparation of copper-cadmium column.
1. Use good quality cadmium filings, 25 to 60 mesh size.
2. Clean 10 g of cadmium with 50 mL of 6N HCI for 1 min. Decant the HCI and repeat the process one
more time.
3. Decant the HCI and wash the cadmium several times with distilled water.
4
-------
METHOD NO. A-NITROGEN-15
4. Decant the distilled water and add 50 mL of 2 percent (w/v) CuSO4-5H2O. Wash the cadmium until
no blue color remains in solution, then repeat the process one more time.
5. Decant and wash-thoroughly (approximately 10 times) with deionized water:
6. Fill the reductor column (a piece of 0.011 in. ID tubing, 22-cm long) with ammonium chloride reagent
and transfer the prepared cadmium particles to the column using a Pasteur pipette. Be careful not to
ow any air bubbles to be trapped in the column.
7. When the entire column is fairly well packed with granules, insert glass wool plugs at both ends of
the column. With reagents running through the system, attach the column to the intake side of the
valve first. Ensure that there are no air bubbles in the valve.
8. Check for good flow characteristics (e.g., consistent bubble pattern). If the column is packed too
tightly, an inconsistent flow pattern will result.
9. Prior to sample analysis, condition the column with approximately 100 mg N (as nitrate)/L for 5 min,
followed by 100 mg N (as nitrite)/L for 10 min.
3.4 Equipment/instrument calibration. The AutoAnalyzer is calibrated with each run. Refer to Section
4.3.3 for calibration procedures, and Section 6.2 for calculations.
4. PROCEDURE
4.1 Sample handling and preservation. After collection, water samples are filtered through GF/F filters
(nominal pore size 0.7 /im) and are placed in either polypropylene bottles or directly into 4-mL
Autoanalyzer cups. The samples are then stored frozen at > 20 °C until analysis (up to 28 days).
4.2 Interferences. Metal ions may produce a positive error if present in sufficient concentrations. The
presence of large concentrations of sulfide and/or sulfate will result in a loss of sensitivity to the
copper-cadmium column.
4.3 Sample analysis.
4.3.1 Glassware. Prior to use, wash all glassware with 1 N HCI followed by numerous rinses with
deionized water.
4.3.2 Instrument specifications.
1. Manifold assembly. Refer to Figure 1 for manifold diagram
2. Standard calibration settings. When using yellow/orange sample tubes-2.0,1.0, and 0.5; when
using black/black sample tubes-9.0, 6.0, and 0.5
3. Damp. Normal
4. Sampling rate. 40 samples/h; 9:1 sample/wash ratio
5. Filter. 550 nm
5
-------
Cadmium
Redactor
Tube_
METHOD NO. A-NITROGEN-15
MANIFOLD CONFIGURATION FOR NTCRAIE
To Sampler Wash Receptacle-^ GRN/GRN (Water)
A2
5 Turns
22 Turns
Debubbler
Waste —
waste
COLORIMETER
550 nm
50 ITTO F/C x 1.5 mm H>
199-8021-01 Phototube
BLK/BLK (Air)
YEL/YEL (Ammonium Chloride)
BLK/BLK (Sample)
BLK/BLK (Air)
BLK/BLK (Color Reagent)
WHT/WHT-
GRX/GRY (From F/C)
Note: If sample concentration >.56
substitute:YEL/BLU for Anmonium Chloride
ORN/YEL for Sanple
Rgure 1. Manifold for Nitrate plus Nitrite Nitrogen Analysis
6
-------
METHOD NO. A-NJTROGEN-15
6. Phototube. 199-B021-01
7. Flowcell. 50mm
4.3.3 Operating procedures.
1. Colorimeter. Turn the power on and allow 10 min for warm-up. Check standard calibration
setting for the desired determination.
2. Recorder. Turn the power on and allow 10 min for warm-up; check recorder paper supply.
3. Water reservoirs. Check and fill the deionized water reservoirs.
4. Pump tubes. Connect pump tubes and attach platen to pump.
5. Pump. Start the pump with deionized water flowing through the system. Check for leaks in
tubes at the connections and for a regular bubble pattern in the manifold.
6. Recorder. Turn the recorders on (chart paper should start moving).
7. Colorimeter. Check ZERO and FULL SCALE on the recorder. ZERO simulates a zero output so
that ZERO adjustment of the recorder can be made with a screwdriver.
8. Baseline control. With deionized water pumping through the system, establish a zero baseline
using the BASELINE CONTROL adjustment at a STD. CAL of 1.0.
9. Reagent blank. Allow reagents to pump through; note any rise in the baseline and readjust to
zero. Refer to this rise as the REAGENT BLANK (at a STD. CAL of 1.0).
10. Standard calibration control. An extremely wide range of nutrient concentrations, both
temporally and spatially, are found in estuarine and marine waters. The standard calibration
control setting (STD. CAL), located on the colorimeter, allows the operator to adjust the electrical
output to the concentration range of the standards or samples. Extremely low concentrations
(M9/L) require high STD. CAL settings, or high sensitivity, whereas high concentrations (mg/L)
require lower STD. CAL settings, or lower sensitivity.
If a calibration curve encompassing a wide range of concentrations is necessary to analyze samples
that would otherwise go off scale, run all calibration standards at STD. CAL settings 9.0,6.0, and
0.5. (There should be no deflection of the pen at zero baseline if the STD. CAL setting is
switched back to 1.0.) Record the peak heights of standards at the various STD. CAL settings,
along with the STD. CAL settings.
11. Sample analysis. After the initial run of calibration standards, intersperse standards in the run
after approximately every 20 samples. Include at least one standard analyzed at each STD. CAL.
setting employed during analysis of the preceding 40 samples. A visual comparison with the day's
initial standard curve should indicate no greater variance than 5 percent of the peak height (e.g., if
the initial standard peak height is 60.0, subsequent standards may vary from 57.0 to 63.0). If the
variance is greater than 5 percent, the source of the problem must be identified and corrected, and
the affected samples must be reanalyzed. The baseline should be adjusted after approximately every
-------
METHOD NO. A-NFTROGEN-IS
20 samples. If an adjustment of more than 1 unit is required, the source of the problem must be
identified and corrected, and the affected samples must be reanalyzed.
4.3.4 Shutdown procedure.
1. At the end of the run. disconnect the reagents and place the tubes in distilled water.
2. Turn off recorder.
3. Wash the system with 1N HCI for 15 min; place the pump tubes in deionized water and wash
with deionized water for an additional 15 min.
4. Turn off the pump, release the proportioning platen, and loosen the pump tubes.
5. Turn off the colorimeter.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. Precision of this method for nitrate plus nitrite analysis is demonstrated by analysis of
laboratory duplicates. A total of four duplicates are analyzed at random per batch of samples collected
(or per cruise). Duplicate analyses are performed during the course of a run. After a sample is
analyzed, the same sample cup is removed from its position in the tray, and placed further along the
sample tray to be reanalyzed. The mean of the two values is reported as the concentration of that
sample.
Results of the duplicate analyses are placed in a separate QA/QC data file along with the sample
number, sample date, and analysis date.
5.2 Accuracy. Accuracy of this method for nitrate plus nitrite analysis is demonstrated by analysis
laboratory spiked samples. A total of four spikes are analyzed at random per batch of samples collected
(or per cruise)-one each for the first and third sample collection days, and two for the second day. A
spike is prepared by adding a known volume of standard to a known volume of sample. This sample is
then analyzed and calculated as if it were a normal sample. A comparison is then made of the
determined value of the spiked sample and its expected value (calculated as the original sample
concentration plus the concentration of the spike).
These three concentrations (original, determined, and expected) are placed in a separate QA/QC data file
along with the sample number, sample date, and analysis data.
& RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve in which the concentrations of the standards are entered
as the independent variables, and their corresponding peak heights are the dependent variable.
Concentrations of nitrite in samples are calculated from the linear regression obtained from the standard
curve; nitrate is obtained by subtracting NC-2 values from NOa + NOa. Concentrations are reported in
units of mg N/L
8
-------
METHOD NO. A-NfTROGEN-15
When a broad range of sample concentrations requires that several standard calibration settings be
employed during a run, a separate regression must be determined for calculating concentrations from
peak heights read at each standard calibration setting. All standards analyzed during the run at a
particular STD. CAL-setting are included in the calculations for that regression; and only samples whose
peak heights were measured at the same STD. CAL setting are calculated using that regression. For
example, peak heights taken from standards analyzed at STD. CAL 1.0 are used to determine the linear
regression at STD. CAL 1.0, and only concentrations of samples analyzed at STD. CAL 1.0 are
calculated using the regression at STD. CAL 1.0.
6.2 Data handling procedures.
1. The data are input to a predetermined format onto floppy disks via LOTUS 1-2-3 and a Compaq 386
microcomputer.
2. Printouts of the data are then verified by laboratory personnel, corrections are made, and all files
are sorted by date and sample number in ascending order.
3. The nitrate and nitrite analysis data, along with other nutrient data, are then checked via a series
of computer programs to ascertain information such as:
NOa, NO2, and NH4 < Total dissolved nitrogen
NO2 < NOa + NO2
4. Print files are created from the LOTUS files.
5. If more than one data set is collected per month, the data are combined and sorted by date.
6.3 Data reporting. All analysis documents are kept in bound notebooks with a carbon copy given to
the investigator or granting agency. Information includes the name of analysis, collection date, source
of samples, analyst, analysis date, sample number, peak height, STD. CAL setting, sample concentration,
standard concentrations, standard peak heights, standard peak heights interspersed through the run,
regression statistics, results of duplicate analyses, results of spike analyses, and reagent blank readings.
7. SPECIAL PRECAUTIONS
7.1 Hearth and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
-------
METHOD NO. A-NITROGEN-15
8. REFERENCES
Sanborn, H. and J. Larrance. 1972. An operations manual of the AutoAnalyzer for seawater nutrient
analysis. NIOAA/NMFS, Seattle, Washington. 44 pp.
10
-------
METHOD NO. A-NrTBOGEN-16
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter: Kjeldahl nitrogen
1. METHOD TITLE
Determination of Kjeldahl Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. D'Elia. C. F...N. L Kaumeyer, C.W. Keefe, K. V. Wood, C.F. Zimmerman. 1988.
Nutrient Analytical Services Laboratory Standard Operating Procedures. Kjeldahl Nitrogen. Biological
Laboratory (CBL), University of Maryland. Box 38, Solomons, Maryland 20688. Tel. (301) 326-4281.
This CBL method is based on EPA, 1979, Chemical Analysis of Water and Wastes, USEPA-600/4-79-020.
Method no. 351.2.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This method for the determination of Kjeldahl nitrogen in seawater is used by
the Nutrient Analytical Services Laboratory at the Chesapeake Biological Laboratory for analyses
conducted as part of the Chesapeake Bay Program.
The sample is heated with a Teflon boiling ball in the presence of sulfuric acid, potassium sulfate,
and mercuric sulfate for 3.5 h. The residue is cooled, diluted to the original volume, and is then
analyzed for ammonium. The ammonium determination is based on a colorimetric method in which
and emerald green color is formed by the reaction of ammonia with sodium salicylate, sodium
nitroprusside, and sodium hypochlorite in a buffered alkaline medium at a pH of 12.8 to 13.0. The
ammonia salicylate complex is read at 660 nm using an automated analyzer.
The limit of detection (the lowest concentration of an analyte that the analytical procedure can
reliably detect) is defined as three times the standard deviation of the mean of a minimum of seven
replicate analyses of one sample. At concentrations less than 0.25 mg/L, the detection limit for
ammonium is 0.0051 mg/L; at concentrations greater than 0.25 mg/L, the detection limit for
ammonium is 0.0100 mg/L
This method requires the use of a segmented continuous flow analyzer, such as the AutoAnalyzer II,
where samples and reagents are continuously added in a specific sequence along a path of glass
'tubing and mixing coils. Air bubbles are injected at precise intervals to sweep the walls of the
tubing and to help prevent diffusion between successive samples. The reactions in the
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NfTROGEN-16
AutoAnalyzer do not develop to completion as in manual methods, by reach identical stages of
development in each sample, because every sample follows the same path, timing, and exposure to
specific reagents.
The basic function of each component of the segmented continuous flow analyzer is briefly
discussed in Section 3.1. The explanation is similar to that of Sanborn and Larrance (1972).
2.3.2 Reference to compatible sampling procedures. Surface, bottom, and water samples from above
and below the pycnocline are collected via a submersible pump system.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
Technicon AutoAnalyzer II system, consisting of the following components:
1. Sampler. A sampler probe alternately draws fluid from a tray of discrete samples and then from a
wash-fluid receptacle. The probe dips into the sample to be extracted, and at a timed interval, moves to
a wash solution while a tray of samples advances one position. A bubble of air, which acts as a
diffusion barrier, is aspirated into the sample stream between sample and wash. The ratio of sample to
wash time, as well as the number of samples analyzed per hour, are controlled by a cam located in the
top well of the sampler assembly. Cams are easily changed and are available for a varied range of
sampling rates.
The wash solution separates successive samples by alternating minima (wash) and maxima (sample). The
sample probe is connected to a stream divider that delivers identical samples simultaneously to each
manifold via the pump.
2. Proportioning pump. The proportioning pump is a peristaltic-type pump that continuously delivers
air, reagents, and samples to the manifold. Plastic pump tubes of various diameters are pressed between
a series of moving rollers and a platen. The motion of the rollers along the tubes delivers a continuous
flow. The delivery rate is determined by the inside diameter of the tube, because the rollers move at a
constant rate. These pump tubes are available in a large assortment of delivery rates. The pump will
hold a maximum of 28 tubes and has an air bar that mechanically measures and injects identical air
bubbles into the analytical stream. The pump tubes, which deliver reagents, air, and samples, are
connected to appropriate manifolds.
3. Manifold. Each analysis requires a manifold specifically designed for the chemical method being
used. The manifolds are composed of a series of horizontal glass coils, injection fittings, and heating
baths arranged for the proper sequence of reactions leading to color development. The samples and the
reagents mix within the glass coils. As two solutions with different densities travel around each turn of
the mixing coil, the denser solution falls through the less dense one, causing mixing and resulting in a
homogenous mixture of the two solutions. The length of the coil determines the amount of time allowed
for chemical reaction between the addition of successive reagents, injection fittings for each of the
reagents are placed between mixing coils. Thus, a sample enters one end of the manifold, a reagent is
-------
METHOD NO. A-NITROGEN-16
added, the solution is mixed and given time to react and then another reagent is added and mixed.
After all reagents have been added and an adequate reaction time has passed, the solution flows into a
colorimeter.
4. Colorimeter. -Ihe-colorimeter measures the absorption of monochromatic light by the solution in the
flow cell. Light from a single source passes through two separate but identical interference filters that
emit light within a narrow spectral band, then through the appropriate flow cell, and finally projects
onto a phototube which generates an electrical signal in response to the intensity of the impinging light.
The output from each phototube is a measure of transmittance and is converted electronically by the
colorimeter to a signal proportional to absorbance. The relationship between transmittance and
absorbance is given by the equation A = log I/T, where A = absorbance and T = transmittance. The
resulting signal is linear in absorbance and is directly proportional to concentration. As each sample
passes through the cell, the signals are sent to a recorder.
5. Recorder. Results of the analyses are continuously recorded by strip chart recorders. Each recorder
simultaneously monitors two separate analyses. The output of the colorimeter is proportional to
absorbance, and standards of known concentrations must be analyzed to relate absorbance to
concentration on the chart. The analog signals can be converted to absorbance values by referring to
the Technicon reference curve and the standard calibration control.
3.2 This method requires the following reagents:
1. Deionized water. Throughout this method, deionized water is defined as 18.3 megohm water. NOTE:
CBL uses a Barnstead Nanopure II System that produces Type 1 reagent grade water equal to or
exceeding the standards established by ASTM. Water is first filtered through a string prefilter and then
goes through a reverse-osmosis membrane. Final product water then passes through a series of five
filters (organic colloid, two mixed-bed cartridges, organic-free cartridge, and a 0.3 /*m final filter).
DIGESTION REAGENTS
2. Stock mercuric sulfate.
8 g Mercuric oxide, red (HgO)
10 mL Sulfuric acid, concentrated (H2SO4)
Dilute to 100 mL with deionized water.
3. Digestion solution.
135g Potassium sulfate (K2SO4)
200 mL Sulfuric acid, concentrated
25 mL Stock mercuric sulfate
Dissolve 135 g of K2SO4 in approximately 500 mL deionized water and slowly add 200 mL concentrated
H2SO4. Add 25 mL mercuric sulfate solution, let cool, and dilute to 1000 mL with deionized water.
-------
METHOD NO. A-NfTROGEN-16
ANALYSIS REAGENTS
4. Sulfuric acid sampler wash solution.
34 g Potassium sulfate («2SO4)
50 mL Sulfuric acid
To approximately 800 mL deionized water, add 34 g «2SO4 and dissolve. Slowly add 50 mL concentrated
H2SO4 and dilute to 1 L with deionized water.
5. Sodium chloride diluent solution. Dissolve 10 g sodium chloride in deionized water and dilute to 1 L
with deionized water.
6. Sodium hydroxide solution. To approximately 600 mL deionized water, carefully and slowly add 200 g
NaOH. NOTE: Wear goggles-a great deal of heat will be liberated. After the solution has cooled.
dilute to 1 L with deionized water.
7. Sodium salicylate/Sodium nitroprusside solution.
70.0 g Sodium salicylate
0.3 g Sodium nitroprusside
0.5 mL Brij-35 (a wetting agent recommended and supplied by Technicon Corp. for use in
Auto Analyzers).
Dissolve 70.0 g sodium salicylate and 0.3 g sodium nitroprusside in deionized water. Dilute with
deionized water to 1 L and add 0.5 mL Brij-35.
8. Sodium hypochlorite solution. Dilute 12 mL sodium hypochlorite (Chlorox) to 200 mL with deionized
water.
9. Stock buffer solution.
134.0 g Sodium phosphate, dibasic (Na2HPO4-7H2O)
20.0 g Sodium hydroxide
Heat to dissolve 134.0 g of sodium phosphate, dibasic, in approximately 800 mL deionized water. Add
20.0 g of sodium hydroxide and dilute to 1 L
10. Working buffer.
50 g Sodium potassium tartrate
200 mL Stock buffer solution
100 mL NaOH solution (20 percent, w/v)
-------
METHOD NO. A-NITROGEN-16
0.3 mL Brij-35
Add 50 g of sodium potassium tartrate to approximately 600 mL deionized water. (This is added as a
solid to avoid the rapid formation of mold during storage of a 20 percent w/v sodium potassium tartrate
stock solution.) -Add 200 mL of stock buffer and 100 mL of sodium hydroxide solution. Dilute to 1 L
with deionized water and add 0.3 mL Brij-35.
3.4 Equipment/instrument calibration. The AutoAnalyzer is calibrated with each run. Refer to Section
4.3.3 for calibration procedures, and Section 6.2 for calculations.
4. PROCEDURE
4.1 Sample handling and preservation. After collection, water samples are filtered through GF/F filters
(nominal pore size 0.7 j«m) and are placed in either polypropylene bottles or directly into 4 mL
Autoanalyzer cups. The samples are then stored frozen at > 20 °C until analysis (up to 28 days).
4.2 Glassware. Prior to use, wash all glassware with 1N HCI followed by numerous rinses with
deionized water.
4.3 Digestion procedure.
1. Prepare standards and blanks in exactly the same manner ad the samples, taking them all through
the digestion procedure.
2. Add a 25-mL sample to each digestion tube.
3. Add 5 mL of digestion solution and two Teflon boiling balls to each digestion tube and mix on a
vortex mixer.
4. Insert silicone airtight plugs in the digestion tubes whenever they are not being heated.
5. Heat the digestion tubes in a block digestor at 200 °C for 1 h, then at 360 °C for 2.5 h.
6. After heating, remove the tubes from the digestor and allow to cool for 15 min. Add approximately
15 mL of deionized water to each tube to dissolve any precipitate, cap each tube, and allow to stand
overnight.
7. The following day, dilute up to a volume of 25 mL with deionized water. (Digestion tubes must be
pre-marked.)
8. Cleaning digestion tubes. Add 25 mL of deionized water to each tube, and boil at 200 °C until dry.
The tubes may need to be rinsed with 20 percent NaOH, followed by numerous rinsed with deionized
water.
4.4 Analysis procedure.
4.4.1 Instrument specifications.
1. Manifold assembly. Refer to Figure 1 for manifold diagram.
5
-------
METHOD NO. A-NITROGEN-16
Manifold Configuration for Kjeldahl Nitrogen
To sampler wash receptacle
5 turns
OPOO a
heating coll
Colorimeter |
660 nm filters
50 mm x 1.5 mm flow cell
199-B021-01 phototubes
or
199-B021-04 phototubes
10 turns
GRN/GRN (H2S04 Sampler wash solution)
BLK/BLK (air)
RED/RED (NaCl Diluent Solution)
ORN/WHT (Sample)****'*
Sampler
30/hr
12:1
10 turns
20 turns
Waste
BLK/BLK (air)
PRO/RED (working huffer)
RLK/BLK (Rcsample)
Waste
BLK/BLK (Sal1cylate/N1tropruss1<1e)
ORN/YEL (Sodium HypochloHte Solution)
GRY/GRY (From F/C)
Figure 1. Manifold for Kjeldahl Nitrogen Analysis
6
-------
METHOD NO. A-NITROGEN-16
2. Standard calibration settings. 6.0
3. Damp. Normal
4. Sampling rate. 40 samples/h; 9:1 sample/wash ratio
5. Filter. 650 nm
6. Phototube. 199-B021-04
7. Flowcell. 50 mm
4.4.2 Operating procedures.
1. Colorimeter. Turn the power on and allow 10 min for warm-up. Check standard calibration
setting for the desired determination.
2. Recorder. Turn the power on and allow 10 min for warm-up; check recorder paper supply.
3. Water reservoirs. Check and fill the deionized water reservoirs.
4. Pump tubes. Connect pump tubes and attach platen to pump.
5. Pump. Start the pump with deionized water flowing through the system. Check for leaks in
tubes at the connections and for a regular bubble pattern in the manifold.
6. Recorder. Turn the recorders on (chart paper should start moving).
7. Colorimeter. Check ZERO and FULL SCALE on the recorder. ZERO simulates a zero output so
that ZERO adjustment of the recorder can be made with a screwdriver.
8. Baseline control. With deionized water pumping through the system, establish a zero baseline
using the BASELINE CONTROL adjustment at a STD. CAL of 1.0.
10. Reagent lines. With the system pumping and deionized water flowing through the system, add
all the reagent lines except the salicylate/nitroprusside line. After approximately 10 min, add the
salicylate/nitroprusside line. If the pH of the flow stream is low, the sodium salicylate line will
precipitate. Presence of precipitate requires diagnosis of the problem followed by corrective
action-remake reagents, check for clogged lines, and replace worn pump tubes.
11. Reagent blank. Allow reagents to pump through; note any rise in the baseline and readjust to
zero. Refer to this rise as the REAGENT BLANK (at a STD. CAL. of 1.0).
12. Standard calibration control. An extremely wide range of nutrient concentrations, both
temporally and spatially, are found in estuarine and marine waters. The standard calibration
control setting (STD. CAL.), located on the colorimeter, allows the operator to adjust the electrical
output to the concentration range of the standards or samples. Extremely low concentrations
(^g/L) require high STD. CAL settings, or high sensitivity, whereas high concentrations (mg/L)
require lower STD. CAL. settings, or lower sensitivity.
-------
METHOD NO. A-NITROGEN-16
Record the peak heights of standards along with the STD. CAL setting.
13. Sample analysis. After the initial run of calibration standards, intersperse standards in the run
after approximately every 20 samples. Include at least one standard analyzed at each STD. CAL.
setting employed during analysis of the preceding 40 samples. A visual comparison with the day's
initial standard curve should indicate no greater variance than 5 percent of the peak height (e.g., if
the initial standard peak height is 60.0, subsequent standards may vary from 57.0 to 63.0). If the
variance is greater than 5 percent, the source of the problem must be identified and corrected, and
the affected samples must be reanalyzed. The baseline should be adjusted after approximately every
20 samples. If an adjustment of more than 1 unit is required, the source of the problem must be
identified and corrected, and the affected samples must be reanalyzed.
4.4.3 Shutdown procedure.
1. At the end of the run, disconnect the reagents and place the tubes in distilled water.
2. Turn off recorder.
3. Wash the system with 1N HCI for 15 min; place the pump tubes in deionized water and wash
with deionized water for an additional 15 min.
4. Turn off the pump, release the proportioning platen, and loosen the pump tubes.
5. Turn off the colorimeter.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. Precision of this method for Kjeldahl nitrogen analysis is demonstrated by analysis of
laboratory duplicates. A total of four duplicates are analyzed at random per batch of samples collected
(or per cruise). Duplicate analyses are performed during the course of a run. After a sample is
analyzed, the same sample cup is removed from its position in the tray, and placed further along the
sample tray to be reanalyzed. The mean of the two values is reported as the concentration of that
sample.
Results of the duplicate analyses are placed in a separate QA/QC data file along with the sample
number, sample date, and analysis date.
5.2 Accuracy. Accuracy of this method for Kjeldahl nitrogen analysis is demonstrated by analysis
laboratory spiked samples. A total of four spikes are analyzed at random per batch of samples collected
(or per cruise)-one each for the first and third sample collection days, and two for the second day. A
spike is prepared by adding a known volume of standard to a known volume of sample. This sample is
then analyzed and calculated as if it were a normal sample. A comparison is then made of the
determined value of the spiked sample and its expected value (calculated as the original sample
concentration plus the concentration of the spike).
These three concentrations (original, determined, and expected) are placed in a separate QA/QC data file
along with the sample number, sample date, and analysis data.
8
-------
METHOD NO. A-NnnOGEN-16
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve in which the concentrations of the standards are entered
as the independent variables, and their corresponding peak heights are the dependent variable.
Concentrations of ammonium in samples are calculated from the linear regression obtained from the
standard curve. Concentrations are reported in units of mg N/L
When a broad range of sample concentrations requires that several standard calibration settings be
employed during a run, a separate regression must be determined for calculating concentrations from
peak heights read at each standard calibration setting. All standards analyzed during the run at a
particular STD. CAL setting are included in the calculations for that regression; and only samples whose
peak heights were measured at the same STD. CAL setting are calculated using that regression. For
example, peak heights taken from standards analyzed at STD. CAL 1.0 are used to determine the linear
regression at STD. CAL 1.0, and only concentrations of samples analyzed at STD. CAL. 1.0 are
calculated using the regression at STD. CAL. 1.0.
6.2 Data handling procedures.
1. The data are input to a predetermined format onto floppy disks via LOTUS 1-2-3 and a Compaq 386
microcomputer.
2. Printouts of the data are then verified by laboratory personnel, corrections are made, and all files
are sorted by date and sample number in ascending order.
3. The Kjeldahl nitrogen data, along with other nutrient data, are then checked via a series of
computer programs to ascertain information such as dissolved Kjeldahl N < total Kjeldahl N.
4. Print files are created from the LOTUS files.
5. If more than one data set is collected per month, the data are combined and sorted by date.
6.3 Data reporting. All analysis documents are kept in bound notebooks with a carbon copy given to
the investigator or granting agency. Information includes the name of analysis, collection date, source
of samples, analyst, analysis date, sample number, peak height, STD. CAL. setting, sample concentration,
standard concentrations, standard peak heights, standard peak heights interspersed through the run,
regression statistics, results of duplicate analyses, results of spike analyses, and reagent blank readings.
7. SPECIAL PRECAUTIONS
7.1 Health and safely considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing> be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
-------
METHOD NO. A-NfTROGEN-16
8. REFERENCES
Sanborn, H. and J. Larrance. 1972. An operations manual of the AutoAnalyzer for seawater nutrient
analysis. NOAA/NMFS, Seattle, Washington. 44 pp.
10
-------
METHOD NO. A-NfTROGEN-17
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Ammonia nitrogen
1. METHOD TITLE
Determination of Ammonia
2. BACKGROUND AND APPLICATION
2.1 Source of method. Determination of Ammonia. Method II.9. In: J.D.H. Strickland and T.R.
Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries Research Board of Canada,
Ottawa.
22. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. Seawater is treated with an alkaline citrate medium with sodium hypochlorite
and phenol in the presence of a catalyst (sodium nitroprusside). The blue indophenol formed with
ammonia is measured using a 10-cm cell. This method should be used when an estimate of the
concentration of ammonia alone is desirable.
This method will detect ammonia nitrogen with certainty in a single determination in the range of
0.1 A*g-at/L
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method. Comparability studies using this method with a sea
water matrix have not been cited; this method will be updated to include this information as soon as
data are available. This method states that precision at the 3 /*g-at/L level lies in the range of n
determinations ±0.15/n /«g-at/L; at the 1.0 /ig-at/L level, the range of the mean of n determinations is
±0.lO/n/*g-at/L
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. 125-mL Erlenmeyer flasks, cleaned with distilled water and drained.
2. Spectrophotometer.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEN-17
3.2 This procedure requires the following reagents.
NOTE: All solutions must be made with ammonia-free water.
1. Deionized Water. Pass distilled water through a small column (for example, 30 cm long and 1-2 cm
in diameter) of cation exchange resin in the hydrogen form just before use. Store the processed water
in a tightly stoppered flask.
NOTE: This specially treated distilled water should be used for making all solutions and for the
determination of standards and blanks. Do not use ordinary distilled water.
2. Sodium hypochlorite solution. Dissolve 12.5 g of good-quality sodium thiosulfate (Na2S2O3-5H2O) in
500 mL of deionized water. Add a few crystals (approximately 2 g) of potassium iodide (Kl) to 50 ml
of deionized water in a small flask. Pipet into the Kl solution 1.0 mL of hypochlorite solution. (Use a
solution of commercial hypochlorite (e.g. Chlorox) which should be approximately 1.5 N (this solution
decomposes slowly and should be checked periodically). Add 5-10 drops of concentrated hydrochloric
acid and titrate the liberated iodine with the thiosulfate solution until no yellow color remains. Discard
the hypochlorite when less than 12 mL of thiosulfate is used.
3. Phenol solution. Dissolve 20 g of crystalline analytical reagent grade phenol in 200 mL of 95 percent
(v/v) ethyl alcohol.
4. Sodium nitroprusside solution. Dissolve 1.0 g of sodium nitroprusside (Na2Fe(CN)sNO-2H2O) in
200 mL of deionized water. Store the solution in an amber bottle. The solution will be stable for one
month.
5. Alkaline reagent. Dissolve 100 g of sodium citrate and 5 g of analytical reagent grade sodium
hydroxide in 500 mL deionized water. This solution remains stable indefinitely.
6. Ox'dizing solution. Mix 100 mL of alkaline reagent (see above) and 25 mL of sodium hypochlorite
solution (see above). Tightly stopper when not in use. Prepare fresh solution daily.
3.3 Equipment/instrument calibration.
NOTE: Carry out the following calibration procedures using filtered sea water.
1. Standard ammonia solution. Dissolve 0.100 g of analytical reagent quality ammonium sulphate in 1000
mL of distilled water. Add 1 mL of chloroform and store sheltered from strong light. If tightly
stoppered, the solution is stable for several months. Pipet 1.00 mL (1 mL = 1.5 /«g-at N) of this solution
into a 500-mL volumetric flask and complete with sea water to the mark. The resulting ammonium
concentration is equivalent to 3.0 ^g-at N/L
2. Procedure. Measure out 50 mL portions of the dilute ammonia solution (see above) into each of four
clean 125-mL Erlenmeyer flasks. Prepare two blank solutions with no added ammonia as described in
Section 4.4.1. Carry out the determinations as described in Sections 4.3.1 and 4.3.2. Read the extinction
after 60 min. Calculate the F factor as described in Section 6.1.1.
4. PROCEDURE
4.1 Sample handling and preservation. Samples may be temporarily stored in either glass or
polyethylene. Samples should be analyzed within 2 h of collection; samples should be frozen solid in a
-------
METHOD NO. A-NITROGEN-17
deep-freeze if a longer storage time is required. There are indications that, even with refrigeration,
sample degradation may be significant after more than a few days.
4.2 Interferences.
1. The quantity and strength of sodium hypochlorite have a significant effect in the reaction in sea
water. When more dilute hypochlorite is used, a correspondingly higher volume can be added, but care
must be taken that the pH does not exceed 9.8. At a higher pH, the reaction is faster but produces a
slight blue coloration (associated with the presence of nitro-prusside and not due to the presence of
amino compounds) is suppressed when working with distilled water. This results in a false blank and
erroneously high values for the ammonia content of sea water. At the pH used in this method, or pH of
9.8 in seawater and 10.4 in distilled water, the blue coloration form the nitro-prusside is formed equally
in samples and blanks.
2. Sodium nitroprusside in concentrations of 0.5 percent is sufficient to catalyze the reaction and
produce a stable and low blank. Higher concentrations produce high and unstable blanks; this increases
with time.
3. Urea and several amino acids, including glycine, D-L Alanine, L-Arginine, D-L Histidine, L-Tyrosine,
L-Lysine, and L-Glutamine, in concentrations of 3 /*g-at N/L in filtered seawater gave negligible
response to this treatment.
4.3 Sample analysis.
NOTE: Section 4.4 should be consulted before commencing analysis.
1. Place 50 mL of sample into an Erlenmeyer flask. Add 2 ml_ of phenol solution and swirl the
solution. Sequentially add 2 ml of sodium nitroprusside solution and 5 mL of oxidizing solution, mixing
after each addition. Allow the flask to stand at room temperature (20 to 27°C) for 1 h (the reaction
requires a full 60 min for completion and produces a color which is stable for at least 24 h). Cover the
top of the flask with aluminum foil to lessen contamination by atmospheric ammonia.
2. Using 10-cm cells in a spectrophotometer, read the extinction at 6400 A against distilled water.
3. Correct the extinction of the sample by the extinction of the reagent blank. Calculate the ammonia-
nitrogen using the formula given in Section 6.1.
4.4 Preparation of quality control blanks.
1. Reagent blanks. Prepare these blanks as described in Sections 4.3.1 and 4.3.2 using distilled water.
2. Precautions to reduce contamination. Great care must be taken to prevent laboratory contamination
of ammonia, carried as gas or particles of ammonia salts, in samples and blanks. Solutions should be
kept in tightly stoppered bottles and samples should be stored in well stoppered containers until analysis
is begun. Under no circumstances should bottles of ammonium hydroxide be opened in the laboratory
while samples are being analyzed. All glassware should be prewashed with dilute acid and thoroughly
rinsed with distilled water immediately before each use (ordinary distilled water may be used).
-------
METHOD NO. A-NITROGEN-17
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. Reject duplicate determinations if extinction values differ by more than 0.015 in the
extinction range 0.1 to 0.2, and by more than 0.025 in the extinction range 0.2 to 0.5. If duplicate
extinction values differ by less than these limits, take a mean value.
5.2 Accuracy. Reagent blanks extinctions using a 10-cm cell should not exceed 0.075.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations.
1. F factor.
F = 3.0 / (Es - Eb)
where F =F factor
Es =mean extinction of the standards.
Eb =mean extinction of the blanks.
2. Concentration of ammonia-nitrogen.
jjg-at N/L = F x E
where F =F factor
E = corrected extinction
6.2 Reporting units. Concentrations of ammonia-nitrogen in unknown samples are reported in
/*g-at N/L Results are reported to two significant figures.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions; wear protective eyewear and clothing, be familiar with laboratory safety devices, understand
the proper handling of acids, solvents, and other reagents.
72. Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the equipment. Personnel conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency in the laboratory
techniques described in this method.
8. REFERENCES
Strickland, J.D.H. and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries
Research Board of Canada, Ottawa.
-------
METHOD NO. A-NITROGEN-18
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter: Ammonia plus amino acids
1. METHOD TITLE
Determination of Ammonia Plus Amino Acids
2. BACKGROUND AND APPLICATION
2.1 Source of method. Determination of Ammonia Plus Amino Acids. Method U.S. In: J.D.H.
Strickland and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries
Research Board of Canada, Ottawa.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. The ammonia in sea water is oxidized to nitrite by hypochlorite in alkaline
medium. The excess oxidant is destroyed by the addition of arsenite. The nitrite is determined by
allowing the sea water to react with sulphanilamide in an acid solution. The resulting diazo
compound reacts with N-(l-naphthyl)-ethylenediamine and forms a highly colored azo dye, the
extinction of which is measured using 10-cm cells.
This method will detect ammonia nitrogen in the range of 0.1 to 10 j*g-at/L The smallest quantity
of ammonia nitrogen that can be detected by this method is 0.1
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method. Comparability studies using this method with a sea
water matrix have not been cited; this method will include this information as soon as data are available.
This method states that precision at the 3 j*g-at/L level lies in the range of the mean of n
determinations ± 0.25/nV2^g-at/L; precision at the 1.0 /ig-at/L lies in the range of the mean of n
determinations ± 0.11/n1/2jjg-at/L
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. 125-mL Erlenmeyer flasks, prerinsed with warm hydrochloric acid (10 percent), thoroughly rinsed with
distilled water, and drained completely before use.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NfTROGEN-18
2. Spectrophotometer.
3. Automatic pipettor.
3.2 This procedure requires the following reagents.
NOTE: All solutions must be made with ammonia-free water.
1. Deionized water. Just before use, remove ammonia from distilled water by passing it through a small
column (for example, 30 cm long and 1 to 2 cm ID) of cation exchange resin in the hydrogen form.
Store the water in a tightly stoppered glass flask.
2. Alkaline sodium citrate. Dissolve 700 g of sodium citrate and 40 g of sodium hydroxide in 2000 ml
of deionized water. Store the solution in a tightly stoppered polyethylene bottle. The solution will be
stable for several months.
3. Sodium hypochlorite solution. Use a solution of commercial hypochlorite (for example, Chlorox)
which should be approximately 1.5 N. The solution decomposes slowly and should be checked
periodically. Dissolve 12.5 g of good-quality sodium thiosulfate (NaaSaOa-SHaO) in 500 mL of water.
Add a few crystals (approximately 2 g) of potassium iodide (Kl) to about 50 mL of water in a small
flask. Pipet 1.0 mL of hypochlorite solution into the flask. Add 5 to 10 drops of concentrated
hydrochloric acid and titrate the liberated iodine with the thiosulfate solution until no yellow color
remains. Discard the hypochlorite when less than 12 mL of thiosulfate is used.
4. Sodium arsenite solution. Dissolve 20 g of analytical reagent sodium meta-arsenite (Na2AsO2) in
1000 mL of deionized water. Store the solution in a well-stoppered polyethylene bottle. The solution
will remain stable indefinitely.
5. Sodium bromide solution. Dissolve 1.5 g analytical reagent quality sodium bromide (NaBn) in 250 mL
of deionized water.
6. Oxidizing reagent. Add 0.5 mL of sodium hypochlorite solution to 100 mL of alkaline sodium citrate.
This solution should be prepared immediately before use and not stored for more than 3 h. Prepare
multiples of the above solutions according to the number of samples to be analyzed per batch (10 mL of
oxidizing reagent per sample).
7. Acidifying solution. Dilute analytical reagent quality concentrated hydrochloric acid with an equal
volume of deionized water. Add to a 125-mL Erlenmeyer flask approximately 50 mL of distilled water,
pipet into this 10.0 mL of oxidizing agent (see above), and add 2 mL of sodium arsenite solution. Add
one or two drops of thymol blue indicator solution (0.1 percent solution in distilled water) and titrate
the mixture carefully with the diluted hydrochloric solution until the color changed from blue to pink
(pH approximately 1.7). Carry out titrations in duplicate. Titrations should agree to better than 0.1 mL
of acid and the mean volume should be recorded to the nearest 0.05 mL If x mL of acid is used (about
5 to 6 mL) dilute 200x mL of acid to exactly 2000 mL with deionized water using a measuring flask.
This solution must be prepared fresh whenever a new alkaline sodium citrate solution is used.
8. Sulphanilamide solution. Dissolve 5 g of sulphanilamide in a mixture of 50 mL of concentrated
hydrochloric acid (sp gr 1.18) and approximately 300 mL of distilled water. Dilute to 500 mL with water.
The solution will remain stable for several months.
-------
METHOD NO. A-NITROGEN-18
9. N-(1-naphthyl)-ethylenediamine dihydrochloride solution. Dissolve 0.50 g of the dihydrochloride in
500 mL of distilled water. Store the solution in a dark bottle. The solution should be renewed once a
month; a strong brown coloration develops in solutions held too long.
3.3 Equipment/instrument calibration.
NOTE: Carry out the calibration using filtered sea water.
1. Standard ammonia solution. Dissolve 0.100 g of analytical reagent quality ammonium sulphate in 1000
mL of distilled water. Add 1 ml of chloroform and store sheltered from strong light. If tightly
stoppered, the solution will remain stable for several months.
2. Diluted standard ammonia solution. Pipet 1 mL of standard ammonia solution (1 mL = 1.5 ^g-at N)
into a 500-mL volumetric flask and fill with sea water to the mark. The resulting ammonium
concentration will be 3.0 /*g-at N/L
3. Calibration procedure. Place 50-mL aliquots of the dilute ammonia solution (see above) into each of
four clean 125-mL Erlenmeyer flasks. To two additional 125-mL flasks, add 50 mL of the sea water used
to make the diluted standard solution. Carry out the determinations on all six solutions, according to
the procedures described in Section 4.3. Calculate the F factor according to the formula given in
Section 6.1.
4. PROCEDURE
4.1 Sample handling and preservation. Temporarily store the samples in either glass or polyethylene
bottles; analyze samples within 1 to 2 h at the longest. If longer storage is necessary, freeze the
samples solid in a deep-freeze. There are indications that, even if frozen solid, samples may degrade
within a few days.
4.2 Interferences.
1. A correction for nitrite initially present in the sample must be made in the calculation, as indicated
in Section 6.1.2. This correction may be significant in water containing relatively large amounts of
nitrite but little ammonia.
2. The interference from urea and amino acids may be up to 40 percent of the amino nitrogen of some
compounds.
4.3 Sample analysis.
NOTE: Section 4.4.2 should be consulted before commencing analysis.
1. Add 50 mL of sample to an Erlenmeyer flask from a 50 mL measuring cylinder. Add 10 mL of
oxidizing reagent from a pipet, swirl the solution, and allow the flask to stand at a temperature between
20 and 25 °C for 10 min. NOTE: The time required for maximum oxidation depends on salinity and
temperature. The oxidation requires a full 10 min after which stable results are obtained. The
sensitivity in distilled water is slightly less than in sea water but the difference is sufficiently small
that it can be neglected when making a blank determination. Bromide acts as a catalyst and must be
added to distilled water or synthetic bromide-free water.
2. Add 2 mL of sodium arsenite solution from an automatic pipet and mix the contents of the flask.
-------
METHOD NO. A-NfTROGEN-18
NOTE: Arsenite is added to destroy excess hypochlorite without reducing nitrite. The reaction is very
rapid but for safety about 2 min should be allowed for the reaction to be completed before adding the
acidifying solution.
3. Add 10mL of acidifying solution form a pipet and mix. NOTE: If sulphanilamide is present at this
stage, an appreciable fraction of nitrite is decomposed and the method becomes less sensitive and more
erratic.
4. After as short an interval as possible, to lessen the chances of atmospheric contamination, add 1 mL
of sulphanilamide solution from an automatic pipet. Swirl the contents of the flask. After 3 to 8 min,
add 1.0 mL of naphthylethelynediamine solution from an automatic pipet and mix immediately.
NOTE: The determination is from now on the same as that for nitrite, except that both sample volume
and acidity are greater.
5. Between 10 min and 2 h later, measure the extinction of the solution in a 10-cm cell against
distilled water at a wavelength of 5430 A. If the extinction exceeds about 1.3 (rarely), measure the
extinction with a 5-cm cell and double the reading so obtained. Unless adjacent samples are known to
have extinction values within about 25 percent of one another, the absorptiometer cell should be rinsed
with each new solution before filling.
6. Correct the measured extinction by that of a reagent blank. Calculate the ammonia-nitrogen
concentration (plus some amino-acid nitrogen) from the formula given in Section 6.1.2.
4.4 Preparation of quality control blanks.
1. Reagent blanks. Substitute 50 mL of freshly deionized water and 1 mL of sodium bromide as a
catalyst in the procedure described in Section 4.3.
2. Precautions to reduce contamination. Great care must be taken to prevent laboratory contamination
of ammonia, carried as gas or particles of ammonia salts, in samples and blanks. Solutions should be
stored in tightly stoppered bottles until analysis is begun. Under no circumstances should bottles of
ammonium hydroxide be opened in the laboratory while samples are being analyzed. All glassware should
be prewashed with dilute acid and thoroughly rinsed with distilled water immediately before each use
(ordinary distilled water may be used).
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. Reagent blanks will be analyzed in triplicate. There are no recommendations regarding
duplicate sample analyses.
5.2 Accuracy. Analyze reagent blanks in triplicate with each batch of samples analyzed. Blank
extinctions using a 10-cm cell should not exceed 0.15.
-------
METHOD NO. A-NITROGEN-18
6. RECORDKEEPINQ AND DATA REPORT1NQ REQUIREMENTS
6.1 Calculations
1. F factor
F = 3.0/(Es-Eb)
where F =F factor
Es = extinction of the standards
Eb = extinction of the blanks
2. Concentration of ammonia-nitrogen plus some amino acid nitrogen.
NOTE: Any nitrite initially present in the sample is unchanged by the analytical procedure, so a
correction (F') for its presence can be made in the manner shown. In this calculation, allowance is
made for the fact that the sample is diluted from 52 to 74 mL by reagents before extinction
measurements are made and that only a fraction of the ammonia is converted to nitrite. This correction
can be quite significant in water containing relatively large amounts of nitrite but little ammonia.
Mg-at N/L = F [E - ((0.70 x C) / F')]
where F =F factor
E = corrected extinction
C concentration of nitrite in the same seawater (j»g-at N/L)
F' =assumed to be 2.1 with little error if a spectrophotometer is used (see NOTE above)
6.2 Reporting units. Data will be reported in /tg-at N/L.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions; wear protective eyewear and clothing, be familiar with laboratory safety devices, understand
the proper handling of acids, solvents, and other reagents.
72 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the equipment. Personnel conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency in the laboratory
techniques described in this method.
8. REFERENCES
Strickland, J.D.H. and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries
Research Board of Canada, Ottawa.
-------
METHOD NO. A-NITROGEN-19
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Nitrate nitrogen
1. METHOD TITLE
Determination of Reactive Nitrate
2. BACKGROUND AND APPLICATION
2.1 Source of method. Determination of Reactive Nitrate. Method II.6. In: J.D.H. Strickland and T.R.
Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries Research Board of
Canada, Ottawa.
22. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1. Description. Cadmium filings are loosely coated with metallic copper and transferred into a
reductor column. Seawater is passed through the column, where the nitrate in the water is almost
quantitatively reduced to nitrite. The nitrite produced in this manner is determined by diazotizing
with sulphanilamide and coupling with N-(1-naphthyl)-ethylenediamine to form a highly colored azo
dye. The extinction of this azo dye is measured, and a correction may be m'ade for any nitrite
initially present in the sample.
This method will detect nitrate nitrogen in the range of 0.05 to 45 /*g-at/L The detection limit is
0.05 M9/L using 10-cm cells.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method. From sea-sampling bottles, collect
100±2 mL subsamples in a 100-mL graduated cylinder and decant into a 125-mL Erlenmeyer flask.
2.4 Standardization /validation status of method. At the 20 /jg-at/L level, precision lies in the range of
the mean of n determinations ± 0.50/nV2^g-at/L (using 1-cm cells). At the 1 pg-at/L level, precision
lies in the range of the mean of n determinations ± 0.50/n1/2^g-at/L (using 10-cm cells).
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. Reduction columns. To assemble the reduction columns, join 10 cm of 5-cm ID glass tubing onto
30 cm of 10-mm ID glass tubing (this 30-cm length is to contain the metal filings; refer to Section
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NFTROGEN-19
3.2.7). Join a 35 cm length of 2-mm ID glass tube to the 30-cm length. Bend the 35-cm length just
below the joint into a U, so that it runs parallel to the 5-cm length tube, and bend the end over to
form an inverted U siphon. When the assembly is held upright, the last bend should be level with the
top of the 5-cm length. If the assembly is correctly constructed, liquid placed in the top reservoir tube
should flow out of the system and stop when the level of liquid is such that it just covers the metal
filings. To protect the reduction columns, place the columns inside large cylinders (glass or plastic). To
the outside of the cylinders, affix a small glass cylinder drawn to a tube at one end and sealed with a
rubber tube and pinch clamp. This cylinder should hold about 75 ml_ and be arranged under the exits of
the reduction columns to collect effluents. Mark the 40- and 50-mL levels on the cylinder.
2. 50-mL graduated cylinders.
3. 125-mL Erlenmeyer flasks that are grease free.
4. Absorptiometer.
3.2 This procedure requires the following reagents.
NOTE: All solutions must be made with ammonia-free water.
1. Concentrated ammonium chloride solution. Dissolve 125 g of ammonium chloride (analytical reagent
quality) in 500 ml_ distilled water. Store solution in a glass or plastic bottle.
2. Dilute ammonium chloride solution. Dilute 50 mL of concentrated ammonium chloride solution (see
above) to 2000 mL with distilled water. Store the solution in either a glass or plastic bottle.
3. Sulfanilamide solution. Dissolve 5 g of sulfanilamids in a mixture of 50 mL concentrated
hydrochloric acid (sp gr 1.18) and approximately 300 mL distilled water. Dilute to 500 mL with distilled
water. This solution will remain stable for several months.
4. n-(1-naphthyl)-ethylenediamine dihydrochloride solution. Dissolve 0.50 g of dihydrochloride in
500 mL distilled water. Store the solution in a dark bottle. The solution should be renewed once a
month or a strong brown coloration develops.
t
5. Synthetic sea water. Dissolve 310 g of analytical reagent quality sodium chloride (NaCI), 100 g of
analytical reagent quality magnesium sulphate (MgSO4-7H2O), and 0.50 g of sodium bicarbonate
(NaHCOs-H2O) in 10 L of distilled water.
6. Standard nitrate solution. Dissolve 1.02 g of analytical reagent quality potassium nitrate (KNOs) in
1000 mL of distilled water. The solution is stable indefinitely in the absence of evaporation. One
milliliter (1 mL) of this solution equals 10.0 /*g-at N/L Dilute 4.00 mL of this solution to 2000 mL with
synthetic sea water. The concentration of nitrate in this solution is 20 ^g-at N/L Prepare a fresh
solution immediately before use, and store in a dark bottle.
7. Cadmium copper filings. In a 18 x 150-mm pyrex test tube buried in dry sand, melt cadmium metal
(99.9 percent purity is satisfactory) and allow the metal to solidify. Using a coarse wood rasp, file off
the required amount of metal (110 g of filings is enough for 2 columns). Collect the fraction of the
filings that passes through a 1.0-mm mesh sieve and is retained on a 0.5-mm mesh sieve. Stir
approximately 100 g of filings (enough for two columns) with 500 mL of a 2 percent w/v solution of
copper sulphate pentahydrate, CuSO4-5H2O, until all blue color is gone and semicolloidal copper particles
begin to appear in the supernatant. Roll very fine copper turnings between fingers and thumb to
-------
METHOD NO. A-NITROGEN-19
produce a small plug, and insert the plug into the bottom of a redactor column (glass wool does not
work as well and should be used only if very fine copper "wool" turnings cannot be obtained). Fill the
reductor column with dilute ammonium chloride solution and pour in enough cadmium-copper mixture to
produce a column approximately 30 cm in length. Slowly add the filings to the column, lightly tapping
the column after each addition to ensure that the filings are well settled. Thoroughly wash the column
with dilute ammonium chloride solution. Check the flow rate of the column; 100 mL of the solution
should take between 8 and 12 min to completely flow through the column. If the flow rate is too great
(100 mL flows through in less than 8 min), slow rt by restricting the outlet of the siphon or by packing
more copper or glass wool at the bottom. If the flow is too slow (100 mL of solution takes more than
12 min to flow through the column), loosen the packing at the base of the column. Once an acceptable
flow rate is achieved, add a small plug of copper "wool" to the top of the column. When the columns
are not in use, the metal filings in the columns must be completely covered with dilute ammonium
chloride solution.
If the efficiency of the reduction is suspect, empty the filings into a beaker and wash them. Filings
from four columns should be washed in 300 mL 5 percent (v/v) hydrochloric acid solution, stirring
vigorously. Decant the acid rinse once more with hydrochloric acid. Wash the filings with several 200-
300-mL aliquots distilled water until the wash is no longer acidic (pH > 5). Decant the liquid and allow
the filings to dry completely. Treat the filings with copper sulfate solution, as described above. The
regenerated cadmium-copper mixture should be sufficient for three columns.
3.3 Equipment/instrument calibration.
1. There is a slight salt effect in this method and calibration should be performed using synthetic sea
water (see description above) or natural sea water with a nitrate concentration less than 1 /*g-at N/L
When analyzing for very small concentrations of nitrate (less than 0.5 >*g-at N/L), determine the factor
on spiked sea water; however, this may not be necessary. To check for deactivation of the column,
process a standard through the column at the beginning of each day. Typically, there should be no
significant difference between the factors for all columns, but the accumulative mean for each one may
be used to minimize any errors that would arise should a slight difference occur.
2. Add approximately 110 mL of diluted standard nitrate solution (see Section 3.1.6) to clean dry 125-mL
Erlenmeyer flasks. Process the solutions described in Section 4.3 and measure the extinction in a 1-cm
cell. Perform this process in triplicate for each column, calculate the mean of the three extinctions,
and correct the mean by the blank extinction. Subsequent checks each day need only one determination
for each column. Calculate the F factor as described in Section 6.1.1.
4. PROCEDURE
4.1 Sample handling and preservation. Store samples for several hours cold and in the dark; sample
analysis should not be delayed any longer than 12 h. If sample analysis will not be done within 12 h,
store the samples frozen (-20°C) in a deep freezer where no detectable changes occur for several weeks.
4.2 Interferences.
1. Continual use of a column leads to deactivation. The slight acidification of the sample before
processing greatly reduces the deactivation, and a well-made column should be capable of reducing at
least 100 samples. The volume of the samples is not critical up to 5 mL
-------
METHOD NO. A-NITROGEN-19
2. Samples containing sulfide in concentrations of up to 2 mg S2/L may be analyzed by this method
without interference by the sulfide. However, repeated analysis of such samples will lead to deactivation
of the columns by the production of cadmium sulfide.
4.3 Sample analysis.
1. Add 2.0 mL concentrated ammonium chloride to the sample in the Erlenmeyer flask. NOTE: The
acidification of the sample by the addition of ammonium chloride greatly slows the deactivation of the
column.
Thoroughly mix the solution and pour approximately 5 mL onto the top of the column and allow the
sample to pass through the column. NOTE: This initial addition ensures that the liquid in the top of
the column is similar to the sample. By doing this, no error will result when the remainder of the
sample is added and some of the interstitial liquid in the top of the column mixes with the sample. For
example, if a sample were poured through a column immediately after a blank had passed through and
this preliminary aliquot had not been poured through, some dilutions of the sample could occur. This
precaution is particularly important when processing consecutive samples containing varying
concentrations of nitrate.
2. Add the remainder of the sample to the column and place the empty Erlenmeyer flask under the
collection tube at the bottom of the reduction column. When approximately 40 mL of liquid has passed
through the column and into the collection tube, drain the collection tube into the flask, rinse the flask
with the effluent, drain, and place the flask under the collection tube. NOTE: The passage of at least
40 mL of solution is necessary to completely flush the column. This is especially important when
processing samples of varying nitrate concentrations.
3. Collect approximately 50 mL in the collection tube and quickly transfer the volume into the
Erlenmeyer flask. Allow the column to drain until flow ceases. NOTE: The volume in the flask should
be within a few milliliters of 50 mL, but it is not critical that the flask be completely drained of the
column washings. Under the conditions detailed thus far, reduction will be approximately 93 percent;
temperature variations between 10 and 35°C will have no effect. NOTE: It is not necessary to wash
the columns between samples; however, if columns will not be reused for 1 or 2 hours or longer, pour 50
mL of dilute ammonium chloride through the columns. Store the columns completely covered with the
liquid.
4. Add 1.0 mL of sulphanilamide solution to the flask as soon as possible after reduction. Allow the
reagent to react for at least 2 min but no longer than 8 min. The diazotizing reaction requires at least
2 min for completion, but undesirable side reactions and decomposition become significant after 10 min.
NOTE: Although reduced nitrate solution may be stable for several hours, analysis should be completed
as soon as possible, particularly in hot weather. Temperature is not critical but must fall between 15
and 30° C.
5. Add 1.0 mL of naphthylethylenediamine solution and mix immediately. After 10 min b'ut within 2 h,
measure the extinction of the solution, against distilled water, in a 1-cm cell (wavelength 5430 A).
NOTE: Complete color development requires 10 min, and is stable for up to 2 h. NOTE: If the
extinction exceeds 1.25 (approximately 30 /*g-at N/L), measure again in either a 0.5-cm cell or add
25.0 mL of distilled water to 25.0 mL of solution in a clean dry flask, and double the value used in the
calculations. If the extinction value is less than 0.1 in a 1-cm cell, measure again in a 10-cm cell. If
the extinction is between 0.1 and 0.2 in a 1-cm cell, measure again in a 5-cm cell. NOTE: Rinse the
absorptiometer cell with each new solution before filling. The cell does not have to be rinsed between
adjacent samples if the extinction values are within 25 percent of one another.
-------
METHOD NO. A-NITROGEN-19
6. Using a reagent blank (see Section 4.4.2), correct the observed extinction. Calculate the nitrate
present according to the formula given in Section 6.1.3). NOTE: Using good columns, nitrite is reduced
to the extent of 5 percent and a correction times the nitrite concentration is made. This percentage
may exceed 20 percent when deactivated columns are used.
4.4 Preparation of quality control blanks.
1. Cell-to-cell blanks. Use distilled water as a cell-to-cell blank. Replace the distilled water daily to
prevent the development of significant turbidities.
2. Reagent blanks. Process the reagent blanks in the same method used for samples (se Section 4.3).
In a clean Erlenmeyer flask, add concentrated ammonium chloride solution to 100 mL of redistilled water.
Process the sample in a column previously flushed with at least 50 mL of ammonium chloride solution.
In the instances where samples contain very low nitrate levels and are being analyzed using 10-cm cells,
reagent blanks made using ordinary distilled water may contain significant levels of nitrate. Redistill the
distilled water from a little alkaline permanganate (reject the first few milliliters of the distillate). It
should be assumed that the redistilled water contains no nitrate.
3. Turbidity blanks. If the nitrate concentration is low enough to warrant the use of a 10-cm cell (the
extinction is less than 0.1 in a 1-cm cell), the turbidity should be checked. Samples must be filtered if
the turbidity is significant.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. Initially, for each column, perform the analysis in triplicate. Calculate a mean of the
three extinctions.
5.2 Accuracy.
1. Compare the extinctions of two or more cell-to-cell blanks. The differences between the cells should
be 0.000. Slight optical defects may produce a slightly positive or negative value.
2. Reagent blanks should be checked occasionally. Though the blank is barely significant when 1-cm
cells are used, the blank becomes very significant when 10-cm cells are used. Process the reagent
blanks by the same method used for samples (see Section 4.3), using 1-, 5, or 10-cm cells, as
appropriate. The blank extinction corrected by any cell-to-cell blank should not exceed approximately
0.1 using a 10-cm cell.
3. If the turbidity is high in samples with low nitrate concentrations, the samples must be filtered
before analysis in a 10-cm cell.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations
1. Corrected extinction. Calculate as described in Section 4.3.6.
-------
METHOD NO. A-NITROGEN-19
2. F factor.
F = 20.0/E
where F =F factor
E =Mean extinction of the three values for each column (corrected for a blank)
3. Nitrate present.
A = (Corrected extinction X F) - 0.95 C
where A = concentration of nitrate present (/tg-at N/L)
F = F factor (see Section 6.1.2)
C = concentration of nitrite present (pg-at N/L)
6.2 Reporting units. Data will be reported in /tg-at N/L
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of all equipment. Personnel conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency in the laboratory
techniques described in this method.
8. REFERENCES
Strickland, J.D.H. and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries
Research Board of Canada, Ottawa.
-------
METHOD NO. A-NITROGEN-20
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter: Nitrite nitrogen
1. METHOD TITLE
Determination of Reactive Nitrite
2. BACKGROUND AND APPLICATION
2.1 Source of method. Determination of Reactive Nitrite. Method II.7. In: J.D.H. Strickland and T.R.
Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries Research Board of
Canada, Ottawa.
2.2 Regulatory status. This method has not been cited by any regulation.
2.3 Principle and application.
2.3.1 Description. In an acid solution, the nitrite in the seawater is allowed to react with
sulphanilamide. The resulting diazo compound reacts with N-(1-naphthyl)-ethylenediamine to form a
azo dye. The extinction of the azo dye is measured using 10-cm cells.
This method will deteict nitrite nitrogen in the range of 0.01 to 2.5 //g-at/L.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method. From sea-sampling bottles, collect 50
mL of sample into a 50-mL graduated cylinder and transfer the sample from the graduate to a 125-
mL Erlenmeyer flask.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a sea water matrix have
not been cited. This method will be updated to include this information as soon as data are
available.
2.4.2 Precision and accuracy.
1. Precision. At the 1 g-at/L level, precision lies in the range of the mean of n determinations ±
0.032/nl/2^g-at/L Precision at the 0.3 /*g-at/L level lies in the range of the mean of n
determinations ± 0.023/n1/2^g-at/L
2. Accuracy. Three types of blanks; cell-to-cell blanks, reagent blanks, and seawater turbidity
blanks, will be processed during this procedure.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NfTROGEN-20
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. 125-mL Erlenmeyer flasks
2. Absorptiometer
3.2 This procedure requires the following reagents.
NOTE: All solutions must be made with ammonia-free water.
1. Sulphanilamide solution. Dissolve 5 g of sulphanilamide in a mixture of 50 mL concentrated
hydrochloric acid sp gr 1.18) and approximately 300 mL distilled water. Dilute to 500 mL with distilled
water. This solution will remain stable for several months.
2. N-(1-naphthyl)-ethylenediamine dihydrochloride solution. Dissolve 0.50 g of dihydrochloride in
500 mL distilled water. Store the solution in a dark bottle. The solution should be renewed once a
month or a strong brown coloration develops.
3. Standard nitrite solution. Use anhydrous analytical reagent quality sodium nitrite (NaNO2) for
calibration. To ensure that the reagent is sufficiently pure for calibration, dry a little of the salt at
110 °C for 1 h. Dissolve 0.345 g of the dried salt in 1000 mL of distilled water. (1 mL = 5 /tg-at N).
Store the solution in a dark bottle with 1 mL of chloroform as a preservative. (The solution is stable
for at least 1 to 2 months). Dilute 10.0 mL of this solution to 1000 mL with distilled water and use the
same day (1 mL = 5 X 10-2 ^g-at N and 1 mL = 1.0 /*g-at N/L in 50 mL of seawater sample).
3.3 Equipment calibration. Prepare four standard solutions (see Section 3.2.3 for instructions for the
preparation of the standard nitrite solution) consisting of 2.00 mL (measured with a 2.00 mL graduated
pipet) of the dilute nitrite solution made to a volume of 50 mL in a graduated flask or 50-mL measuring
cylinder. Transfer the solutions to four dry 125-mL Erlenmeyer flasks and place 50 mL of distilled
water in two more flasks to act as blanks. Carry out the nitrite determination as described in Section
4.2.3. Calculate the F factor as described in Section 6.1.1. NOTE: The F factor will not vary with
time or over a wide range of analytical conditions, and is near 2.1 when a spectrophotometer is
employed.
4. PROCEDURE
4.1 Sample handling and preservation. Rinse the glassware used for sample collection should be rinsed
twice with the sample and drained. Measure 50 mL of sample into the flask, and analyze the samples
within 10 h (samples will remain stable if subdued light at room temperature, but they should not be
held for more than 10 h). If samples are to be held longer than 10 h, place them in a freezer and
analyze as soon as possible. Prolonged storage is not recommended. Collect a separate 30-mL sample
for turbidity measurements for highly precise inshore investigations.
42 Interferences. Numerous compounds can interfere with this method but none of them will be
present in significant amounts in ocean, inshore, or estuarine waters unless excessive pollution by land
drainage is encountered.
-------
METHOD NO. A-NITROGEN-20
4.3 Sample analysis.
1. Measure the extinction of samples to derive the turbidity corrections, as described in Section 4.4.3.
2. Add 1.0 mL of sulphanilamide solution to each 50(±5)-mL sample. Mix thoroughly and allow the
reagent to react for at least 2 min but not longer than 8 min (The temperature should be between 15
and 25 °C). NOTE: The diazotizing reaction requires a minimum of 2 min , but undesirable side
reactions and decomposition may become significant if the reaction is allowed to continue longer than
10 min.
3. Add 1.0 mL of naphthylethylenediamine solution and mix immediately. After 10 min but within 2 h,
measure the extinction in a 10-cm cell against distilled water. (10 min is required for complete color
development, and the color is stable for at least 2 h thereafter. However, two hours is the maximum
safe limit, and no significant error will occur for 1 or 2 h after that if the solutions are stored out of
direct sunlight). Measure the extinction at a wavelength of 5430 A; if a filter-type absorptiometer is
used, choose a filter with a peak transmission as close to 5400 A as possible. Unless samples are known
to have extinction values within 25 percent of one another, rinse the absorptiometer cell with new
solution before filling.
4. Correct the measured extinction by subtracting the extinction of the turbidity and reagent blanks.
Calculate the nitrite-nitrogen concentration (microgram-atoms of nitrogen per liter, or /ig-at N/L) as
described in Section 6.1.
4.4 Preparation of quality control blanks.
1. Cell-to-cell blanks. Fill both the distilled water cell and the sample cell with distilled water. The
difference in the extinction between the two cells should be no more than 0.000. However, slight
optical defects may produce a slight positive or negative value. This is compensated for when turbidity
blanks are subtracted but the value should be found when determining the reagent blank. The distilled
water should be changed daily to prevent an increase in turbidity.
2. Reagent blanks. In a 125-mL Erlenmeyer flask, add 1.0 mL sulphanilamide solution to 50 mL distilled
water. (The temperature should be between 15 and 25°C. The exact volume of the distilled water is not
critical, and the volume may be between 45 and 55 mL). Mix thoroughly, and allow the reagent to
react for at least 2 min but not longer than 8 min (the diazotizing reaction requires at least 2 min for
completion and, if allowed to continue beyond 10 min, will lead to significant undesirable side reactions
and decomposition).
Add 1.0 mL naphthylethylenediamine solution and mix immediately. Within 10 min to 2 hr after the
addition of the naphthylethylenediamine solution, measure the extinction of the solution, in a 10-cm cell,
against distilled water. (Complete color development requires at least 10 min and is be stable for up to
2 h. After 2 h, the color begins to fade. No great error will occur if the blank is held 1 to 2 h
beyond that time as long as the blank is stored out of direct sunlight.). Use a wavelength of 5430 A.
If a filter absorptiometer is used, choose a filter with a peak transmission as close as possible to
5400 A. Correct the resulting extinction by the extinction of the cell-to-cell blank. The reagent blank
extinction should not exceed 0.03. NOTE: The preparation and analysis of the reagent blank is the
same as the preparation and analysis of samples, except distilled water is used instead of the sample
(seawater).
-------
METHOD NO. A-NITROGEN-20
3. Seawater turbidity blanks. In turbid inshore waters, extinction of seawater turbidity blanks may be a
significant fraction of total extinction (which rarely exceeds 0.3 in nitrite determinations). Turbidity
blanks should be determined on the surface and 10-m samples for each cast. Measure at progressively
greater depths until the value becomes appreciably constant. This value (generally less than 0.01 at
below 25 m in offshore waters) is then roughly equal to the cell-to-cell blank and may even be slightly
negative. Turbidity blanks should be measured on separate 30-mL samples of the sea water to which
1 mL of sulphanilamide reagent has been added.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. The extinction of the reagent blank should be determined in duplicate with each batch
of samples processed. The limit of detection of this method is in the range 0.01 to 2.5 pg-at/L
Reject duplicate determinations if the extinction values differ by more than the following values in the
given ranges: more than 0.03 in the extinction range 0.5 to 1.0, more than 0.02 in the extinction range
0.1 to 0.5, and more than 0.005 in the extinction range 0.03 to 0.1. If duplicate extinction values differ
by less than these limits, calculate a mean value.
5.2 Accuracy. The difference between the extinctions of two distilled water cell-to-cell blanks should
not exceed 0.000. The extinction for reagent blanks, after correction for cell-to-cell extinctions, should
not exceed 0.03. In seawater turbidity blanks, the extinction for the final constant value should be
roughly equal to the extinction of the cell-to-cell blank.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations.
1. F factor.
F = 2.00/(Es-Eb)
where ES = mean extinction of the four standards
Eb =mean extinction of the two blanks (not corrected for cell-to-cell blanks)
2. Nitrite-nitrogen concentration.
/*g-at N/L = corrected extinction x F
where F =F factor (see Section 6.1.1)
6.2 Reporting units. Report the nitrite-nitrogen concentration in /*g-at N/L
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
-------
METHOD NO. A-NfTROGEN^O
72. Training/level of expertise. Analysts using this method, should be proficient in the operation and
maintenance of the equipment. Persons conducting this analysis should initially work under the guidance
of an experienced supervisor until he/she can demonstrate proficiency in the laboratory techniques
described in this method.
8. REFERENCES
Strickland, J.D.H. and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries
Research Board of Canada, Ottawa.
-------
METHOD NO. A-NrTROGEN-21
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter. Soluble organic nitrogen
1. METHOD TTTUE
Determination of Soluble Organic Nitrogen by Kjeldahl Digestion
2. BACKGROUND AND APPLICATION
2.1 Source of method. Determination of Soluble Organic Nitrogen, Methods III.3.I (Kjeldahl Digestion).
In: J.D.H. Strickland and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis. 2nd ed.
Fisheries Research Board of Canada, Ottawa.
22. Regulatory status. This method has not been cited by any regulation.
2.3 Principle and application.
2.3.1 Description. Filtered seawater is evaporated to dryness with excess sulfuric acid and any
organic nitrogen is converted to ammonia by Kjeldahl digestion. The residue is dissolved in water,
neutralized, and the ammonia is determined by using the method for determination of ammonia plus
amino acids.
This method will detect soluble organic nitrogen in the range of 0.8 to 15 /*g-at/L The smallest
amount that can be detected with certainty is approximately 0.8 /*g-at/L
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method. Sampling equipment should be
thoroughly cleaned with detergent and rinsed with distilled water to ensure the absence of organic
matter.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a sea water matrix have
not been cited. This method will be updated to include this information as soon as data are
available.
2.4.2 Precision. At the 8 //g-at/L level, the correct value lies in the range of the mean of n
determinations ± 1.2/nV2^g-at/L
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NITROGEN-21
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. 30-mL pyrex Kjeldahl flasks. Initially, steep the flasks in near-boiling sulfuric acid for several
hours. Rinse thoroughly with distilled water before use. If the flasks are washed with distilled water
immediately after use and stored with their mouths covered with aluminum foil, the sulfuric acid cleaning
need only be carried out at infrequent intervals and when contamination is suspected.
2. Ice or cold water bath.
3. Micro-Kjeldahl rack and vent to accommodate 30-mL Kjeldahl flasks and give suitably controlled
heating.
4. 125-mL Erlenmeyer flasks. Immediately before use, rinse these flasks with copious amounts of
distilled water and drain.
3.2 This procedure requires the following reagents.
NOTE: All solutions must be made with ammonia-free water.
1. Deionized water. Remove the ammonia from distilled water by passing distilled water through a small
column (for example, 30 cm long and 1 to 2 cm ID) of cation exchange resin in the hydrogen form just
before use and store the water in a tightly stoppered glass flask. NOTE: This deionized water should
be used to prepare solutions, for the determination of blanks, and for standardization.
2. Digestion mixture. Dissolve 0.1 g of analytical reagent quality selenium dioxide (SeO2) in 500 mL of
distilled water. Add 500 mL of special sulfuric acid and cool the mixture to room temperature. Make to
a volume of exactly 1000 mL with distilled water and store in a tightly stoppered glass bottle. Discard
only if contamination is suspected. NOTE: The quality of the sulfuric acid is extremely important in
this method. Analytical reagent quality concentrated acid (sp gr 1.84) must be used but not all such
acid, even in bottles from the same supplier, may be satisfactory. If special "low nitrogen" acid (less
than 2 ppm of N) is available use this, otherwise check several bottles of high grade acid until one is
found which gives blanks (duplicates) with extinctions less than 0.35. Once a satisfactory bottle of acid
is found it should be set aside, well stoppered, and used only for this determination. Distillation of
concentrated sulfuric acid, rejecting the first and last 10 percent of the distillate, greatly improves its
quality for this purpose by reducing its blank value.
3. Sodium hydroxide solution. Rinse 80 g of analytical reagent quality sodium hydroxide, in pellet form,
very rapidly with approximately 20 mL of water, so that only a few percent of the alkali is lost by
dissolution. Discard the rinsing. Dissolve the washed pellets in deionized water and make the volume to
500 mL Store in a well-stoppered polyethylene bottle. The solution is stable in the absence of
excessive carbonation but should be renewed if contamination is suspected.
4. Dilute sulfuric acid. Add 50 mL of special sulfuric acid (see Section 3.2.2) to 500 mL of deionized
water. Store in a well-stoppered glass bottle and transfer a few milliliters at a time to a clean glass
dropping bottle for use.
5. Bromthymol blue indicator. Dissolve 0.1 g of the solid indicator in 100 mL of distilled water. Store
in a dropping bottle.
-------
METHOD NO. A-NITROGEN-21
6. Anti-bumping granules. Pretreat granules (for example, Hengar granules or Fisher's "Boileasers" are
satisfactory) by heating them in boiling or near-boiling concentrated sulfuric acid for 2 to 3 h. After
the acid treatment the granules should be boiled with two or three changes of water, rinsed thoroughly,
and oven-dried. The latter treatment is essential if their anti-bumping properties are to be restored.
7. Alkaline sodium citrate. Dissolve 700 g of sodium citrate and 40 g of sodium hydroxide in 2000 mL
of deionized water. Store the solution in a tightly stoppered polyethylene bottle. The solution will be
stable for several months.
8. Sodium hypochlorite solution. Use a solution of commercial hypochlorite (for example, Chlorox)
which should be approximately 1.5 N. The solution decomposes slowly and should be checked
periodically. Dissolve 12.5 g of good-quality sodium thiosulphate (Na2S2O3-5H2O) in 500 mL of water.
Add a few crystals (approximately 2 g) of potassium iodide (Kl) to about 50 mL of water in a small
flask. Pipet 1.0 mL of hypochlorite solution into the flask. Add 5 to 10 drops of concentrated
hydrochloric acid and titrate the liberated iodine with the thiosulphate solution until no yellow color
remains. Discard the hypochlorite when less than 12 mL of thiosulfate is used.
9. Sodium arsenite solution. Dissolve 20 g of analytical reagent sodium meta-arsenite (Na2AsO2) in 1000
Ml of deionized water. Store the solution in a well-stoppered polyethylene bottle. The solution will
remain stable indefinitely.
10. Sodium bromide solution. Dissolve 1.5 g analytical reagent quality sodium bromide (NaBn) in 250 mL
of deionized water.
11. Oxidizing reagent. Add 0.5 mL of sodium hypochlorite solution to 100 mL of alkaline sodium
citrate. This solution should be prepared immediately before use and not stored for more than 3 h.
Prepare multiples of the above solutions according to the number of samples to be analyzed per batch
(1C mL of oxidizing reagent per sample).
12. Acidifying solution. Dilute analytical reagent quality concentrated hydrochloric acid with an equal
volume of deionized water. Add to a 125-mL Erlenmeyer flask approximately 50 mL of distilled water,
pipet into this 10.0 mL of oxidizing agent (see above), and add 2 mL of sodium arsenite solution. Add
one or two drops of thymol blue indicator solution (0.1 percent solution in distilled water) and titrate
the mixture carefully with the diluted hydrochloric solution until the color changed from blue to pink
(pH approximately 1.7). Carry out titrations in duplicate. Titrations should agree to better than 0.1 mL
of acid and the mean volume should be recorded to the nearest 0.05 mL If x mL of acid is used (about
5 to 6 mL) dilute 200x mL of acid to exactly 2000 mL with deionized water using a measuring flask.
This solution must be prepared fresh whenever a new alkaline sodium citrate solution is used.
13. Sulphanilamide solution. Dissolve 5 g of sulphanilamide in a mixture of 50 mL of concentrated
hydrochloric acid (sp gr 1.18) and approximately 300 mL of distilled water. Dilute to 500 mL with water.
The solution will remain stable for several months.
3.3 Equipment calibration.
1. Standard nitrogen solution. The efficiency of the Kjeldahl oxidation in this method appears to be as
great as in any other so that there is little point in using an organic nitrogen source as a standard.
Dissolve 0.330 g of good quality dry ammonium sulphate in 100 mL of water in a measuring flask. Store
this solution in a well-stoppered glass bottle with about 1 mL of chloroform as a preservative. Dilute
5.00 mL of this solution (1 mL = 50 /*g-at N) to 1000 mL with distilled water. Use at once for
-------
METHOD NO. A-NHROGEN-21
calibration and do not store. Equivalencies of this solution are as follows: 1 ml_ = 0.26 /*g-at N and
1 mL= 10 /iQ-at N/L in 25 mL of seawater.
2. Calibration procedure. Add 25 mL of deionized water, 2.0 mL of the digestion mixture (see Section
3.2.2), and an anti-bumping granule (see Section 3.2.6) to each of five flasks. To three of the flasks
add 1.0 mL of dilute standard nitrogen solution (see Section 3.3.1) and carry out the determination as
for samples (see Section 4.3). Calculate the F factor as described in Section 6.1.1. To allow for slight
losses and other slight discrepancies, it is best to determine the F factor by taking solutions through
the entire procedure in this manner. For surety, the F factor should be determined whenever a new
batch of reagents is prepared but experience may show that a less frequent determination is adequate.
4. PROCEDURE
4.1 Sample handling and preservation. Samples should be filtered within one hour or so of being
collected (see Section 2.3.2). Filtered solutions should be stored in clean glass bottles and frozen at
-20° C if the analysis has to be delayed for more than a few hours, but because of the danger of
contamination by ammonia we suggest that the storage time be left to a minimum.
4.2 Interferences.
The interference from urea and amino acids may be up to 40 percent of the amino nitrogen of some
compounds.
4.3 Sample analysis.
1. Thaw the sample, if necessary, and pipet 25 mL directly into a Kjeldahl flask. Add 2.0 mL of
digestion mixture from a pipet or buret and one anti-bumping granule.
2. Heat the flask on the Kjeldahl heating rack until all water is removed and then digest the residue
for a further 60 min at about 200 °C to complete the reaction. Approximately 30 min is required to
evaporate the sample. After hydrochloric acid has been evolved sulfuric acid fumes will appear. Adjust
the temperature so that the acid refluxes for 1 h near the top of the bulb of the Kjeldahl flask. There
should be very little loss of acid vapor from the mouth of the flask.
3. Cool the tube slightly and dissolve the residue in approximately 20 mL of deionized water, warming,
if necessary. Finally cool the solution in cold water, preferably in an ice bath (the solution is cooled to
prevent appreciable loss of ammonia during the brief period in which the liquid is alkaline). Add one
drop of bromthymol blue indicator.
4. Titrate the solution in the flask with sodium hydroxide solution. Approach the end point (yellow to
blue) with caution and try not to overshoot by more than a few drops of alkali. Rinse down the neck
of the flask with deionized water and titrate, dropwise, with dilute sulfuric acid until the solution is
just yellow again. NOTE: As mentioned in Section 4.3.3, the time during which solutions are alkaline
should be kept to a minimum. Ensure that all alkali is washed down from the neck of the flask before
the dilute acid is added. Similarly make sure that no acid is held up in the neck of the flask. Rinse
down the neck and check on the stability of any end point.
5. Transfer the solution from the Kjeldahl flask 4o a clean, drained-dry, 50 mL measuring cylinder.
Rinse the flask once or twice and make the total volume in the cylinder to exactly 50 mL Transfer the
-------
METHOD NO. A-NITROGEN-21
solution to a well-drained 125-mL Erlenmeyer flask. Drain the cylinder thoroughly but do not wash.
Add 1 mL of potassium bromide solution.
6. Add 50 ml of sample to an Erlenmeyer flask from a 50 mL measuring cylinder. Add 10 mL of
oxidizing reagent from a pipet, swirl the solution, and allow the flask to stand at a temperature between
20 and 25 °C for 10 min. NOTE: The time required for maximum oxidation depends on salinity and
temperature. The oxidation requires a full 10 min after which stable results are obtained. The
sensitivity in distilled water is slightly less than in sea water but the difference is sufficiently small
that it can be neglected when making a blank determination. Bromide acts as a catalyst and must be
added to distilled water or synthetic bromide-free water.
7. Add 2 mL of sodium arsenite solution from an automatic pipet and mix the contents of the flask.
NOTE: Arsenite is added to destroy excess hypochlorite without reducing nitrite. The reaction is very
rapid but for safety about 2 min should be allowed for the reaction to be completed before adding the
acidifying solution.
8. Add 10 mL of acidifying solution from a pipet and mix. NOTE: If sulphanilamide is present at this
stage, and appreciable fraction of nitrite is decomposed and the method becomes less sensitive and more
erratic.
9. After as short an interval as possible, to lessen the chances of atmospheric contamination, add 1 mL
of sulphanilamide solution from an automatic pipet. Swirl the contents of the flask. After 3 to 8 min,
add 1.0 mL of naphthylethelynediamine solution from an automatic pipet and mix immediately. NOTE:
The determination is from now on the same as that for nitrite, except that both sample volume and
acidity are greater.
10. After 2.5 h, measure the extinction of the solution in a 5-cm cell against distilled water at a
wavelength of 5430 A. Unless adjacent samples are known to have extinction values within about 25
percent of one another, the absorptiometer cell should be rinsed with each new solution before filling.
11. Correct the measured extinction by subtracting a reagent blank obtained as described in Section 4.4.
Calculate the soluble organic nitrogen per liter (/*g-at N/L) from the formula given in Section 6.1.2.
4.4 Preparation of quality control banks.
Reagent blank. Carry out the method as described in Section 4.3.1 through 4.3.10, but omit the seawater
and replace with approximately 5 mL of deionized water (the ammonia of which is assumed to be
negligible).
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Accuracy.
1. A blank determination will be carried out in duplicate with each batch of samples being analyzed.
As many samples as practical may be evaporated and oxidized at one time, but the flasks should be
covered by foil until ready for analysis. The extinction of the deionized water blank snould not exceed
0.3 to 0.35 on a 5-cm cell. For this blank to be representative of samples of all salinities the
assumption must be made that the sodium hydroxide solution introduces a negligible amount of ammonia.
-------
METHOD NO. A-NITROGEN-21
This appears to be justified with good quality alkali but the solution can always be boiled to remove
ammonia if its presence is suspected.
2. Sulfuric acid blanks will be run in duplicate.
3. Determine the F factor for each new batch of reagents prepared.
5.2 Precautions to reduce contamination. Great care must be taken to prevent laboratory contamination
of ammonia, carried as gas or particles of ammonia salts, in samples and blanks. Solutions should be
stored in tightly stoppered bottles until analysis is begun. Under no circumstances should bottles of
ammonium hydroxide be opened in the laboratory while samples are being analyzed. All glassware should
be prewashed with dilute acid and thoroughly rinsed with distilled water immediately before each use
(ordinary distilled water may be used).
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS.
6.1 Calculations.
1. F factor.
NOTE: The F factor should be determined with each new batch of standards prepared.
F = lO.O/(Es-Eb) = ca. 15
where Es =mean extinction of the three standards
Eb =mean extinction of the two blanks.
2. Soluble organic nitrogen concentration.
/ig-at N/L = (corrected extinction x F) - (/*g-at NH3-N/L)
where F =F factor (Section 6.1.1)
ng-ai NH3-N/L = concentration of ammonia found in another aliquot of the sample
analyzed as described in Section 4.3. The correction for ammonia may
be unnecessary except for the most precise work where ammonia
concentrations are exceptionally high (above 1 |ig-at N/L)
6.2 Reporting units. Report the soluble organic nitrogen concentration in /*g-at N/L
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions; wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the equipment. Persons conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency int he laboratory
techniques described in this method.
-------
METHOD NO. A-NITROGEN-21
8. REFERENCES
Strickland, J.D.H. and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries
Research Board of Canada, Ottawa.
-------
METHOD NO. A-NITROGEN-22
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter: Soluble organic nitrogen
1. METHODTITLE
Determination of Soluble Organic Nitrogen by Ultraviolet Oxidation
2. BACKGROUND AND APPLICATION
2.1 Source of method. Determination of Soluble Organic Nitrogen by Ultraviolet Oxidation. Method
III.3.II. In: J.D.H. Strickland and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd
ed. Fisheries Research Board of Canada, Ottawa.
2.2 Regulatory status. This method has not been cited by any regulation.
2.3 Principle and application.
2.3.1 Description. Nitrate (plus nitrite) is determined on the sample using the method for
determination of reactive nitrate before and after irradiation with light of a wavelength less than
2500 A. Organic nitrogen compounds (and ammonia, for which a correction must be made) are
oxidized to nitrate plus nitrite. The difference between the value for nitrate plus nitrite on
samples before and after irradiation is therefore a measure of the amount of nitrogen initially
present in organic combination.
This method will detect soluble organic nitrogen in the range of 0.25 to 15 A*g-at L
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited. This method will be updated to include this information as soon as data are
available.
2.4.2 Precision and accuracy. This is a "difference" method with no blank as such.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NrTROGEN-22
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1.110-mL capacity fused silica tubes.
2. Reduction columns. To assemble the reduction columns, join 10 cm of 5-cm ID glass tubing on to
30 cm of 10-mm ID glass tubing (this 30-cm length is to contain the metal filings (Section 3.2.7)). Join
a 35 cm length of 2-mm ID glass tube to the 30-cm length. Bend the 35-cm length just below the joint
into a U. so that it runs parallel to the 5-cm length tube, and bend the end over to form an inverted U
siphon. When the assembly is held upright, the last bend should be level with the top of the 5-cm
length. If the assembly is correctly constructed, liquid placed in the top reservoir tube should flow out
of the system and stop when the level of liquid is such that it just covers the metal filings. To protect
the reduction columns, place the columns inside large cylinders (glass or plastic). To the outside of the
cylinders, affix a small glass cylinder drawn to a tube at one end and sealed with a rubber tube and
pinch clamp. This cylinder should hold about 75 mL and be arranged under the exits of the reduction
columns to collect effluents. Mark the 40- and 50-mL levels on the cylinder.
3. 50-mL graduated cylinders.
4. 125-mLErlenmeyer flasks.
5'. Spectrophotometer and cells.
3.2 This procedure requires the following reagents.
NOTE: All solutions must be made with ammonia-free water.
1. Concentrated ammonium chloride solution. Dissolve 125 g of ammonium chloride (analytical reagent
quality) in 500 mL distilled water. Store solution in a glass or plastic bottle.
2. Dilute ammonium chloride solution. Dilute 50 mL of concentrated ammonium chloride solution (see
above) to 2000 mL with distilled water. Store the solution in either a glass or plastic bottle.
3. Sulfanilamide solution. Dissolve 5 g of sulfanilamide in a mixture of 50 mL concentrated
hydrochloric acid (sp gr 1.18) and approximately 300 mL distilled water. Dilute to 500 mL with distilled
water. This solution will remain stable for several months.
4. N-(1-naphthyl)-ethylenediamine dihydrochloride solution. Dissolve 0.50 g of dihydrochloride in
500 mL distilled water. Store the solution in a dark bottle. The solution should be renewed once a
month or a strong brown coloration develops.
5. Cadmium copper filings. In a 18 x 150-mm pyrex test tube buried in dry sand, melt cadmium metal
(99.9 percent purity is satisfactory) and allow the metal to solidify. Using a coarse wood rasp, file off
the required amount of metal (110 g of filings is enough for 2 columns). Collect the fraction of the
filings that passes through a 1.0-mm mesh sieve and is retained on a 0.5-mm mesh sieve. Stir
approximately 100 g of filings (enough for two columns) with 500 mL of a 2 percent (w/v) solution of
copper sulphate pentahydrate, CuSO/t-SHaO, until all blue color is gone and semicolloidal copper particles
begin to appear in the supernatant. Roll very fine copper turnings between fingers and thumb to
produce a small plug, and insert the plug into the bottom of a reductor column (glass wool does not
work as well and should be used only if very fine copper "wool" turnings cannot be obtained). Fill the
-------
METHOD NO. A-NfTROGEN-22
redactor column with dilute ammonium chloride solution and pour in enough cadmium-copper mixture to
produce a column approximately 30 cm in length. Slowly add the filings to the column, lightly tapping
the column after each addition to ensure that the filings are well settled. Thoroughly wash the column
with dilute ammonium chloride solution. Check the flow rate of the column; 100 mL of the solution
should take between 8 and 12 min to completely flow through the column. If the flow rate is too great
(100 mL flows through in less than 8 min), slow it by restricting the outlet of the siphon or by packing
more copper or glass wool at the bottom. If the flow is too slow (100 mL of solution takes more than
12 min to flow through the column), loosen the packing at the base of the column. Once an acceptable
flow rate is achieved, add a small plug of copper "wool" to the top of the column. When the columns
are not in use, the metal filings in the columns must be completely covered with dilute ammonium
chloride solution.
If the efficiency of the reduction is suspect, empty the filings into a beaker and wash them. Filings
from four columns should be washed in 300 mL 5 percent (v/v) hydrochloric acid solution, stirring
vigorously. Decant the acid rinse once more with hydrochloric acid. Wash the filings with several 200-
300-mL aliquots distilled water until the wash is no longer acidic (pH > 5). Decant the liquid and allow
the filings to dry completely. Treat the filings with copper sulfate solution, as described above. The
regenerated cadmium-copper mixture should be sufficient for three columns.
6. Hydrogen peroxide. Use an analytical reagent quality 30 percent solution.
7. Synthetic sea water. Dissolve 310 g of analytical reagent quality sodium chloride (NaCI), 100 g of
analytical reagent quality magnesium sulphate (MgS04-7H20), and 0.50 g of sodium bicarbonate
in 1 0 L of distilled water.
8. Standard nitrate solution. Dissolve 1 .02 g of analytical reagent quality potassium nitrate (KNO3) in
1 000 mL of distilled water. The solution is stable indefinitely in the absence of evaporation. One
milliliter (1 mL) of this solution equals 10.0 /*g-at N/L Dilute 4.00 mL of this solution to 2000 mL with
synthetic sea water. The concentration of nitrate in this solution is 20 pg-sA N/L Prepare a fresh
solution immediately before use, and store in a dark bottle.
3.3 Equipment/instalment calibration.
1 . There is a slight salt effect in this method and calibration should be performed using synthetic sea
water (see description above) or natural sea water with a nitrate concentration less than 1 /
-------
METHOD NO. A-NITROGEN-22
nitrogen blank must be made on the synthetic seawater. Add the a.a'dipyridyl as 1.0 mL stock solution
to 99 mL of seawater. Prepare the stock by dissolving 0.117 g of base in 400 mL of water with stirring,
and dilute the solution to 1000 mL (1 mL = 1.5 mg-at N). The lamp is suspect if this rough check leads
to less than 90 percent of a theoretical recovery (15 /*g^at N/L).
4. PROCEDURE
4.1 Sample handling and preservation. Store samples for several hours cold and in the dark; sample
analysis should not be delayed any longer than 12 h. If sample analysis will not be done within 12 h,
store the samples frozen (-20°C) in a deep freezer where no detectable changes occur for several weeks.
4.2 Interferences.
1. Continual use of a column leads to deactivation. The slight acidification of the sample before
processing greatly reduces the deactivation, and a well-made column should be capable of reducing at
least 100 samples. The volume of the samples is not critical up to 5 mL
2. Samples containing sulfide in concentrations of up to 2 mg S2-/L may be analyzed by this method
without interference by the sulfide. However, repeated analysis of such samples will lead to deactivation
of the columns by the production of cadmium sulfide.
4.3 Sample analysis.
1. Rinse a silica tube with a few milliliters of the filtered sample. Add approximately 1.5 mL to the
tube and at the same time rinse a 100-mL measuring cylinder and a 125-mL Erlenmeyer flask with the
sample, shake out excess water, and measure out 100 mL of sample into the flask. Set the flask aside
in a cool place. Do not delay irradiation of the other aliquot.
2. Add 1 to 2 drops of hydrogen peroxide to the silica tube, stopper, and mix. Oxidation requires
oxygen gas dissolved in the sample and is markedly more rapid at high oxygen concentrations. This is
ensured by the addition of hydrogen peroxide. The oxidation rate is pH-dependent and is at about a
maximum at seawater pH. If fresh water samples are ever analyzed a little bicarbonate should be added
to ensure that the pH is in the range of 6 to 8.5.
3. Irradiate the tube and its contents for 3 h. (The oxidation of organic nitrogen compounds and
ammonia to nitrate is slower than the rate of orthophosphate liberation.) NOTE: Strickland and
Parsons state: "A large number of organic nitrogen compounds tested by us have been quantitatively
converted to nitrate by this method, including several heterocyclic molecules. It seems fairly certain,
therefore, that most of the metabolic products of the plankton will be measured but urea is surprisingly
resistant and one is left suspecting that other materials, which do not react, may exist in seawater. We
do not believe this is likely to introduce a serious error, however, and the Kjeldahl method and UV
methods when compared directly indicate no systematic differences. Ammonia is qualitatively oxidized in
3 h and a correction must be made for the most precise work (see Section 4.3. below). As "ammonia" is
often determined as ammonia plus amino acids and is generally only a small fraction of the total
organic nitrogen, we have rarely made this correction."
4. Cool the tube to room temperature and use a few milliliters to rinse the 100-mL cylinder and a
second Erlenmeyer flask. Add 100 mL of irradiated sample to the flask.
-------
METHOD NO. A-NfmOGEN-22
5. Measure the nitrate content of the irradiated and untreated samples, as described in the method for
determination of reactive nitrate and in Sections 4.3.6 through 4.3.13 below, using the same redactor
column for both solutions. (The use of the same column ensures maximum precision because the factor
will not change between the two determinations, as might be the case if two different columns were
used.).
6. Add 2.0 mL concentrated ammonium chloride to the sample in the Erlenmeyer flask. NOTE: The
acidification of the sample by the addition of ammonium chloride greatly slows the deactivation of the
column.
7. Thoroughly mix the solution and pour approximately 5 mL onto the top of the column and allow the
sample to pass through the column. NOTE: This initial addition ensures that the liquid in the top of
the column is similar to the sample. By doing this, no error will result when the remainder of the
sample is added and some of the interstitial liquid in the top of the column mixes with the sample. For
example, if a sample were poured through a column immediately after a blank had passed through and
this preliminary aliquot had not been poured through, some dilutions of the sample could occur. This
precaution is particularly important when processing consecutive samples containing varying
concentrations of nitrate.
8. Add the remainder of the sample to the column and place the empty Erlenmeyer flask under the
collection tube at the bottom of the reduction column. When approximately 40 mL of liquid has passed
through the column and into the collection tube, drain the collection tube into the flask, rinse the flask
with the effluent, drain, and place the flask under the collection tube. NOTE: The passage of at least
40 mL of solution is necessary to completely flush the column. This is especially important when
processing samples of varying nitrate concentrations.
9. Collect approximately 50 mL in the collection tube and quickly transfer the volume into the
Erlenmeyer flask. Allow the column to drain until flow ceases. NOTE: The volume in the flask should
be within a few milliliters of 50 mL, but it is not critical that the flask be completely drained of the
column washings. Under the conditions detailed thus far, reduction will be approximately 93 percent;
temperature variations between 10 and 35°C will have no effect. NOTE: It is not necessary to wash
the columns between samples; however, if columns will not used for 1 or 2 hours or more, pour 50 mL
of dilute ammonium chloride through the columns. Store the columns completely covered with the liquid.
10. Add 1.0 mL of sulphanilamide solution to the flask as soon as possible after reduction. Allow the
reagent to react for at least 2 min but no longer than 8 min. The diazotizing reaction requires at least
2 min for completion, but undesirable side reactions and decomposition become significant after 10 min.
NOTE: Although reduced nitrate solution may be stable for several hours, analysis should be completed
as soon as possible, particularly in hot weather. Temperature is not critical but must fall between 15
and 30°C.
11. Add 1.0 mL of naphthylethylenediamine solution and mix immediately. After 10 min but within 2 h
(complete color development requires 10 min, and is stable for up to 2 h.), measure the extinction of the
solution, against distilled water, in a 1-cm cell (wavelength 5430 A).
12. If the extinction exceeds 1.25 (approximately 30 /*g-at N/L), measure again in either a 0.5-cm cell
or add 25.0 mL of distilled water to 25.0 mL of solution in a clean dry flask, and double the value used
in the calculations. If the extinction value is less than 0.1 in a 1-cm cell, measure again in a 10-cm
cell. If the extinction is between 0.1 and 0.2 in a 1-cm cell, measure again in a 5-cm cell.
-------
METHOD NO. A-NrTROGEN-22
13. Rinse the absorptiometer cell with each new solution before filling. The cell does not have to be
rinsed between adjacent samples if the extinction values are within 25 percent of one another.
14. Fill the 1-cm or 0.5-cm spectrophotometer cell normally used for the distilled water blank with the
solution from the sample not irradiated. Measure the extinction of the irradiated sample against this
solution. NOTE: This method of extinction measurement ensures a high precision on the Beckman and
similar spectrophotometers because small differences are read directly on the most open part of the
extinction scale. It will generally be necessary to open the slit width on the spectrophotometer to get
an adequate sensitivity. In any case, if the extinction of the irradiated solution against water exceeds
about 1.0 (25 /«g-at NOa-N/L) then 0.5-cm cells should be used and the measured extinction multiplied
by 2 before calculations are made.
15. Calculate organic nitrogen present from the expression given in Section 6.1.2.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. The precision and range of the method is dependent upon the amount of nitrate in the
water. The ranges given in Section 2.3.1 apply to near-surface waters with less than about 10 /ig-at/L
In deeper waters the precision is much poorer. In deep Pacific waters the value for precision (95
percent confidence) is little better than 0.75 ^g-at N/L
5.2 Accuracy. This is a "difference" method with no blank as such.
6. REC03DKEEPING AND DATA REPORTING REQUIFiEMENTS
6.1 Calculations.
1. F factor.
F = 20.0/E
where F =F factor
E =mean extinction of the three values for each column (corrected for a blank)
2. Concentration of Organic Nitrogen.
/ig-at N/L = (E x F) - (C + X)
where F =F factor (see Section 6.1.1)
E =extinction of solution not irradiated when measured against the irradiated solution
C = concentration of ammonia in the sample
X =0.5, 0.75, or 1 when the nitrate content of the solution not irradiated is in the range
15-25, 25-35, or 35-45 /ig-at N/L, respectively
NOTE: Strickland and Parsons state: "For reasons we do not understand about one half the nitrate in
a sample is reduced to nitrite when samples of high nitrate and low organic nitrogen content are
irradiated. Because nitrite is destroyed to the extent of about 5 percent in the cadmium column (refer
to method for determination of reactive nitrate) a slight error will result, as this nitrite will not be
-------
METHOD NO. A-NrTROGEN-22
present in the untreated sample. The quantity X is used in an attempt to correct this trouble. It is
not a very precise correction but, in deep water, it is probably better to use it than make no correction
at all. For near-surface samples with high organic nitrogen and nitrate contents less than about 10 to
20 ng-at N/L no such correction should be attempted."
6.2 Reporting units. Concentrations of soluble organic nitrogen are reported in j»g-at N/L
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
72. Training/level of expertise Analysts using this method should be proficient in the operation and
maintenance of all equipment. Personnel conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency in the laboratory
techniques described in this method.
8. REFERENCES
Strickland, J.D.H. and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries
Research Board of Canada, Ottawa.
-------
METHOD NO. A-NITROGEN-23
INDEX INFORMATION
Matrices: Seawater, waste
Categories: Estuarine and marine seawater and sea-surface microlayer, wastewater/effluents
Parameter Ammonia nitrogen
1. METHOD TITLE
Distillation Method for the Determination of Ammonia Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrogen, Ammonia. Method 350.2 (Colorimetric. Titrimetric, Potentiometric,
Distillation Procedure) Storet Nos.-Total 00610, Dissolved 00608. In: Methods for Chemical Analysis of
Water and Wastes. U.S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory (EMSL), Cincinnati, OH. March 1979. EPA^600/4-79-020.
22 Regulatory status. This method is approved for NPDES.
2.3 Principle and application.
2.3.1 Description. This method is approved by EPA for the determination of ammonia in marine
and estuarine water and domestic and industrial wastes. It is the method of choice where
economics and sample load do not warrant the use of automated equipment.
The method covers the range from about 0.05 to 1.0 mg NHa-N/L for the colorimetric procedure;
from 1.0 to 25 mg NH3-N/L for the titrimetric procedure; and from 0.5 to 1400 mg/L for the
electrode method.
This method is designed for macro glassware; however, micro distillation equipment may also be
used.
The sample is buffered at a pH of 9.5 with a borate buffer in order to decrease hydrolysis of
cyanates and organic nitrogen compounds. It is then distilled into a solution of boric acid. The
ammonia in the distillate can be determined colorimetrically by nesslerization; titrimetrically with
standard sulfuric acid and a mixed indicator; or potentiometrically by the ammonia electrode. The
choice between the first two procedures depends on the concentration of the ammonia.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method. Collect samples for ammonia analysis
in glass or plastic bottles. Each bottle and cap should be rinsed thoroughly with the sample water
prior to collection.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NFTROGEN-23
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
2.4.2 Precision and accuracy. Twenty-four analysts in 16 laboratories analyzed natural water
samples containing exact increments of ammonium salt (FWPCA Method Study 2, Nutrient Analyses).
with the following results:
Increment as
Nitrogen, Ammonia
mgN/L
Precision as
Standard Deviation
mgN/L
Accuracy as
Bias, percent
Bias, mgN/L
0.21
0.26
1.71
1.92
0.122
0.070
0.244
0.279
-5.54
- 18.12
+ 0.46
- 2.01
- 0.01
- 0.05
+ 0.01
- 0.04
3. SPECIFICATIONS
3.1 This procedure requires the following equipment:
1. Distilling apparatus. All glass, with an 800- to 1000-mL flask.
2. Spectrophotometer or filter photometer. Suitable for use at 425 nm and providing a light patch of
1 cm or more.
3. Messier tubes. Matched Nessler tubes (APHA Standard); about 300 mm long, 17 mm ID, and marked
at 225 mm ±1.5 mm inside measurement from the bottom.
4. Erlenmeyer flasks. The distillate is collected in 500 mL glass stoppered flasks. These flasks should
be marked at the 350- and 500-mL volumes. With such marking, it is not necessary to transfer the
distillate to volumetric flasks.
3.2 This procedure requires the following reagents:
1. Distilled water. Distilled water should be free of ammonia. Such water is best prepared by passage
through an ion exchange column containing a strongly acidic cation exchange resin mixed with a strongly
basic anion exchange resin. Regeneration of the column should be carried out according to the
manufacturer's instructions. NOTE: All solutions must be made with ammonia-free water.
2. Ammonium chloride stock solution. Dissolve 3.819 g NH4CI in distilled water and bring to volume in
a 1-L volumetric flask. 1.0 mL = 1.0 mg NH3-N.
3. Ammonium chloride standard solution. Dilute 10.0 mL of ammonium chloride stock solution to 1 L in
a volumetric flask. 1.0 mL = 0.01 mg NH3-N.
-------
METHOD NO. A-NITROGEN-23
4. Boric acid solution. Dissolve 20 g HaBOa in distilled water and dilute to 1 L in a volumetric flask.
5. Mixed indicator. Mix 2 volumes of 0.2 percent methyl red in 95 percent ethyl alcohol with 1 vok me
of 0.2 percent methylene blue in 95 percent ethyl alcohol. This solution should be prepared fresh every
30 days.
6. Nessler reagent. Dissolve 100 g of mercuric iodide and 70 g of potassium iodide in a small amount
of water. Add this mixture, slowly, while stirring, to a cooled solution of 160 g of NaOH in 500 mL of
water. Dilute the mixture to 1 L This reagent will remain stable for up to 1 year if stored in a Pyrex
bottle out of direct sunlight. NOTE: This reagent should give the characteristic color with ammonia
within 20 min after addition, and should not produce a precipitate with small amounts of ammonia
(0.04 mg in a 50-mL volume).
7. Borate buffer. Add 88 mL of 0.1 NaOH solution to 500 mL of 0.025 M sodium tetraborate solution
(5.0 g anhydrous Na2B4O/ or 9.5 g Na2B4Or-10H2O per liter) and dilute to 1 L
8. Sulfuric acid standard solution. Prepare a stock solution of approximately 0.1 N acid by diluting
3 mL of concentrated HaSO4 (sp. gr. 1 .84) to 1 L with CO2-free distilled water. Dilute 200 mL of this
solution to 1 L with CO2-free distilled water. The resultant solution is 0.02 N; 1 mL = 0.028 mg
NH3-N). NOTE: An alternate and perhaps preferable method is to standardize the approximately 0.1 N
H2SO4 solution against a 0.100 N NaaCOs solution. By proper dilution, the 0.02 N acid can then be
prepared. Standardize the approximately 0.02 N acid against 0.0200 N NaaCOa solution. This last
solution is prepared by dissolving 1 .060 g anhydrous Na2C03, oven-dried at 140 °C, and diluting to 1 L
with CO2-free distilled water.
9. Sodium hydroxide. Dissolve 40 g NaOH in ammonia-free water and dilute to 1 L The resultant
solution is 1 N.
10. Dechlorinating reagents. A number of dechlorinating reagents may be used to remove residual
chlorine prior to distillation. These include the following:
• Sodium thiosulfate. Dissolve 3.5 g N32S2O3-5H2O in distilled water and dilute to 1 L The
resultant solution is 1/70 N. One mL of this solution will remove 1 mg/L residual chlorine in
500 mL of sample.
• Sodium arsenite. Dissolve 1 .0 g NaAsO2 in distilled water and dilute to 1 L
4. PROCEDURE
4.1 Sample handling and preservation. Results of ammonia analyses are most reliable when they are
made on fresh samples. However, if analysis must be delayed, samples can be stored for up to 28 days
if they are preserved by acidification to pH < 2 with H2SO4 (add 2 mL concentrated H2SO4 per liter of
sample), and refrigerated at 4 °C until analysis. Record the length of delay before analysis on a sample
log sheet.
4.2 Interferences.
1 . Contamination of ammonia samples can occur easily due to the volatile nature of ammonia. To
prevent potential cross-contamination, reagents used for other analyses that contain ammonia (e.g.,
-------
METHOD NO. A-NITROGEN-23
colorimetric phenol) should be isolated from samples and standards used for ammonia determinations. In
addition, cleaning preparations that contain significant quantities of ammonia (e.g., Pine-Sol or wax
removers) should not be used in the laboratory area where ammonia determinations are performed.
2. Contaminated glassware should be rinsed with 1 +1 HCI, followed by distilled water. To check for
contamination, blanks should be analyzed whenever a new reagent is prepared.
3. A number of aromatic and aliphatic amines, as well as other compounds, both organic and inorganic,
will cause turbidity upon the addition of Nessler reagent, so direct nesslerization (i.e., without
distillation) has been discarded as an official method.
4. Cyanate, which may be encountered in certain industrial effluents, will hydrolyze to some extent
even at the pH of 9.5 at which distillation is carried out. Volatile alkaline compounds, such as certain
ketones, aldehydes, and alcohols, may cause an off-color upon nesslerization in the distillation method.
Some of these, such as formaldehyde, may be eliminated by boiling off at a low pH (approximately 2 to
3) before distillation and nesslerization.
5. Residual chlorine must also be removed by pretreatment of the sample with sodium thiosulfate before
distillation.
4.3 Preparation of equipment Add 500 mL of distilled water to an 800 mL Kjeldahl flask. The addition
of boiling chips that have been previously treated with dilute NaOH will prevent bumping. Steam out
the distillation apparatus until the distillate shows no trace of ammonia with the Nessler reagent.
4.4 Sample preparation. Remove the residual chlorine in the sample by adding dechlorinating agent
equivalent to the chlorine residual. To 400 mL of sample, add 1 N NaOH solution until the pH is 9.5,
checking the pH during addition with a pH meter or by use of short-range pH paper.
4.5 Distillation. Transfer the sample (adjusted to a pH of 9.5) to an 800 mL Kjeldahl flask and add 25
mL of borate buffer. Distill 300 mL at the rate of 6 to 10 mL/min into 50 mL of 2 percent boric acid
contained in a 500-mL Erlenmeyer flask. NOTE: The condenser tip or an extension of the condenser
tip must extend below the level of the boric acid solution. Dilute to distillate to 500 mL with distilled
water and nesslerize and aliquot to obtain an approximate value of the ammonia-nitrogen concentration.
For concentrations above 1 mg/L, the ammonia should be determined titrimetrically. For concentrations
below this value, ammonia should be determined colorimetrically. The electrode method may also be
used.
4.6 Determination of ammonia in distillata Determine the ammonia content of the distillate
titrimetrically, colorimetrically, or potehtiometrically using one of the following procedures.
4.6,1 Titrimetric determination. Add 3 drops of the mixed indicator to the distillate and titrate
the ammonia with 0.02 N H2SO4, matching the end point against a blank containing the same
volume of distilled water and HsBOs solution.
4.6.2 Colorimetric determination.
1. Prepare a series of Nessler tube standards as follows:
-------
METHOD NO. A-NITROGEN-23
mL of Standard
1.0 mL = 0.01 mg NH^-N mg NHa-N/50.0 mL
0.0 0.0
0.5 0.005
1.0 0.01
2.0 0.02
3.0 0.03
4.0 0.04
5.0 0.05
8.0 0.08
10.0 0.10
2. Dilute each tube to 50 mL with distilled water, add 2.0 mL of Messier reagent, and mix. After
20 min, read the absorbance at 425 nm against the blank. From the values obtained, plot
absorbance against mg NHs-N for the standard curve. Determine the ammonia in the distillate by
nesslerizing 50 mL or an aliquot diluted to 50 mL and reading the absorbance at 425 nm as
described for the standards. Ammonia-nitrogen content is read from the standard curve.
4.6.3 Potentiometric determination. Consult EPA Method No. 350.3, Nitrogen, Ammonia: Selective
Ion Electrode Method.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. The analyst should demonstrate the ability to generate acceptable precision with this
method using replicate sample analyses. Duplicate analyses should be conducted on a minimum of 5
percent of the total number of samples.
5.2 Accuracy. The analyst should demonstrate the ability to generate acceptable accuracy with this
method using laboratory recovery samples and blank samples. A spiked sample should be analysis should
be conducted on a minimum of 5 percent of the total number of samples; a blank should be analyzed
with each batch of samples; a U.S. EPA performance evaluation sample should be analyzed at least once
per quarter.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations.
6.1.1 Titrimetric determination.
mg/L NHa-N = (A x 0.28 x 1,000)/S
where A =mL 0.02 N H2SO4 used
S =mL sample
-------
METHOD NO. A-NHROGEN-23
6.1.2 Spectrophotometric determination.
mg/LNH3-N = [(A x 1 ,000)/D] (B/C)
where A =mg NHs-N read from standard curve
B = total distillate collected, including boric acid and dilution
C =mL distillate taken for nesslerization
D =ml_ of original sample taken
6.1.3 Potentiometric determination.
mg/L NHa-N = (500/D) x A
where A =mg NH3-N/L from electrode method standard curve
D =mL of original sample taken
6.2 Reporting units. Concentrations of ammonia in unknown samples are reported in units of mg
a maximum of three significant figures.
Results should be reported for all determinations, including QA replicates and spiked samples. Any
factors that may have influenced sample quality should also be reported.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of all equipment used for this procedure. Personnel conducting this analysis should initially
work under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
8. REFERENCES
U.S. EPA. 1985. Interim guidance on quality assurance/quality control (QA/QC) for the field and laboratory
methods. U.S. EPA Office of Marine and Estuarine Protection. Washington, D.C.
U.S. EPA. 1986. Handbook of methods for estuarine environmental monitoring. U.S. EPA Office of Marine
and Estuarine Protection. Washington, D.C.
-------
METHOD NO. A-NITROGEN-24
INDEX INFORMATION
Matrices: Seawater, waste
Categories: Estuarine and marine seawater and sea-surface microlayer, wastewater/effluents
Parameter Ammonia nitrogen
1. METHOD TITLE
Potentiometric Method for the Determination of Ammonia Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrogen, Ammonia. Method 350.3 (Potentiometric, Ion Selective Electrode)
Storet Nos.-Total 00610, Dissolved 00608. In: Methods for Chemical Analysis of Water and Wastes.
U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory (EMSL),
Cincinnati, OH. March 1979. EPA-600/4-79-020.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This method is approved by EPA for the determination of ammonia-nitrogen in
marine and estuarine water and domestic and industrial wastes. (It is also approved for
determination of ammonia in surface and drinking waters.)
This method covers the range from 0.03 to 1400 mg NH3-N/L Color and turbidity have no effect
on the measurements; thus distillation may not be necessary..
The ammonia is determined potentiometrically using an ion selective ammonia electrode and a pH
meter equipped with an expanded millivolt scale of a specific ion meter. The ammonia electrode
uses a hydrophobic gas-permeable membrane to separate the sample solution from an ammonium
chloride internal solution. Ammonia in the sample diffuses through the membrane and alters the pH
of the internal solution, which is sensed by the pH electrode. The constant level of chloride in
the internal solution is sensed by a chloride selective ion electrode which acts as the reference
electrode.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method. Collect samples for ammonia analysis
in glass or plastic bottles. Each bottle and cap should be rinsed thoroughly with the sample water
prior to collection.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NfTROGEN-24
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
2.4.2 Precision and accuracy.
1. Precision. In a single laboratory (EMSL), using surface water samples at concentrations of
1.00, 0.77,0.19, and 0.13 mg NHa-N/L, standard deviations were ± 0.038, ± 0.017, ± 0.007, and
± 0.003, respectively.
2. Accuracy. In a single laboratory, (EMSL), using surface water samples at concentrations of 0.19
and 0.13 mg NH3-N/L, recoveries were 96 percent and 91 percent, respectively.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment:
1. pH meter. (Electrometer) with expanded mV scale or a specific ion meter.
2. Ammonia selective electrode. Orion Model 95-10, EIL Model 800202, or equivalent.
3. Magnetic stirrer. Thermally insulated, with Teflon-coated stirring bar.
3.2 This procedure requires the following reagents:
1. Water. Distilled water should be free of ammonia. Such water is best prepared by passage through
an ion exchange column containing a strongly acidic cation exchange resin mixed with a strongly basic
anion exchange resin. Regeneration of the column should be carried out according to the
manufacturer's instructions.
2. Sodium hydroxide. (10N)-Dissolve400gof sodium hydroxide in 800 mL of distilled water. Cool
and dilute to 1 L with distilled water.
3. Ammonium chloride stock solution. Dissolve 3819 g NH4CI in distilled water and bring to volume
with distilled water in a 1-L volumetric flask. 1.0 ml = 1.0 mg NH3-N.
4. Ammonium chloride standard solution. Dilute 10.0 mL of the ammonium chloride stock solution to
1 L with distilled water in a volumetric flask. 1.0 mL = 0.01 mg NHa-N. NOTE: When analyzing saline
waters, standards must be made up in synthetic ocean water (SOW). Prepare SOW with the following
concentrations of compounds:
-------
METHOD NO. A-NFTROGEN-24
Substitute Ocean Water (SOW
NaCI 4.53 g/L NaHCOa 0.20 g/L
MgCIa 5.20 g/L KBr 0.10 g/L
N32SO4 4.09 g/L HaBOa 0.03 g/L
CaCl2 1.16 g/L SrCl2 0.03 g/L
KCI 0.70 g/L NaF 0.003 g/L
3.3 Equipment/instrument calibration.
1. The pH meter (electrometer) or specific ion meter should be calibrated according to the directions of
the manufacturer for the operation of the instrument.
2. Refer to Sections 4.3 and 4.4 for instructions for preparation of calibration standards and a
calibration curve. Concentrations of the calibration standards should bracket the sample concentrations.
A calibration curve should be prepared for each day of sample analysis. If a sample is outside the range
of calibration, then an additional calibration standard should be analyzed to check if the result is within
linear range of the method. Alternatively, the sample should be diluted to within the calibration range
and then reanalyzed.
4. PROCEDURE
4.1 Sample handling and preservation. Results of ammonia analyses are most reliable when they are
made on fresh samples. However, if analysis must be delayed, samples can be stored for up to 28 days
if they are preserved by acidification to pH < 2 with H2SO4 (add 2 mL concentrated H2S04 per liter of
sample), and refrigerated at 4 °C until analysis. Record the length of delay before analysis on a sample
log sheet.
4.2 Interferences.
1. Contamination of ammonia samples can occur easily due to the volatile nature of ammonia. To
prevent potential cross-contamination, reagents used for other analyses that contain ammonia (e.g.,
colorimetric phenol) should be isolated from samples and standards used for ammonia determinations. In
addition, cleaning preparations that contain significant quantities of ammonia (e.g., Pine-Sol or wax
removers) should not be used in the laboratory area where ammonia determinations are performed.
2. Contaminated glassware should be rinsed with 1 +1 HCI, followed by distilled water. To check for
contamination, blanks should be analyzed whenever a new reagent is prepared.
3. Volatile amines act as a positive interference.
4. Mercury interferes by forming a strong complex with ammonia. Thus, the samples cannot be
preserved with mercuric chloride.
4.3 Preparation of standards. Prepare a series of standard solutions covering the concentration range
of the samples by diluting either the stock of standard solutions of ammonium chloride.
-------
METHOD NO. A-NfTROGEN-24
4.4 Calibration
4.4.1 Electrometer
1. Place 100 mL of each standard solution in clean 150 mL beakers. Immerse the electrode into
the standard of lowest concentration and add 1 mL of 10 N sodium hydroxide solution while mixing.
Keep electrode in the solution until a stable reading is obtained. NOTE: Sodium hydroxide must
not be added prior to electrode immersion, because ammonia may be lost from a basic solution.
NOTE: The pH of the solution after the addition of NaOH must be above 11.
2. Repeat this procedure with the remaining standards, going from lowest to highest
concentrations. Using semilogarithmic graph paper, plot the concentration of ammonia in mg
NH3-N/L on the log axis against the electrode potential developed in the standard on the linear
axis, starting with the lowest concentration at the bottom of the scale.
4.4.2 Specific ion meter. Follow the directions of the manufacturer for the operation of the
instrument.
4.5 Sample measurement
1. Place 100 mL of each sample in clean 150 mL beakers. Immerse the electrode into the sample and
add 1 mL of 10 N sodium hydroxide solution while mixing. Keep electrode in the solution until a stable
reading is obtained. NOTE: Sodium hydroxide must not be added prior to electrode immersion, because
ammonia may be lost from a basic solution. NOTE: The pH of the solution after the addition of NaOH
must be above 11.
2. Record the stabilized potential of each unknown sample and convert the potential reading to the
ammonia concentration using the standard curve.
3. If a specific ion meter is used, read the ammonia level directly in mg NHs-N/L
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. The analyst should demonstrate the ability to generate acceptable precision with this
method using replicate sample analyses. Duplicate analyses should be conducted on a minimum of 5
percent of the total number of samples.
5.2 Accuracy. The analyst should demonstrate the ability to generate acceptable accuracy with this
method using laboratory recovery samples and blank samples. A spiked sample should be analysis should
be conducted on a minimum of 5 percent of the total number of samples; a blank should be analyzed
with each batch of samples; a U.S. EPA performance evaluation sample should be analyzed at least once
per quarter.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
Concentrations of ammonia in unknown samples are reported in units of mg NHs-N/L to a maximum of three
significant figures. Results should be reported for all determinations, including QA replicates and spiked
samples. Any factors that may have influenced sample quality should also be reported.
-------
METHOD NO. A-NITROGEN-24
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertisa Analysts using this method should be proficient in the operation and
maintenance of the all equipment used for this procedure. Personnel conducting this analysis should
initially work under the guidance of an experienced supervisor until he/she can demonstrate proficiency
in the laboratory techniques described in this method.
8. REFERENCES
U.S. EPA. 1979. Methods for chemical analysis of water and wastes. U.S. EPA Environmental Monitoring
and Support Laboratory. Cincinnati, OH.
U.S. EPA. 1985. Interim guidance on quality assurance/quality control (QA/QC) for the field and laboratory
methods. U.S. EPA Office of Marine and Estuarine Protection. Washington, D.C.
U.S. EPA. 1986. Handbook of methods for estuarine environmental monitoring. U.S. EPA Office of Marine
and Estuarine Protection. Washington, D.C.
-------
METHOD NO. A-NITROGEN-25
INDEX INFORMATION
Matrices: Seawater, waste
Categories: Estuarine and marine seawater and sea-surface microlayer, wastewater/effluents
Parameter: Total Kjeldahl nitrogen
1. METHOD TITLE
Colorimetric. Automated Phenate Method for the Determination of Total Kjeldahl Nitrogen
2. BACKGROUND AND APPLICATION
2.1 Source of method. Nitrogen, Kjeldahl, Total. Method 351.1. (Colorimetric, Automated Phenate)
Storet No. 00625. In: Methods for Chemical Analysis of Water and Wastes. U.S. Environmental
Protection Agency, Environmental Monitoring and Support Laboratory (EMSL), Cincinnati, OH. March
1979. EPA-600/4-79-020.
2.2 Regulatory status. This method is approved for NPDES.
2.3 Principle and application.
2.3.1 Description. This automated method is approved for use in determining total Kjeldahl
nitrogen (TKN) in marine and estuarine waters. The applicable range of this method is 0.05 to
2.0 mg N/L Approximately 20 samples/h can be analyzed.
The sample is automatically digested with a sulfuric acid solution containing potassium sulfate and
mercuric sulfate as a catalyst to convert organic nitrogen to ammonium sulfate. The solution is
then automatically neutralized with sodium hydroxide solution and treated with alkaline phenol
reagent and sodium hypochlorite reagent. This treatment forms a blue color designated as
indophenol. Sodium nitroprusside, which increases the intensity of the color, is added to obtain the
necessary sensitivity for measurement of low level nitrogen.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.3.3 Definitions.
1. Total Kjeldahl Nitrogen is defined as the sum of free-ammonia and organic nitrogen compounds
which are converted to ammonium sulfate, (NH4)2S04, under the conditions of digestion described
by this method.
2. Organic Kjeldahl Nitrogen is defined as the difference obtained by subtracting the free-ammonia
value from the TKN value. Also, organic Kjeldahl nitrogen may be determined directly by removal
of ammonia before digestion.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-NrTROGEN-25
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
2.4.2 Precision and accuracy. Six laboratories analyzed four natural water samples containing
exact increments of organic nitrogen compounds, with the following results:
Increment as Precision as
KjekJahf-Nitrogen Standard Deviation Accuracy as
mgN/L mg N/L Bias, percent Bias,mgN/L
1.89
2.18
5.09
5.81
0.54
0.61
1.25
1.85
- 24.6
- 28.3
- 23.8
- 21.9
- 0.46
- 0.62
- 1.21
- 1.27
3. SPECIFICATIONS
3.1 This procedure requires the following equipment:
Technicon AutoAnalyzer consisting of the following components:
1. Sampler II, equipped with a continuous mixer
2. Two proportioning pumps
3. Manifold I
4. Manifold II
5. Continuous digester
6. Planetary pump
7. Five-gallon carboy fume-trap
8. 80°C heating bath
9. Colorimeter equipped with 50 mm tubular flow cell and 630 nm filters
10. Recorder equipped with range expander
11. Vacuum pump
3.2 This method requires the following reagents:
NOTE: All solutions must be made using ammonia-free water.
-------
METHOD NO. A-NITROGEN-25
1. Distilled water. Special precaution must be taken to insure that distilled water is free of ammonia.
Such water is prepared by passage of distilled water through an ion-exchange column consisting of a
mixture of both strongly acidic cation and strongly basic anion-exchange resins. Furthermore, since
organic contamination may interfere with this analysis, use of the resin Dowex XE-75 or equivalent,
which also tends to remove organic impurities, is advised. The regeneration of the ion-exchange column
should be carried out according to the manufacturer's instructions.
2. Sulfuric acid. Because sulfuric acid readily absorbs ammonia, special precaution must also be taken
with respect to its use. Do not store bottles reserved for this determination in areas of potential
ammonia contamination.
3. Sodium hydroxide. (30 percent solution)-Dissolve 300 g NaOH in 1 L of distilled water. NOTE:
The 30 percent NaOH should be sufficient to neutralize the digestate. In rare cases it may be necessary
to increase the concentration of NaOH in this solution to insure neutralization of the digested sample in
the manifold at the water jacketed mixing coil.
4. EDTA solution. (2 percent)-Dissolve 20 g disodium ethylenediamine tetraacetate in 1 L of distilled
water. Adjust pH to 10.5 to 11 with 30 percent NaOH solution.
5. Sodium nitroprusside. (0.05 percent solution)-Dissolve 0.5 g Na2Fe(CN)sNO-2H2O in 1 L of distilled
water.
6. Alkaline phenol reagent. Pour 550 ml_ liquid phenol (88 to 90 percent) slowly, while mixing, into 1 L
of 40 percent (400 g/L) NaOH. Cool and dilute to 2 L with distilled water.
7. Sodium hypochlorite. (1 percent solution)-Dilute 200 ml_ of commercial Chlorox to 1 L with distilled
water. The available chlorine level should be approximately 1 percent. Because of the instability of this
product, storage over and extended period of time should be avoided.
8. Digestant mixture. Place 2 g red HgO in a 2-L container. Slowly add, with stirring, 300 ml_ of acid
water (100 mL H2S04 = 200 ml H2O) and stir until cool. Add 100 mL 10 percent (10 g/100 mL) K2SO4.
Dilute to 2 L with concentrated sulfuric acid (approximately 500 mL at a time, allowing time for
cooling.) Allow 4 h for the precipitate to settle or filter through glass fiber filter paper.
9. Stock solution. Dissolve 4.7193 g of pre-dried (1 h at 105 °C) ammonium sulfate in distilled water
and dilute to 1.0 L in a volumetric flask. 1.0 mL = 1.0 mg N.
10. Standard solution. Dilute 10.0 mL of stock solution to 1 L 1.0 mL = 0.01 mg N.
11. Calibration standards. Using the standard solution, prepare the following standards in 100 mL
volumetric flasks:
Concentration,
mgN/L
0.00
0.05
0.10
0.20
mL Standard Solution/
100 mL
0.0
0.5
1.0
2.0
-------
METHOD NO. A-NITROGEN-25
0.40 4.0
0.60 6.0
0.80 8.0
1.00 10.0
1.50 15.0
2.00 20.0
3.3 Equipment/instrument calibration. A calibration curve should be prepared for each day of sample
analysis using the calibration standards described in Section 3.2, Step 11. Concentrations of the calibration
standards should bracket the sample concentrations. If a sample is outside the range of calibration, then an
additional calibration standard should be analyzed to check if the result is within linear range of the
method. Alternatively, the sample should be diluted to within the calibration range and then reanalyzed.
4. PROCEDURE
4.1 Sample handling and preservation. Analysis should be conducted as soon as possible after sample
collection. If samples cannot be analyzed immediately, samples can be preserved by the addition of 2 ml_
of concentrated H2SO4 per liter of sample and stored under refrigeration at 4 °C. However, even when
preserved in this manner, conversion of organic nitrogen to ammonia may occur.
4.2 Interferences.
1. Iron and chromium ions tend to catalyze indophenol color reaction; copper ions tend to inhibit
indophenol color reaction.
2. Because ammonia is a component of TKN, precautions against potential cross-contamination should
include isolation of reagents for TKN analysis from reagents used for other analyses (e.g., colorimetric
phenol). In addition, cleaning preparations that contain significant quantities of ammonia (e.g., Pine-Sol
or wax removers) should not be used in the laboratory area where TKN determinations are performed.
3. Contaminated glassware should be rinsed with 1 + 1 HCI, followed by distilled water. To check for
contamination, blanks should be analyzed whenever a new reagent is prepared.
4.3 Manifold set up.
1. Set up manifolds as shown in Figures 1,2, and 3. In the operation of manifold No. 1, the control of
four key factors is required to enable manifold No. 2 to receive the mandatory representative feed.
First, the digestant flowing into the pulse chamber (PC-1) must be bubble-free; otherwise, air will
accumulate in A-7, thus altering the ratio of sample to digestant in the digestor. Second, in maintaining
even flow from the digestor helix, the peristaltic pump must be adjusted to cope with differences in
density of the digestate and the wash water. Third, the sample pick-up rate from the helix must be
precisely adjusted to insure that the entire sample is aspirated into the mixing chamber, and finally,
the contents of the mixing chamber must be kept homogeneous by the proper adjustment of the air
bubbling rate.
2. In the operation of manifold No. 2, it is important in the neutralization of the digested sample to
adjust the concentration of the NaOH so that the waste from the C-3 debubbler is slightly acid to
Hydrion B paper.
-------
METHOD NO. A-NITROGEN-25
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-------
METHOD NO. A-NHROGEN-25
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-------
METHOD NO. A-NfTROGEN-25
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Figure 3. Manifold 2 for KjeUahl Nitrogen Determination (AAJ)
7
-------
METHOD NO. A-NITROGEN-25
3. The digestbr temperature is 390 °C for the first stage and 369 °C for the second and third stages.
4.4 Operation.
1. Allow both colorimeter and recorder to warm up for 30 min. Run a baseline with all reagents,
feeding distilled water through the sample line. Adjust the dark current and operative opening on
colorimeter to obtain a stable baseline.
2. Set the sampling rate of Sampler II at 20 samples/h, using a sample to wash ration of 1:2 (1 min
sample, 2 min wash).
3. Arrange various standards in sampler cups in order of increasing concentration. Complete loading of
sampler tray with unknown samples.
4. Switch sample line from distilled water to sampler and begin analysis.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. The analyst should demonstrate the ability to generate acceptable precision with this
method using replicate sample analyses. Duplicate analyses should be conducted on a minimum of 5
percent of the total number of samples.
5.2 Accuracy. The analyst should demonstrate the ability to generate acceptable accuracy with this
method using laboratory recovery samples and blank samples. A spiked sample should be analysis should
be conducted on a minimum of 5 percent of the total number of samples; a blank should be analyzed
with each batch of samples; a U.S. EPA performance evaluation sample should be analyzed at least once
per quarter.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations.
1. Prepare a standard curve by plotting peak heights of processed standards against concentration
values. Compute concentration of samples by comparing sample peak heights with the standard curve.
2. Any sample that has a computed concentration that is less than 10 percent of the sample run
immediately prior to it must be rerun.
6.2 Reporting units. Report TKN in units of mg N/L to a maximum of three significant figures.
Results should be reported for all determinations, including QA replicates and spiked samples. Any
factors that may have influenced sample quality should also be reported.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
8
-------
METHOD NO. A-NITROGEN-25
7.2 Training/level of expertise Analysts using this method should be proficient in the operation and
maintenance of the all equipment used for this procedure. Personnel conducting this analysis should
initially work under the guidance of an experienced supervisor until he/she can demonstrate proficiency
in the laboratory techniques described in this method.
8. REFERENCES
U.S. EPA. 1979. Methods for chemical analysis of water and wastes. U.S. EPA Environmental Monitoring
and Support Laboratory. Cincinnati, OH.
U.S. EPA. 1985. Interim guidance on quality assurance/quality control (QA/QC) for the field and laboratory
methods. U.S. EPA Office of Marine and Estuarine Protection. Washington. D.C.
U.S. EPA. 1986. Handbook of methods for estuarine environmental monitoring. U.S. EPA Office of Marine
and Estuarine Protection. Washington, D.C.
-------
METHOD NO. A-PHOS-1
INDEX INFORMATION
Matrices: Seawater, waste
Categories: Estuarine and marine seawater and sea-surface microlayer, wastewater/effluents
Parameter: Total phosphorus
1. METHOD TITLE
Colorimetric Automated Block Digestor Method for the Determination of Total Phosphorus
2. BACKGROUND AND APPLICATION
2.1 Source of method. Phosphorus, Total. Method 365.4 (Colorimetric, Automated, Block Digestor. AAII;
Storet No. 00665) In: Methods for Chemical Analysis of Water and Wastes. U.S. Environmental
Protection Agency, Environmental Monitoring and Support Laboratory (EMSL), Cincinnati, OH. March
1979. EPA-600/4-79-020.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This method has been approved by EPA for the determination of total
phosphorus in drinking water, surface water, and domestic and industrial wastes. It has not been
approved by EPA for analysis of saline water, but has been used successfully on seawater samples
(U.S. Army Corps of Engineers Waterways Experiment Station, Vicksburg, Mississippi).
The sample is heated in the presence of H2SO4, K2S04, and HgS04 for 2.5 h. The residue is
cooled, diluted to 25 mL, and placed on the Technicon Autoanalyzer for phosphorus determination.
The applicable range of this method is 0.01 to 20 mg P/L
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method. Sample containers may be of plastic
material, such as a cubitainer, or of Pyrex glass.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-PHOS-1
2.4.2 Precision and accuracy.
1. Precision. In a single laboratory (EMSL), using sewage samples containing total P at
concentrations of 0.23,1.33, and 2.0 mg P/L, the precision was ± 0.01, ± 0.04, and ± 0.06,
respectively.
2. Accuracy. In a single laboratory (EMSL), using sewage samples with concentrations of 1.84 and
1.89 mg P/L, the recoveries were 95 and 98 percent, respectively.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. Block Digestor-40
2. Technicon Method No. 327-74W for phosphorus
3.2 This method requires the following reagents:
1. Mercuric sulfate. Dissolve 8 g red mercuric oxide (HgO) in 50 mL of 1:4 H2SO4 (10 mL concentrated
H2SO4:40 mL distilled water) and dilute to 100 mL with distilled water.
2. Digestion solution-Sulfuric acid/mercuric sulfate/potassium sulfate solution. Dissolve 133 g of K2SO4
in 600 mL of distilled water and 200 mL of concentrated H2SO4. Add 25 mL of mercuric sulfate solution
and dilute to 1 L
3. Sulfuric acid solution. 0.72N. Add 10 mL of concentrated H2SO4 to 800 mL of distilled water, mix,
and dilute to 1 L
4. Molvbdate/antimony solution. Dissolve 8 g of ammonium molybdate and 0.2 g of antimony potassium
tartrate in about 800 mL of distilled water and dilute to 1 L
5. Ascorbic acid solution. Dissolve 60 g of ascorbic acid in about 600 mL of distilled water. Add 2 mL
of acetone and dilute to 1 L
6. Diluent water. Dissolve 40 g of NaCI in about 600 mL of distilled water and dilute to 1 L
7. Sulfuric acid solution, 4 percent. Add 40 mL of concentrated H2SO4 to 800 mL of distilled water,
cool, and dilute to 1 L
4. PROCEDURE
4.1 Sample handling and preservation. Analysis should be conducted as soon as possible after sample
collection. If samples cannot be analyzed immediately, samples can be preserved by the addition of 2 mL
of concentrated H2SO4 per liter of sample and stored under refrigeration at 4 °C.
-------
METHOD NO. A-PHOS-1
4.2 Sample analysis.
4.2.1 Digestion.
1. To 20 or 25 mL of sample, add 5 mL of digestion solution and mix, using a vortex mixer.
2. Add several (4-8) Teflon boiling chips. (Too many boiling chips may cause the sample to boil
over.)
3. With the Block Digester in its manual mode, set both the low and high temperatures at 160 °C
and preheat the unit to 160 °C. Place the tubes in the digester and switch to automatic mode.
Set the low temperature timer for 1 h. Reset the high temperature to 380 °C and set the timer
for 2.5 h.
4. After digestion, cool the sample and dilute to 25 ml with distilled water.
4.2.2 Colorimetric analysis.
1. Check the levels of all reagent containers to ensure an adequate supply.
2. Excluding the molybdate/antimony line, place all reagent lines in their respective containers,
connect the sample probe to the Sampler IV, and start the proportioning pump.
3. Flush the Sampler IV wash receptacle with about 25 mL of 4.0 percent H2SO4.
4. When reagents have been pumping for at least 5 min, place the molybdate/antimony line in its
respective container and allow the system to equilibrate.
5. After a stable baseline has been obtained, start the sampler.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. The analyst should demonstrate the ability to generate acceptable precision with this
method using a laboratory control standard. Analysts should be able to meet the precision criteria
obtained by EMSL-using sewage samples with concentrations of 0.23,1.33, and 2.0 mg P/L, the precision
was ± 0.01, ± 0.04, and ± 0.06, respectively.
5.2 Accuracy. The analyst should demonstrate the ability to generate acceptable accuracy with this
method using a laboratory control standard. Analysts should be able to meet the accuracy criteria
obtained by EMSL-using sewage samples with concentrations of 1.84 and 1.89 mg P/L, the recoveries
were 95 and 98 percent, respectively.
6. RECORDKEEPINQ AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve by plotting peak heights of processed standards against
concentration values. Calculate concentrations by comparing sample peak heights with the standard
curve.
-------
METHOD NO. A-PHOS-1
6.2 Reporting units. Concentrations of total phosphorus in unknown samples are reported in units of
mgP/L
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices.
understand the proper handling of acids, solvents, and other reagents.
72. Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
8. REFERENCES
Gales, M.E. and R.L Booth. Evaluation of the Technicon Block Digester System for the measurement of
total Kjeldahl nitrogen and total phosphorus. EPA-6001/4-78-015, Environmental Monitoring and Support
Laboratory, Cincinnati, OH.
Gales, M.E. and R.L Booth. 1974. Simultaneous and automated determination of total phosphorus and total
Kjeldahl nitrogen. Methods Development and Quality Assurance Laboratory.
Gales, M.E. and R.L Booth. 1972. Evaluation of organic nitrogen methods. EPA Office of Research and
Monitoring.
McDaniel, W.H., R.N. Hemphill, and W.T. Donaldson. 1967. Automatic determination of total Kjeldahl
nitrogen in estuarine water. Technicon Symposia, Vol. 1. pp. 362-367.
Technicon Corp. 1974. Total Kjeldahl nitrogen and total phosphorus BD-40 digestion procedure for water.
-------
METHOD NO. A-PHOS-2
INDEX INFORMATION
Matrices: Seawater, waste
Categories: Estuarine and marine seawater and sea-surface microlayer, wastewater/effluents
Parameter Phosphorus, all forms
1. METHOD TITLE
Colorimetric Automated Ascorbic Acid Method for the Determination of Phosphorus
2. BACKGROUND AND APPLICATION
2.1 Source of method. Phosphorus, .All Forms. Method 365.1 (Colorimetric, Automated, Ascorbic Acid)
In: Methods for Chemical Analysis of Water and Wastes. U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory (EMSL), Cincinnati, OH. March 1979.
EPA-600/4-79-020.
• 2.2 Regulatory status. This method is approved for NPDES.
2.3 Principle and application
2.3.1 Description. This is a method for the determination of specified forms of phosphorus in
saline water and domestic and industrial wastes (also drinking and surface waters).
Ammonium molybdate and antimony potassium tartrate react in an acid medium with dilute solutions
of phosphorus to form an antimony-phospho-molybdate complex. This complex is reduced to an
intensely blue-colored complex by ascorbic acid. The color is proportional to the phosphorus
concentration.
Only orthophosphate forms a blue color with this test. Polyphosphates (and some organic
phosphorus compounds) may be converted to the orthophosphate form by manual sulfuric acid
hydrolysis. Organic phosphorus compounds may be converted to the orthophosphate form by manual
persulfate digestion. The developed color is measured automatically on the Technicon AutoAnalyzer.
The methods are based on reactions that are specific for the orthophosphate ion. Thus, depending
of the prescribed pre-treatment of the sample, the various forms are defined as follows:
1. Total phosphorus (P). All of the phosphorus present in the sample regardless of form, as
measured by the persulfate digestion procedure (Storet No. 00665).
• Total orthophosphate (P-ortho)-inorganic phosphorus [(PO2)~3] in the sample as measured by
the direct Colorimetric analysis procedure (Storet No. 70507).
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-PHOS-2
• Total hydrolyzable phosphorus (P-hydro)-phosphorus in the sample as measured by the
sulfuric acid hydrolysis procedure, and minus predetermined orthophosphate. This
hydrolyzable phosphorus includes polyphosphates [(R2O7)-4, (PsOio)'5, etc.] plus some organic
phosphorus (Storet No. 00669).
• Total organic phosphorus (P-org)-phosphorus (inorganic plus oxidizable organic) in the
sample as measured by the persulfate digestion procedure, and minus hydrolyzable phosphorus
and orthophosphate (Storet No. 00670).
2. Dissolved phosphorus (P-D). All of the phosphorus present in the filtrate of a sample filtered
through a phosphorus-free filter of 0.45 p pore size and measured by the persulfate digestion
procedure (Storet No. 00666).
• Dissolved orthophosphate (P-D, ortho)-as measured by the direct colorimetric analysis
procedure (Storet No. 00671).
• Dissolved hydrolyzable phosphorus (P-D. hydro)-as measured by the sulfuric acid hydrolysis
procedure, and minus predetermined dissolved orthophosphate (Storet No. 00672).
• Dissolved organic phosphorus (P-D. org)-as measured by the persulfate digestion procedure,
and minus dissolved hydrolyzable phosphorus and orthophosphate (Storet No. 00673).
3. The following forms, when sufficient amounts of phosphorus are present in the sample to
warrant such consideration, may be calculated:
• Insoluble phosphorus-(P-l) = (P) - (P-D) (Storet No. 00667).
• Insoluble orthophosphate-(P-l, ortho) = (P-ortho) - (P-D, ortho) (Storet No. 00675).
• Insoluble hydrolyzable phosphorus-(P-l, hydro) = (P-hydro) - (P-D, hydro) (Storet No. 00675).
• Insoluble organic phosphorus-(P-l. org) = (P-org) - (P-D. org) (Storet No. 00676).
The applicable range of this method is 0.001 to 1.0 mg P/L Approximately 20 to 30 samples per
hour can be analyzed.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a seawater matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
2.4.2 Precision and accuracy. Six laboratories, participating in an EPA Method Study, analyzed
four natural water samples containing exact increments of orthophosphate and obtained the
following results (given for the AAI system):
-------
METHOD NO. A-PHOS-2
Increment as
Orthophosphate
(mg P/L)
0.04
0.04
0.29
0.30
Precision as
Standard Deviation
(mg P/L)
0.019
0.014
0.087
0.066
Accuracy as
Bias,
percent
+ 16.7
-8.3
- 15.5
- 12.8
Bias,
mgP/L
+ 0.007
- 0.003
- 0.05
- 0.04
3. SPECIFICATIONS
3.1 This procedure requires the following equipment:
Technicon AutoAnalyzer Unit (AAI or AAII), consisting of the following components:
1. Sampler
2. Manifold (AAI) or Analytical Cartridge (AAII)
3. Proportioning pump
4. Colorimeter equipped with 15 or 50 mm tubular flow cell
5. 650 to 660 or 880 nm filters
6. Recorder
7. Digital printer for AAII (optional)
8. Hot plate or autoclave
9. Acid-washed glassware. All glassware used in the determination of phosphorus should be washed with
hot 1:1 HCI and rinsed with distilled water. The acid-washed glassware should be filled with distilled
water and treated with all the reagents to remove the last traces of phosphorus that might be adsorbed
on the glassware. Preferably, this glassware should be dedicated to this analysis only, and after use, it
should be rinsed with distilled water and kept covered until needed again. If this is done, the treatment
with 1:1 HCI and reagents is only required occasionally. Commercial detergent should never be used.
3.2 This method requires the following reagents:
1. Sulfuric acid solution, 5N. Slowly add 70 ml of concentrated H2SO4 to approximately 400 ml_ of
distilled water. Cool to room temperature and dilute to 500 mL with distilled water.
2. Antimony potassium tartrate solution. Weigh 0.3 g K(SbO)C4H4Oe-1/2H2O, dissolve in 50 mL distilled
water in a 100 mL volumetric flask, and dilute to volume. Store at 4 °C in a dark, glass-stoppered
bottle.
-------
METHOD NO. A-PHOS-2
3. Ammonium molybdate solution. Dissolve 4 g (NH4)eMo7O24-4H2O in 100 ml of distilled water. Store
in a plastic bottle at 4 °C.
4. Ascorbic acid, 0.1 M. Dissolve 1.8 got ascorbic acid in 100 mL of distilled water. This solution is
stable for about a week if prepared with water containing no more than trace amounts of heavy metals
and stored at 4 °C.
5. Combined reagent (AAI). Mix the above reagents in the following proportions for 100 mL of the
mixed reagent:
- 50 mL of 5N H2SO4
5 mL of antimony potassium tartrate solution
- 15 mL of ammonium molybdate solution
- 30 mL of ascorbic acid solution
NOTE: Mix after the addition of each reagent. All reagents must be at room temperature before they
are mixed, and must be mixed in the order given. If turbidity forms in the combined reagent, shake and
let stand for a few minutes until the turbidity disappears before processing. This volume is sufficient
for 4 hours of operation. Since the stability of this solution is limited, it must be freshly prepared for
each run.
A stable solution can be prepared by not including the ascorbic acid in the combined reagent. If this is
done, the mixed reagent (molybdate, tartrate, and acid) is pumped through the distilled water line and
the ascorbic acid solution (30 mL of 0.1 M ascorbic acid diluted to 100 mL with distilled water) is pumped
through the original mixed reagent line.
6. Sulfuric acid solution, 11N. Slowly add 310 mL concentrated H2SO4 to 600 mL distilled water. When
cool, dilute to 1 L
7. Ammonium persulfate.
8. Acid wash water. Add 40 mL of sulfuric acid solution to 1 L of distilled water and dilute to 2 L.
(Not to be used when only orthophosphate is being determined.)
9. Phenolphthalein indicator solution (5 g/L). Dissolve 0.5 g of phenolphthalein in a solution of 59 mL
of ethyl or isopropyl alcohol and 50 mL of distilled water.
10. Stock phosphorus solution. Dissolve 0.4393 g of pre-
-------
METHOD NO. A-PHOS-2
mL of Standard
Phosphorus Solution A
0.0
2.0
5.0
10.0
Concentration,
mgP/L
0.00
0.02
0.05
0.10
mL of Standard Concentration,
Phosphorus Solution B mg P/L
2.0 0.20
5.0 0.50
8.0 0.80
10.0 1.00
3.3 Equipment/instrument calibration. A calibration curve should be prepared for each day of sample
analysis. Concentrations of the calibration standards should bracket the sample concentrations. If a
sample concentration is outside the range of calibration, then an additional calibration standard should
be analyzed to check if the result is within the linear range of the method. Alternatively, the sample
should be diluted to within the calibration range and reanalyzed.
4. PROCEDURE
4.1 Sample handling and preservation.
4.1.2 Sample collection. Samples may be collected in glass or plastic containers; however, samples
expected to have low concentrations should not be stored in plastic containers, as phosphates may
absorb onto the container walls. Containers should be rinsed with 1N HCI followed by several
rinses with distilled water. Detergents containing phosphate should never be used on containers or
labware that is to be used for phosphate analysis. Sample containers and lids should be rinsed
thoroughly with sample water before sample collection.
If benthic deposits are present in the area being sampled, great care should be taken not to include
these deposits.
4.1.3 Sample preservation. Analysis should be conducted as soon as possible after sample
collection. If samples must be stored for more than 24 h, they should be preserved by the addition
of 2 mL H2SO4 per liter of sample, then refrigerated.
4.2 Interferences.
1. No interference is cause by copper, iron, or silicate at concentrations many times greater than their
reported concentrations in seawater. However, high iron concentrations can cause precipitation of and
subsequent loss of phosphorus.
2. The salt error for samples ranging from 5 to 20 percent salt content was found to be less than 1
percent.
-------
METHOD NO. A-PHOS-2
3. Arsenate is determined similarly to phosphorus and should be considered when present in
concentrations higher than phosphorus. However, at concentrations found in seawater, it does not
interfere.
4. Sample turbidity must be removed by filtration prior to analysis for orthophosphate. Samples for
total or total hydrolyzable phosphorus should be filtered only after digestion. Sample color that absorbs
in the photometric range used for analysis will also interfere.
5. Various components of effluent can interfere with analysis. The method should be reviewed for ways
to remove interferences or adjust for interferences that cannot be removed. Silica and arsenic are
possible interferences, and hexavalent chromium can cause low recovery.
4.3 Sample analysis.
4.3.1 Sample preparation for phosphorus.
1. Add 1 mL sulfuric acid solution to a 50 mL sample and/or standard in a 125 mL Erlenmeyer
flask.
2. Add 0.4 g ammonium persulfate.
3. Boil gently on a pre-heated hot plate for approximately 30 to 40 min or until a final volume of
about 10 mL is reached. Do not allow sample to go to dryness. Alternatively, heat for 30 min in
an autoclave at 121 °C (15 to 20 psi).
4. Cool and dilute the sample to 50 mL If the sample is not clear at this point, filter.
4.3.2 Sample preparation for hydrolyzable phosphorus.
1. Add 1 mL sulfuric acid solution to a 50 mL sample and/or standard in a 125 mL Erlenmeyer
flask.
2. Boil gently on a pre-heated hot plate for approximately 30 to 40 min or until a final volume of
about 10 mL is reached. Do not allow sample to go to dryness. Alternatively, heat for 30 min in
an autoclave at 121 °C (15 to 20 psi).
3. Cool and dilute the sample to 50 mL If the sample is not clear at this point, filter.
4.3.3 Sample preparation for orthophosphata Add 1 drop of phenolphthalein indicator solution to
approximately 50 mL of sample. If a red color develops, add sulfuric acid solution drop-wise to
just discharge the color. Acid samples must be neutralized with 1 N sodium hydroxide (40 g
NaOH/L).
4.3.4 Analysis.
1. Set up the manifold as shown in Figure 1 (AAI) or Figure 2 (AAII).
2. Allow both the colorimeter and recorder to warm up for 30 min. Obtain a stable baseline with
all reagents, feeding distilled water through the sample line.
6
-------
METHOD NO. A-PHOS-2
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GE MIXING
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2.9 SAMPLE
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10.42 MIXEP
REAGENT
2.00 W..TI-
RECORDER
50mm FLOW CELL
650-660 or 880nm FILTER
PHOSPHORUS MANIFOLD AA I
Rgure 1. Manifold for Phosphorus Determination (AAI)
7
-------
METHOD NO. A-PHOS-2
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TO SAMPLER
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i
w/
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COLORIMETER
30mm FLOW CELL
6SO-660 nm or
880nm FILTER
PHOSPHORUS MANIFOLD AAII
Rgure 2. Manifold for Phosphorus Determination (AAII)
8
-------
METHOD NO. A-PHOS-2
3. Place the appropriate standards in the sampler in order of decreasing concentration. Complete
the loading of the sampler tray with unknown samples.
4. For the AAI system, sample at a rate of 20/h, 1 min sample, 2 min wash. For the Mil, use a
30/h, 2:1 cam and a common wash.
5. Switch the sample line from distilled water to sampler and start analysis.
5. DATA QUALJTY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. Duplicate analyses should be conducted on a minimum of 5 percent of the total number
of samples. Criteria for precision of analyses are given in Section 2.4.2.
5.1 Accuracy.
1. An additional 5 percent of the samples should be spiked to check for percent recovery. Criteria for
accuracy of analyses are given in Section 2.4.2.
2. A blank should be analyzed with each batch of samples to demonstrate that the analytical system is
in control.
3. The laboratory should analyze a U.S.EPA performance evaluation sample at least once per quarter.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve by plotting peak heights of processed standards against
known concentrations. Calculate concentrations of samples by comparing sample peak heights with the
standard curve. Any sample whose computed value is less than 5 percent of its immediate predecessor
must be rerun.
6.2 Concentrations of phosphorus in unknown samples are reported in units of mg P/L to a maximum of
three significant figures. Results of all determinations should be reported, including quality assurance
replicate samples and spiked samples. Any factors that may have influenced sample quality should also
be reported.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
-------
METHOD NO. A-PHOS-2
8. REFERENCES
APHA. AWWA, WPCF. 1975. Standard Methods for the Examination of Water and Wastewater. 14th ed.
ASTM. 1976. Annual Book of ASTM Standards. Part 31, "Water," Standard D515-72, p. 388.
Gales, M., Jr., E. Julian, and R. Kroner. 1966. Method for quantitative determination of total phosphorus in
water. Jour. AWWA, 58, No. 10,1362.
Lobring, LB. and R.L Booth. 1972. Evaluation of the AutoAnalyzer II; A progress report. Technicon
International Symposium. New York.
Murphy, J. and J. Riley. 1962. A modified single solution for the determination of phosphate in natural
waters. Anal. Chim. Acta., 27,31.
10
-------
METHOD NO. A-PHOS-3
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Phosphate
1. METHOD TITLE
Automated Method for the Determination of Phosphate
2. BACKGROUND AND APPLICATION
2.1 Source of method. Phosphate Analysis. Method 4.1.6.1. Analytical Methods Manual for Bottom
Sediment Analysis (Draft). U.S. Environmental Protection Agency, Office of Research and Development.
July 1974.
22. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This is a method that can be used for the determination of phosphorus in
marine waters.
Whatever form of phosphate is desired, it is first necessary to separate the constituent of interest
and then convert the phosphorus form to orthophosphate and react it with molybdate to form the
phosphomolybdate complex. Ammonium molybdate and potassium antimony! tartrate react in an acid
medium with dilute solutions of phosphorus to form an antimony-phospho-molybdate complex. This
complex is reduced to an intensely blue-colored complex by ascorbic acid. The color is proportional
to the phosphorus concentration.
The arsenic reducing agent is introduced into the analytical scheme before the introduction of the
working reagent.
The range of detection of phosphate for this method is not given.
2.3.2 Reference to compatible Dimpling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-PHOS-3
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
Technicon AutoAnalyzer II (Industrial system), consisting of the following components:
1. Sampler II or IV and pump IV
2. Analytical cartridge, Technicon part no. 116-D241-01 or equivalent
3. Industrial colorimeter, single channel, equipped with 50 mm tubular flow cell and 880 nm filter
4. Recorder
3.2 This method requires the following reagents:
1. Distilled water. Because of possible contamination, this should be prepared by passage through an
ion-exchange column consisting of a mixture of both strongly acidic cation- and strongly basic anion-
exchange resins. The regeneration of the ion-exchange column should be carried out according to
manufacturer's instructions.
2. Reagents for automated analysis.
NOTE: All reagents should be of reagent grade quality or better, and low in phosphate.
• Sulfuric acid solution-Add, while cooling, 110 mL of concentrated H2SO4 to 800 ml of deionized
distilled water. After solution has cooled, dilute to 1 L with deionized distilled water.
• Ammonium molybdate solution-Dissolve 27 g of ammonium molybdate in 800 mL of deionized
distilled water and dilute to 1 L
• Ascorbic acid solution-Dissolve 10 g of U.S.P. quality ascorbic acid in 800 mL of deionized
distilled water and dilute to 1 L. Keep in a tightly-stoppered container. The ascorbic acid
solution is stable for about 2 months if kept in a freezer or refrigerator; or for about 2 weeks if
kept at room temperature.
• Antimony potassium tartrate solution-Dissolve 3.0 g of antimony potassium tartrate in 800 mL of
deionized distilled water and dilute to 1 L
3. Preparation of combined working reagent-To 100 mL of sulfuric acid solution, add, while stirring,
30 mL of ammonium molybdate solution, 60 mL of ascorbic acid solution, and 10 mL of antimony
potassium tartrate solution. This mixed reagent must be prepared daily and the unused portion must be
discarded.
4. Reagents for arsenic interference.
NOTE: All reagents should be of reagent grade quality or better, and low in phosphate.
• Sodium metabisulfite solution-Dissolve 5.6 g of sodium metabisulfite in 25 mL of distilled water
and dilute to 40 mL
-------
METHOD NO. A-PHOS-3
• Sodium thiosulfite solution-Dissolve 0.56 g of sodium thiosulfate in 25 mL of distilled water and
dilute to 40 mL
5. Mixed reagent. Add, while stirring, 20 mL of sulfuric acid solution to 40 mL of sodium metabisulfite
solution and 40 mL of sodium thiosulfate solution. The mixed reagent is stable for up to 24 hours if
kept refrigerated.
6. Dilute water. Dissolve 5 g of sodium chloride in 800 mL of water, add 2 mL of Levor IV (a wetting
agent marketed by Technicon Corporation under part no. T21-0110), and dilute to 1 L Use this as
dilute water.
7. Digestant solution for total phosphate digestion and total dissolved phosphate. Add 40 g of
ammonium persulfate to 250 mL sulfuric acid solution. This solution must be prepared daily.
8. Stock phosphate solution. Dissolve 0.4393 g of potassium dihydrogen phosphate in 800 mL of distilled
water and dilute to 1 L 1 mL = 0.1 mg P.
9. Standard phosphate solutions. Using the stock phosphate solution, prepare appropriate solutions to
cover the range of analysis of interest.
4. PROCEDURE
4.1 Sample handling and preservation. Samples should be analyzed immediately after collection. The
need for immediate analysis or for rapid preservation of phosphate forms is likely more critical for
phosphorus than any of the other nutrient constituents. Samples may be quick-frozen within 30 min
after collection in a glycol-carbon dioxide bath at -20 °C. Mercuric chloride has also been recommended
as an inhibitor of biological activity.
4.2 Interferences. Silicate, copper, and iron do not interfere when present in concentrations normally
found in seawater. Arsenate interferes on a 1:1 basis. Thus, at normal arsenate and phosphate levels
found in seawater, the effect of arsenate approaches the error in the phosphate analysis. However, at
low phosphate concentrations where specialized procedures may be in use, the arsenate interference in
serious. In fresh water, the procedure of D.L Johnson (see references) is used to eliminate arsenate
interference, however, no studies have been conducted to evaluate this procedure in marine waters.
4.3 Sample analysis.
1. Total phosphate and total dissolved phosphate. Combine 0.5 mL of digestant solution per 10 mL of
sample in a screw-top test tube. Place a screw cap with a Teflon R liner on the test tube and tighten.
Autoclave the samples at 132 °C, then place in a tray for analysis.
The manifold arrangement is shown in Figure 1.
* A blank reading for the seawater of interest is run through the manifold while pumping distilled
water through the manifold in place of the combined working reagent line.
2. Orthophosphate phosphorus. Use the manifold arrangement shown in Figure 2. Samples that are
turbid should be filtered. Determine blank correction for the seawater of interest by substituting
distilled water for the combined working reagent line and pumping samples through.
-------
METHOD NO. A-PHOS-3
HATING
b TH
Total Phosphate in Seawater
After Manual Digestion
Range o-l mg/kg
Tube Size
(Inches)
Air (.3?) Blac
SAMPLER IV
RATE :JQ._ per hour
20 turns
fi5£ar..'.M_ tnnp ftcaa
JG
- -• --Si/
5 turns ^
JCC3 v [ k^'1
1 (i)
0)
•- 'Vi/
Sample (. 10) Orn-Green j
Dilution Water (1.0) Grey
Diluent Water (.16)0 rn-Yel low
Arsenic Reducing reagent (. 13) 0
^ Working Reagent (. 32) Blk-Blk
To Waste
From Flow Cell (1. 20) Yellow
To Sampler Wash fi\ Wash Water (3 90) Pur-Wht
PROPORTIONING PUMP
To Waste
R?
COLORIMETER RECORDER
JQ mm Tubular f/c
880 nm Filters
TOTAL PHOSPHATE IN SEAWATER AFTER MANUAL DIGESTION MANIFOLD
Rgure 1. Manifold for Total Phosphate and Total Dissolved Phosphate Analysis
4
-------
METHOD NO. A-PHOW
Orthophosphate Phosphorus :n Sea water
Range 0-1. Grog/kg
20 turns 10 turns 10 turns 5 turns
_„. T.M3 .. OnM _
MIXER MIXERfMIXER
RT?
MIXER
HEATING BATH
ToWasteJZ[H
tube size
(inches)
AirSlack(.32)
Water
/a ~| Sample" Ycl-Blu (1.4)
wI Rrr:^...i im-1-..j .->-- \or
—@
i
SAMPlEil IV
Rate: 50_per
hour
S Rcducting Reagent Qrn-Grn i 10)
Working Rpagent Blnck (.32)
Red (. 80)
Grcv (10)1 Fron Flow Cell
Pro'porlio'ning
Pump
To Waste
COLORIMETER RECORDER
15mmTubularf/c
880 nm Filters
Notes: Numbers in parentheses Indicate flow rates
1n ml/mln.
* Diluent water contains 250 g of 3.5 N
Sulfurlc Add and 5 g of IUCL per liter.
^^Concentration range can be Increased to
0-5 ppro by appropriate change 1n sample
and diluent water pump tube size and
concentration of acid and salt in diluent
water.
ORTHOPIIOSPIWTE PHOSPHORUS IN SEAUATER KAMI FOLD
Rgure2. Manifold for Orthophosphate Analysis
5
-------
METHOD NO. A-PHOS-3
3. Standards are run before and after each group of samples (a group may vary from 20 to 120
samples). A salt water blank is run every 20th sample.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
Precision and accuracy. No requirements for measurement of precision or accuracy are given for this
method, however, analysts should demonstrate the ability to generate acceptable precision with this method
using replicate sample analyses, and demonstrate acceptable accuracy using laboratory recovery samples and
blank samples.
6. RECORDKEEPINQ AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve by plotting peak heights of processed standards against
known concentrations. Calculate concentrations of samples by comparing sample peak heights with the
standard curve.
6.2 Reporting units. Concentrations of phosphorus in unknown samples are reported in units of
/*g/kg P.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothingi be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
8. REFERENCES
Hager, S.W., et al. 1972. A comparison at sea of manual and autoanalyzer analyses of phosphate, nitrate, and
silicate. Limnology and Oceanography. Vol. 17, pp. 931-937.
Jenkins, D. 1965. A study of methods suitable for the analysis and preservation of phosphorus forms in an
estuarine environment. Report to the USPHS Region IX, WSPC Division, SERL NO. 65-13, College of
Engineering and School of Public Health, University of California.
Johnson, D.L 1971. Simultaneous determination of arsenate and phosphate in natural waters. Environmental
Science and Technology. Vol 5. pp. 411-414.
Murphy, J. and J. Riley. A modified single solution for the determination of phosphate in natural waters.
Anal. Chim. Acta., 27, 31. 1962.
-------
METHOD NO. A-PHOS-3
Strickland, J.D.H., and T.R. Parsons. 1968. A Practical Handbook of Seawater Analysis. Fisheries Research
Board of Canada, Ottawa, Canada.
U.S. Environmental Protection Agency. 1971. Methods for Chemical Analysis of Water and Wastes. U.S.
EPA, NERC. AQCL, Cincinnati, OH. pp. 195-197.
Weber, C.I. 1967. The preservation of plankton grab samples. Water Pollution Surveillance System
Applications and Development Report No. 26. FWPCA. Dept. of the Interior.
-------
METHOD NO. A-PHOS-4
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter: Phosphate
1. METHOD TITLE
Manual Method for the Determination of Phosphate
2. BACKGROUND AND APPLICATION
2.1 Source of method. Phosphate Analysis. Method 4.1.6.2. Analytical Methods Manual for Bottom
Sediment Analysis (Draft). U.S. Environmental Protection Agency, Office of Research and Development.
July 1974.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This is a method that can be used for the determination of phosphorus in
marine waters.
Whatever form of phosphate is desired, it is first necessary to separate the constituent of interest
and then convert the phosphorus form to orthophosphate and react it with molybdate to form the
phosphomolybdate complex. Ammonium molybdate and potassium antimonyl tartrate react in an acid
medium with dilute solutions of phosphorus to form an antimony-phospho-molybdate complex. This
complex is reduced to an intensely blue-colored complex by ascorbic acid. The color is proportional
to the phosphorus concentration.
The range of detection of phosphate for this method is not given.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available.
3. SPECIFICATIONS
3.1 Equipment The equipment needed for this method is presented in Strickland and Parsons (1968).
3.2 Reagents. The reagents needed for this method are presented in Strickland and Parsons (1968).
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-PHOS-4
4. PROCEDURE
4.1 Sample handling and preservation. Samples should be analyzed immediately after collection. The
need for immediate analysis or for rapid preservation of phosphate forms is likely more critical for
phosphorus than any of the other nutrient constituents. Samples may be quick-frozen within 30 min
after collection in a glycol-carbon dioxide bath at -20 °C. Mercuric chloride has also been recommended
as an inhibitor of biological activity.
4.2 Interferences. Silicate, copper, and iron do not interfere when present in concentrations normally
found in seawater. Arsenate interferes on a 1:1 basis. Thus, at normal arsenate and phosphate levels
found in seawater, the effect of arsenate approaches the error in the phosphate analysis. However, at
low phosphate concentrations where specialized procedures may be in use, the arsenate interference in
serious. In fresh water, the procedure of D.L Johnson (see references) is used to eliminate arsenate
interference, however, no studies have been conducted to evaluate this procedure in marine waters.
4.3 Sample analysis. Follow instructions for sample analysis presented in Strickland and Parsons (1968).
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
No requirements for demonstration of precision or accuracy are given for this method. However, analysts
should be able to generate acceptable precision and accuracy data using replicate sample analyses, recovery
analyses, and blank samples. Precision information is given in the study conducted and reported by Hager,
et al. (1972). Hager's data were converted from pM PO4 to ng P and are presented below:
Number of
Replicates
6
5
11
Mean Concentration
(M9/L)
37.8
36.5
30.7
Standard Deviation
(M9/L)
0.6
«• 0.9
0.3
6. RECORDKEEPINQ AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve by plotting peak heights of processed standards against
known concentrations. Calculate concentrations of samples by comparing sample peak heights with the
standard curve.
6.2 Reporting units. Concentrations of phosphorus in unknown samples are reported in units of
P.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
-------
METHOD NO. A-PHOS-4
7.2 Training/level of expertisa Analysts using this method should initially work under the guidance of
an experienced supervisor until he/she can demonstrate proficiency in the laboratory techniques
described in this method.
8. REFERENCES
Hager, S.W., et al. 1972. A comparison at sea of manual and autoanalyzer analyses of phosphate, nitrate, and
silicate. Limnology and Oceanography. Vol. 17, pp. 931-937.
Jenkins, D. 1965. A study of methods suitable for the analysis and preservation of phosphorus forms in an
estuarine environment. Report to the USPHS Region IX, WSPC Division, SERL NO. 65-13, College of
Engineering and School of Public Health, University of California.
Johnson, D.L 1971. Simultaneous determination of arsenate and phosphate in natural waters. Environmental
Science and Technology. Vol 5. pp. 411-414.
Murphy, J. and J. Riley. A modified single solution for the determination of phosphate in natural waters.
Anal. Chim. Acta.,27, 31. 1962.
Strickland, J.D.H., and T.R. Parsons. 1968. A Practical Handbook of Seawater Analysis. Fisheries Research
Board of Canada, Ottawa, Canada.
U.S. Environmental Protection Agency. 1971. Methods for Chemical Analysis of Water and Wastes. U.S.
EPA, NERC, AQCL, Cincinnati, OH. pp. 195-197.
Weber, C.I. 1967. The preservation of plankton grab samples. Water Pollution Surveillance System
Applications and Development Report No. 26. FWPCA. Dept. of the Interior.
-------
METHOD NO. A-PHOS-5
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Orthophosphate
1. METHOD TITLE
Determination of Orthophosphate
2. BACKGROUND AND APPLICATION
2.1 Source of method. D'Elia. C. F.. N. L Kaumeyer, C.W. Keefe. K. V. Wood, C.F. Zimmerman. 1988.
Nutrient Analytical Services Laboratory Standard Operating Procedures. Orthophosphate. Chesapeake
Biological Laboratory (CBL), University of Maryland. Box 38, Solomons, Maryland 20688. Tel.
(301) 326-4281.
This CBL method is based on Technicon Industrial Method No. 155-71WEPA. 1979. USEPA-600/4-79-020.
Method No. 365.1.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. This method for the determination of Orthophosphate in seawater is used by the
Nutrient Analytical Services Laboratory at the Chesapeake Biological Laboratory for analyses
conducted as part of the Chesapeake Bay Program.
Ammonium molybdate and antimony potassium tartrate react in an acid medium to form an
antimony-phosphomolybdate complex that is reduced to an intensely colored complex by ascorbic
acid.
The limit of detection (the lowest concentration of an analyte that the analytical procedure can
reliably detect) is defined as three times the standard deviation of the mean of a minimum of seven
replicate analyses of one sample. At concentrations less than 0.05 mg/L the detection limit for
Orthophosphate is 0.0011 mg/L At concentrations greater than 0.05 mg/L, the detection limit for
Orthophosphate is 0.0014 mg/L
This method requires the use of a segmented continuous flow analyzer, such as the AutoAnalyzer II,
where samples and reagents are continuously added in a specific sequence along a path of glass
tubing and mixing coils. Air bubbles are injected at precise intervals to sweep the walls of the
tubing and to help prevent diffusion between successive samples. The reactions in the
AutoAnalyzer do not develop to completion as in manual methods, by reach identical stages of
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-PHOS-5
development in each sample, because every sample follows the same path, timing, and exposure to
specific reagents.
The basic function of each component of the segmented continuous flow analyzer is briefly
discussed in Section 3.1. The explanation is similar to that of Sanborn and Larrance (1972).
2.3.2 Reference to compatible sampling procedures. Surface, bottom, and water samples form above
and below the pycnocline are collected via a submersible pump system.
2.4 Standardization/validation status of method. Comparability studies using this method with a
seawater matrix have not been cited; this method will be updated to include this information as soon as
data are available.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
Technicon AutoAnalyzer II system, consisting of the following components:
1. Sampler. A sampler probe alternately draws fluid from a tray of discrete samples and then from a
wash-fluid receptacle. The probe dips into the sample to be extracted, and at a timed interval, moves to
a wash solution while a tray of samples advances one position. A bubble of air, which acts as a
diffusion barrier, is aspirated into the sample stream between sample and wash. The ratio of sample to
wash time, as well as the number of samples analyzed per hour, are controlled by a cam located in the
top well of the sampler assembly. Cams are easily changed and are available for a varied range of
sampling rates.
The wash solution separates successive samples by alternating minima (wash) and maxima (sample). The
sample probe is connected to a stream divider that delivers identical samples simultaneously to each
manifold via the pump.
2. Proportioning pump. The proportioning pump is a peristaltic-type pump that continuously delivers
air, reagents, and samples to the manifold. Plastic pump tubes of various diameters are pressed between
a series of moving rollers and a platen. The motion of the rollers along the tubes delivers a continuous
flow. The delivery rate is determined by the inside diameter of the tube, because the rollers move at a
constant rate. These pump tubes are available in a large assortment of delivery rates. The pump will
hold a maximum of 28 tubes and has and air bar that mechanically measures and injects identical air
bubbles into the analytical stream. The pump tubes, which deliver reagents, air, and samples, are
connected to appropriate manifolds.
3. Manifold. Each analysis requires a manifold specifically designed for the chemical method being
used. The manifolds are composed of a series of horizontal glass coils, injection fittings, and heating
baths arranged for the proper sequence of reactions leading to color development. The samples and the
reagents mix within the glass coils. As two solutions with different densities travel around each turn of
the mixing coil, the denser solution falls through the less dense one, causing mixing and resulting in a
homogenous mixture of the two solutions. The length of the coil determines the amount of time allowed
for chemical reaction between the addition of successive reagents. Injection fittings for each of the
reagents are placed between mixing coils. Thus, a sample enters one end of the manifold, a reagent is
added, the solution is mixed and given time to react, and then another reagent is added and mixed.
-------
METHOD NO. A-PHOS-5
After all reagents have been added and an adequate reaction time has passed, the solution flows into a
colorimeter.
4. Colorimeter. The colorimeter measures the absorption of monochromatic light by the solution in the
flow cell. Light from a single source passes through two separate but identical interference filters that
emit light within a narrow spectral band, then through the appropriate flow cell, and finally projects
onto a phototube which generates an electrical signal in response to the intensity of the impinging light.
The output from each phototube is a measure of transmittance arid is converted electronically by the
colorimeter to a signal proportional to absorbance. The relationship between transmittance and
absorbance is given by the equation A = log I/T, where A = absorbance and T = transmittance. The
resulting signal is linear in absorbance and is directly proportional to concentration. As each sample
passes through the cell, the signals are sent to a recorder.
5. Recorder. Results of the analyses are continuously recorded by strip chart recorders. Each recorder
simultaneously monitors two separate analyses. The output of the colorimeter is proportional to
absorbance, and standards of known concentrations must be analyzed to relate absorbance to
concentration on the chart. The analog signals can be converted to absorbance values by referring to
the Technicon reference curve and the standard calibration control.
3.2 This method requires the following reagents:
1. Deionized water. Throughout this method, deionized water is defined as 18.3 megohm water. NOTE:
CBL uses a Barnstead Nanopure II System that produces Type 1 reagent grade water equal to or
exceeding the standards established by ASTM. Water is first filtered through a string prefilter and then
goes through a reverse-osmosis membrane. Final product water then passes through a series of five
filters (organic colloid, two mixed-bed cartridges, organic-free cartridge, and a 0.3 /*m final filter).
2. Sulfuric acid (4.9 N). Add 136 ml concentrated sulfuric acid (H2SO4; sp. gr. 1.84) to approximately
800 ml deionized water, while cooling the solution in a cold water bath. After solution has cooled,
dilute to 1 L with deionized water.
3. Ammonium molybdate. Dissolve 40 g ammonium molybdate [NH4)eMo7O24-4H20] in approximately 800
mL of deionized water. Dilute to 1 L with deionized water. Store in plastic bottles away from direct
sunlight.
4. Ascorbic acid. Dissolve 18 g of ascorbic acid in approximately 800 mL of deionized water. Dilute to
1 L with deionized water and dispense approximately 40 mL into clean polybottles and freeze. Thaw
overnight in the refrigerator before use.
5. Antimony potassium tartrate. Dissolve 3.0 g antimony potassium tartrate [(K(SbO)C4H4O6-|H2O] in
approximately 800 mL of deionized water. Dilute to 1 L with deionized water.
6. Sodium lauryl suifate. Dissolve 3.0 g sodium lauryl sulfate (sodium dodecyl sulfate, M.W. = 288.38;
phosphate < 0.0001 percent) in approximately 80 mL of deionized water. Dilute to 100 mL with deionized
water.
7. Working reagent A.
50 mL Sulfuric acid (4.9N)
-------
METHOD NO. A-PHOS-5
15mL Ammonium molybdate solution
5 mL Antimony potassium tartrate solution
1 mL Sodium lauryl sulfate solution
Combine all reagents.
8. Stock standard. Dissolve 1 .632 g KH2PO4 in 1 L of deionized water (1 ml = 1 2 fig-at P). Add 1 .0
mL chloroform to act as a preservative. As a general rule, stock standards should be prepared every 6
months, and the preparation date logged.
9. Secondary standard. Dilute 1 .0 mL of stock standard and dilute to 100 mL with deionized water
(1 mL = 0.12/jg-atP).
10. Working standards. Prepare working standards daily. Concentrations of standards should encompass
the range of the samples.
0.1 mL of secondary standard diluted to 100 mL with deionized water will yield 0.12 /*g-at P/L
(0.00372 mg P/L).
0.25 mL of secondary standard diluted to 100 mL with deionized water will yield 0.3 /*g-at P/L
(0.0093 mg P/L).
0.5 mL of secondary standard diluted to 100 mL with deionized water will yield 0.6 pg-at P/L
(0.0186 mg P/L).
2.5 mL of secondary standard diluted to 1 00 mL with deionized water will yield 1 .2 pg-at P/L
(0.0372 mg P/L).
5 mL of secondary standard diluted to 100 mL with deionized water will yield 6.0 ^g-at P/L
(0.186 mg P/L).
3.3 Equipment/instrument calibration. The AutoAnalyzer is calibrated with each run. Refer to Section
4.2.3 for calibration procedures, and Section 6.2 for calculations.
4. PROCEDURE
4.1 Sample handling and preservation. After collection, water samples are filtered through GF/F filters
(nominal pore size 0.7 /*m) and are placed in either polypropylene bottles or directly into 4 mL
Autoanalyzer cups. The samples are then stored frozen at >20 °C until analysis (up to 28 days).
4.2 Interferences. Silicon at a level of 100 /*g-at Si/L causes and interference equivalent to
approximately 0.04 /ig-at P/L.
4.3 Sample analysis.
4.3.1 Glassware. Prior to use, wash all glassware with 1 N HCI followed by numerous rinses with
deionized water.
-------
METHOD NO. A-PHOS-5
4.3.2 Instrument specifications.
1. Manifold assembly. Refer to Figure 1 for manifold diagram
2. Standard calibration settings. 9.0,6.0, and 3.0
3. Damp. Normal
4. Sampling rate. 40 samples/h; 9:1 sample/wash ratio
5. Filter. 880 nm
6. Phototube. 199-B021-04
7. Flowcell. 50mm
4.3.3 Operating procedures.
1. Colorimeter. Turn the power on and allow lOmin for warm-up. Check standard calibration
setting for the desired determination.
2. Recorder. Turn the power on and allow 10 min for warm-up; check recorder paper supply.
3. Water reservoirs. Check and fill the deionized water reservoirs.
4. Pump tubes. Connect pump tubes and attach platen to pump.
5. Pump. Start the pump with deionized water flowing through the system. Check for leaks in
tubes at the connections and for a regular bubble pattern in the manifold.
7. Recorder. Turn the recorders on (chart paper should start moving).
8. Colorimeter. Check ZERO and FULL SCALE on the recorder. ZERO simulates a zero output so
that ZERO adjustment of the recorder can be made with a screwdriver.
9. Baseline control. With deionized water pumping through the system, establish a zero baseline
using the BASELINE CONTROL adjustment at a STD. CAL of 1.0.
10. Reagent blank. Allow reagents to pump through; note any rise in the baseline and readjust to
zero. Refer to this rise as the REAGENT BLANK (at a STD. CAL of 1.0).
11. Standard calibration control. An extremely wide range of nutrient concentrations, both
temporally and spatially, are found in estuarine and marine waters. The standard calibration
control setting (STD. CAL), located on the colorimeter, allows the operator to adjust the electrical
output to the concentration range of the standards or samples. Extremely low concentrations
(ng/L) require high STD. CAL settings, or high sensitivity, whereas high concentrations (mg/L)
require lower STD. CAL settings, or lower sensitivity.
-------
HAOTPOU) COWIGQMXION TOR PHOSPHME
To Sampler Wash Receptacle-
5 Turn
Heating
Bath
COLORIMETER
880 run filters
50 mm F/C x 1.5 irm ID
199-B021-04 Phototube
5 Turns
Waste
GRN/GRN (Water)
BLK/BLK (Air)
YEI/YEL (Sanple)
ORN/WHT (Reagent A)
ORN/GRN (Reagent B)
WOT/WHT (From F/C)
METHOD NO. A-PHOS-5
Rgurel. Manifold for Orthophosphate Analysis
6
-------
METHOD NO. A-PHOS-5
If a calibration curve encompassing a wide range of concentrations is necessary to analyze samples
that would otherwise go off scale, run all calibration standards at STD. CAL settings 9.0,6.0, and
3.0. (There should be no deflection of the pen at zero baseline if the STD. CAL setting is
switched back to 1.0.) Record the peak heights of standards at the various STD. CAL settings,
along with the STD. CAL settings.
13. Sample analysis. After the initial run of calibration standards, intersperse standards in the run
after approximately every 20 samples. Include at least one standard analyzed at each STD. CAL.
setting employed during analysis of the preceding 40 samples. A visual comparison with the day's
initial standard curve should indicate no greater variance than 5 percent of the peak height (e.g., if
the initial standard peak height is 60.0, subsequent standards may vary from 57.0 to 63.0). If the
variance is greater than 5 percent, the source of the problem must be identified and corrected, and
the affected samples must be reanalyzed. The baseline should be adjusted after approximately every
20 samples. If an adjustment of more than 1 unit is required, the source of the problem must be
identified and corrected, and the affected samples must be reanalyzed.
4.3.4 Shutdown procedure.
1. At the end of the run, disconnect the reagents and place the tubes in distilled water.
2. Turn off recorder.
3. Wash the system with 1N HCI for 15 min; place the pump tubes in deionized water and wash
with deionized water for an additional 15 min.
4. Turn off the pump, release the proportioning platen, and loosen the pump tubes.
5. Turn off the colorimeter.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
5.1 Precision. Precision of this method for orthophosphate analysis is demonstrated by analysis of
laboratory duplicates. A total of four duplicates are analyzed at random per batch of samples collected
(or per cruise). Duplicate analyses are performed during the course of a run. After a sample is
analyzed, the same sample cup is removed from its position in the tray, and placed further along the
sample tray to be reanalyzed. The mean of the two values is reported as the concentration of that
sample.
Results of the duplicate analyses are placed in a separate QA/QC data file along with the sample
number, sample date, and analysis date.
5.2 Accuracy. Accuracy of this method for orthophosphate analysis is demonstrated by analysis
laboratory spiked samples. A total of four spikes are analyzed at random per batch of samples collected
(or per cruise)-one each for the first and third sample collection days, and two for the second day. A
spike is prepared by adding a known volume of standard to a known volume of sample. This sample is
then analyzed and calculated as if it were a normal sample. A comparison is then made of the
determined value of the spiked sample and its expected value (calculated as the original sample
concentration plus the concentration of the spike).
-------
METHOD NO. A-PHOS-5
These three concentrations (original, determined, and expected) are placed in a separate QA/QC data file
along with the sample number, sample date, and analysis data.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations. Prepare a standard curve in which the concentrations of the standards are entered
as the independent variables, and their corresponding peak heights are the dependent variable.
Concentrations of orthophosphate in samples are calculated from the linear regression obtained from the
standard curve. Concentrations of orthophosphate are reported in units of mg P/L
When a broad range of sample concentrations requires that several standard calibration settings be
employed during a run, a separate regression must be determined for calculating concentrations from
peak heights read at each standard calibration setting. All standards analyzed during the run at a
particular STD. CAL setting are included in the calculations for that regression; and only samples whose
peak heights were measured at the same STD. CAL setting are calculated using that regression. For
example, peak heights taken from standards analyzed at STD. CAL 1.0 are used to determine the linear
regression at STD. CAL 1.0, and only concentrations of samples analyzed at STD. CAL 1.0 are
calculated using the regression at STD. CAL 1.0.
6.2 Data handling procedures.
1. The data are input to a predetermined format onto floppy disks via LOTUS 1-2-3 and a Compaq 386
microcomputer.
2. Printouts of the data are then verified by laboratory personnel, corrections are made, and all files
are sorted by date and sample number in ascending order.
3. Print files are created from the LOTUS files.
4. If more than one data set is collected per month, the data are combined and sorted by date.
6.3 Data reporting. All analysis documents are kept in bound notebooks with a carbon copy given to
the investigator or granting agency. Information includes the name of analysis, collection date, source
of samples, analyst, analysis date, sample number, peak height, STD. CAL setting, sample concentration,
standard concentrations, standard peak heights, standard peak heights interspersed through the run,
regression statistics, results of duplicate analyses, results of spike analyses, and reagent blank readings.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the Technicon AutoAnalyzer. Personnel conducting this analysis should initially work
under the guidance of an experienced supervisor until he/she can demonstrate proficiency in the
laboratory techniques described in this method.
8
-------
METHOD NO. A-PHOS-5
8. REFERENCES
Sanborn, H. and J. Larrance. 1972. An operations manual of the AutoAnalyzer for seawater nutrient
analysis. NOAA/NMFS, Seattle, Washington. 44 pp.
-------
METHOD NO. A-CHLOR-1
INDEX INFORMATION
Matrices: Seawater, waste
Categories: Estuarine and marine seawater and sea-surface microlayer, wastewater/effluents
Parameter. Chlorophyll
1. METHOD TITLE
Fluorometric Determination of Chlorophyll a
2. BACKGROUND AND APPLICATION
2.1 Source of method. Fluorometric Determination of Chlorophyll a. Method 1002.G.2. In: Standard
Methods for the Examination of Water and Wastewater, 16th Edition. 1985. APHA, AWWA, WPCF. p.
1071-1072.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. The fluorometric method for chlorophyll a is more sensitive than the
spectrophotometric method and thus smaller samples can be used. Phaeophytin a also can be
determined fluorometrically.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method. Comparability studies using this method with a sea
water matrix have not been cited; this method will be updated to include this information as soon as
data are available.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. Fluorometer (Model 111. Sequoia-Turner Corp.). equipped with a high-intensity F4T.5 blue lamp,
photomultiplier tube R-446 (red-sensitive), sliding window orifices 1X, 3X, 10X, and SOX. and filters for
light emission (CS-2-64) and excitation (CS-5-60). A high sensitivity door is preferable.
2. Spectrophotometer, with a narrow band (pass) width (0.5 to 2.0 nm) because the chlorophyll
absorption peak is relatively narrow. At a spectral band width of 20 nm the chlorophyll a concentration
may be underestimated by as much as 40 percent.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-CHLOR-1
3. Cuvettes, with 1-. 4-, and 10-cm path lengths.
4. Clinical centrifuge.
5. Pipets, 0.1 and 5.0 ml_
6. Tissue grinder (Kontes Glass Co.. Glass/glass grinder model 8855; glass/TFE grinder model 886000; or
equivalent). Successfully macerating glass fiber filters in tissue grinders with grinding tube and pestle
of conical design may be difficult. Preferably use round-bottom grinding tubes with a matching pestle
having grooves in the TFE tip.
7. Centrifuge tubes, 15-mL graduated, screw cap.
8. Filtration equipment, filters, glass fiber (GF/C or GF/A 4.5-cm diameter, or equivalent) membrane
(0.45-/im porosity, 47-mm diameter); vacuum pump; solvent-resistant disposable filter assembly, 1.0-^m
pore size (Gelman Acrodisc or equivalent); 10-mL solvent-resistant syringe.
3.2 This procedure requires the following reagents.
1. Saturated magnesium carbonate solution. Add 1.0 g finely powdered MgCOs to 100 mL distilled
water.
2. Aqueous acetone solution. Mix 90 parts acetone (reagent grade BP 56° C) with 10 parts saturated
magnesium carbonate solution.
3. Chlorophyll extract. Pure chlorophyll a, or a plankton chlorophyll extract with a spectrophotometric
before-and-after acidification ratio of 1.70 containing no chlorophyll b.
3.3 Equipment/instrumentation calibration.
1. Calibrate the fluorometer spectrophotometrically with a sample from the same source to achieve
acceptable results. Optimum sensitivity for chlorophyll a extract measurements is obtained at an
excitation wavelength of 430 nm and an emission wavelength of 663 nm. (A method for the continuous
measurement of chlorophyll a is available, but is reported to be less efficient than the in-vitro method
given here, yielding about one-tenth as much fluorescence per unit weight as the same amount in
solution.)
2. Calibrate the fluorometer with a chlorophyll solution of known concentration prepared as follows.
Prepare chlorophyll extract and analyze spectrophotometrically. Prepare serial dilutions of the extract
to provide concentrations of approximately 2,6,20, and 60 /*g chlorophyll a/L Make fluorometric
readings for each solution at each sensitivity setting (sliding window orifice): 1X, 3X, 10X, and 30X.
Using the values obtained, derive calibration factors to convert fluorometric readings in each sensitivity
level to concentrations of chlorophyll a, presented in Section 6.1.1.
3. Determine extract fluorescence at each sensitivity setting before and after acidification.
-------
METHOD NO. A-CHLOR-1
4. PROCEDURE
4.1 Sample handling and preservation. Whole water samples may be stored up to 2 " eeks in the dark
at 4°C. Use opaque bottles because even brief exposure to light during storage will alter chlorophyll
values. Samples on filters collected form water having pH 7 or higher may be placed in airtight plastic
bags and stored frozen for 3 weeks. Samples from acidic water must be processed promptly to prevent
chlorophyll degradation. Use glassware and cuvettes that are clean and acid-free.
4.2 Sample analysis.
1. Place the sample (the filter) in a tissue grinder, cover with 2 to 3 mL 90 percent aqueous acetone
solution, and macerate at 500 rpm for 1 min. Use TFE/glass grinder for a glass-fiber filter and
glass/glass for a membrane filter.
2. Transfer the sample to screw-cap centrifuge tube, rinse grinder with a few milliliters 90 percent
acetone, and add the rinse to the extraction slurry. Adjust total volume to a constant level, 5 to
10 mL, with 90 percent aqueous acetone. Use solvent sparingly and avoid excessive dilution of pigments.
Steep the samples at least 2 h at 4°C in the dark.
3. Clarify the filtering through a solvent-resistant disposable filter (to minimize retention of extract in.
filter and filter holder, force 1 to 2 mL air through the filter after the extract), or by centrifuging in
closed tubes for 20 min at 500 g. Decant clarified extract into a clean, calibrated, 15-mL, screw-cap
centrifuge tube and measure total volume.
4. Calibrate the fluorometer with a chlorophyll solution of known concentration (see Section 3.3.2).
Determine extract fluorescence at each sensitivity setting before and after acidification. Calcu ate
calibration factors and before-and-after acidification fluorescence ratio by dividing fluorescence reading
obtained before acidification by the reading obtained after acidification. Avoid readings on the 1X
scale and those outside the range of 20 to 80 fluorometric units.
5. Measure sample fluorescence at sensitivity settings that will provide a mid-scale reading. Avoid
using the 1X window because of quenching effects). Convert fluorescence readings to concentrations of
chlorophyll a by multiplying the readings by the appropriate calibration factor.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
No requirements for demonstration of precision or accuracy are given in this method.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations.
1. Calibration factors.
Fs = Ca'/Rs
where FS =calibration factor for sensitivity settings
Ca' = concentration of chlorophyll a determined spectrophotometrically (nQ/L)
-------
METHOD NO. A-CHLOR-1
RS =fluorometer reading for sensitivity setting S
2. Corrected chlorophyll a and phaeophytin a
Ca = Fs[r/(r-1)](Rb-Ra)
Pa = Fs[r/(r-1)](rRa-Rb)
where Ca = concentration of chlorophyll a (mg/m3)
Pa = concentration of phaeophytin a (mg/m3)
FS =conversion factor for sensitivity setting S
(see Section 6.1.1)
r =Rb/Ra. as determined with pure chlorophyll a for the instrument. Redetermine r if
filters or light source are changed.
RO =fluorescence of extract before acidification
Ra ^fluorescence of extract after acidification
6.2 Reporting units. Chlorophyll a and phaeophytin a concentrations will be reported in mg/m3.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions; wear protective eyewear and clothing, be familiar with laboratory safety devices, understand
the proper handling of acids, solvents, and other reagents.
72. Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of the equipment. Personnel conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency in the laboratory
techniques described in this method.
8. REFERENCES
APHA, AWWA, and WPCF. 1985. Standard Methods for the Examination of Water and Wastewater, 16th
Edition. APHA. Washington, D.C. p. 1071-1072.
-------
METHOD NO. A-CHLOR-2
INDEX INFORMATION
Matrices: Seawater, waste
Categories: Estuarine and marine seawater and sea-surface microlayer, wastewater/effluents
Parameter: Pigments-Chlorophylls a, b, and c and total carotenoids
1. METHOD TTTIJE
Spectrophotometric Determination of Chlorophylls and Total Carotenoids
2. BACKGROUND AND APPLICATION
2.1 Source of method.1 Spectrophotometric Determination of Chlorophylls and Total Carotenoids.
Method IV.3.1 and Addendum. In: J.D.H Strickland and T.R. Parsons. 1972. A Practical Handbook of
Seawater Analysis. Fisheries Research Board of Canada, Ottawa.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. At present, the only rapid chemical method known for estimating living plant
matter in the particulate organic matter of seawater is to determine the characteristic plant
pigments-the chlorophylls, carotenes, and xanthophylls. The amount of organic substance
associated with a given quantity of plant pigment is very variable, depending upon the class of the
phytoplankter and its state of nutrition. (The factor of converting chlorophyll a to total plant
carbon can vary between about 25 and 100). The method described herein determines three
chlorophylls (a, b, and c) commonly found in planktonic algae. The carotenoid pigments (carotenes
and xanthophylls) are estimated collectively in somewhat arbitrary units. If the plant population
contains many myxophyceae some forms of phycobilin pigments may extract and interfere with all
determinations except that of chlorophyll a. Fortunately this rarely occurs in truly marine waters.
Chlorophyll degradation products may at times constitute a significant fraction of the total green
pigments in seawater. These degraded forms of inactive chlorophyll interfere with the
Spectrophotometric determination of chlorophylls because they absorb light in the same region of
the spectrum as chlorophyll. Pigment samples from the aphotic zone, sediments, and samples from
areas of high zooplankton grazing are particularly likely to contain inactive chlorophyll products.
Chemically these may consist primarily of phaeophytin and phaeophorbide (phaeo-pigments) but
sometimes large quantities of chlorophyllide may also be present. This method includes procedures
for measuring the total quantity of chlorophyll a and phaeophytin a, but not of chlorophyllide a or
the phaeophytins and phaeophorides of other chlorophylls. For complete analysis of all chlorophylls
and their degradation products there is probably no alternative to chromatographic methods which
are generally too tedious for the routine analysis of a large number of samples. For a routine
observation, however, it is often sufficient to obtain a measure of the amount of non-active
chlorophyll a in terms of the quantity of phaeo-pigments present.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-CHLOR-2
The sensitivity of this method is adequate except where sample volumes are restricted or where the
chlorophyll content of the water is below approximately 0.2 mg/m3. The precision decreases
significantly with concentrations below this level, becoming very poor if less than 0.1 mg/m3 is
present. Under these conditions a fluorometric determination is recommended.
Whole seawater is filtered through a 300-/*m mesh net to remove all large zooplankters. The
remaining filtered sample is filtered through a Millipore AA filter or glass filter. Pigments are
extracted from the algal cells for spectrophotometric analysis. To determine the concentration of
phaeophytin pigments, the extinction of an acetone extract if plant pigment is measured before and
after the addition of dilute acid. The change following acidification is used as a measure of the
quantity of phaeo-pigments in the original sample.
The detection limit of plant pigments in sea water is difficult to present because an unlimited
volume of water may be filtered for analysis, although typically the volume does not exceed 10 L
The lower limit of detection for the filtration of 10 L has not been statistically determined but
may be on the order of 0.02 mg/m3 for chlorophyll a and 0.04 mg/m3 for most other pigments
except chlorophyll c. For phaeophytin a determinations, the limit of detection will depend on the
total amount of sea water filtered but for all measurements the initial extinction at 6650 A should
be greater than 0.2.
232. Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method. The sample is generally 500 mL to 5 L
in volume and filtered through a clean 0.3-mm mesh netting to remove larger zooplankton. For
open ocean samples, filtration of small volumes through a 0.15-mm mesh net will not retain
significant amounts of phytoplankton.
2.4 Standardization/validation status of method.
2.4.1. Comparability studies. Comparability studies using this method with a sea water matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
2.4.2. Precision. At the 5 /ig level, the correct value for chlorophyll a is in the range of the
mean of n determinations ± 0.26/n V2^g chlorophyll a. At the 0.5 /*g level, the correct value for
chlorophyll b is in the range of the mean of n determinations ± 0.21/n V2^g chlorophyll b. At the
1.5 /i-SPU level, the correct value for plant carotenoids is in the range of the mean of n
determinations ± 0.15/nV2^-SPU. the precision of chlorophyll c determinations is variable and very
poor, anywhere between ±10 and ±30 percent of the amount being measured. The results are not
accurate and are nearly always too high. For the determination of phaeophytin a, the precision at
the 0.5 /*g level lies in the range of the mean of n determinations ± 0.05/nV2^g phaeophytin a.
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. Millipore filtration unit designed to hold 47-mm membrane filters.
2. 300-mL polyethylene wash bottle.
-------
METHOD NO. A-CHLOR-2
3. 15-mL graduated centrifuge tubes with both glass and polyethylene stoppers.
4. Small volume (< 10 mL) spectrophotometer cells with path length of 10 cm.
5. Spectrophotometer.
3.2 This procedure requires the following reagents.
1. Redistilled acetone. Distill reagent grade acetone over approximately 1 percent of its weight of both
anhydrous sodium carbonate and anhydrous sodium sulphite. Collect the fraction boiling at a constant
temperature near 56.5°C (uncorrected). Pipet 100 mL of water into a 1000-mL volumetric flask and add
acetone to a volume of exactly 1000 mL Store the redistilled acetone in a tightly stoppered dark glass
bottle and store the 90 percent reagent in moderately small amounts (approximately 1-L at a time) for
use. This reagent is conveniently dispensed from a polyethylene wash bottle which should be kept
nearly full. If good quality reagent acetone is available, it should be shaken with a little granular
anhydrous sodium carbonate and decanted directly for use.
2. Magnesium carbonate suspension. Add approximately 1 g of finely powdered magnesium carbonate
(light weight or "Levis" grade) of analytical reagent quality to 100 mL of distilled water in a stoppered
Erlenmeyer flask. Shake vigorously to suspend the powder immediately before use.
3. Hydrochloric acid. Dilute 50 mL of concentrated hydrochloric acid to 100 mL with distilled water.
3.3 Equipment/instrument calibration.
The wavelength setting of the spectrophotometer used should be checked against a standard hydrogen
or neon line source because the precision of the present method depends upon settings being correct to
better than 20 to 30 A. With quartz prisms at wavelength exceeding 6000 X, very slight movements of
the optical system (brought about by vibrations, etc.) can easily result in errors of 50 A or more in
wavelength settings. If a suitable lamp is not available check the extinction of a suitably concentrated
plant extract and adjust the spectrophotometer until a maximum extinction is obtained at 6300 A.
4. PROCEDURE
4.1 Sample handling and preservation. The required volume of sample is measured using a polyethylene
measuring cylinder into a polyethylene bottle. Two or three drops (ca. 0.1 to 0.2 mL) of magnesium
carbonate suspension (see Section 3.2.2) are added. The sample may then be stored in a cool dark place
for a maximum of about 8 h. It is desirable, however, that samples be filtered through a membrane
filter at the time of collection. Membrane filters can be stored by folding them in half (with the
plankton innermost) and storing them in the dark in a desiccator frozen to -20°C for no more than a
few weeks. This procedure nearly always leads to low results and makes the extraction of chlorophyll
more difficult; filters should be extracted without delay if at all possible.
4.2 Interferences.
Filters. Millipore filters have the advantage that they dissolve in acetone completely, give no
complications at the centrifugation stage, and require no particular precautions during filtration.
However, unless great care is taken, undesirably high blanks will occur when using Millipore filters,
making determination of small concentrations of carotenoids difficult. These filters are expensive. Glass
-------
METHOD NO. A-CHLOR-2
filters are less expensive and their use results in practically no blank interference. They are
recommended if a cell grinding step is required to give better extraction, although care must be taken
when filtering samples through relatively coarse glass filters and trouble is experienced at the
centrifugation stage. A manostat (there are several inexpensive commercial laboratory units based on
the cartesian diver) must be used with glass papers to ensure that the suction never exceeds 1/4 to 1/3
atm. otherwise pigment may pass through the filter. Millipore filters must be used if chlorophyll c is to
be determined on the same extract by the method for determination of chlorophyll c.
4.3 Sample analysis.
1. Place either a either a 47-mm diameter Millipore AA filter or a 4.5 cm Whatman GF/C glass filter
paper onto the base of the Millipore filter apparatus and secure the funnel. Care should be taken that
the Millipore filtration equipment, centrifuge tubes, and spectrophotometer cells are kept free from acid
and that the filter is not touched with acidic fingers. NOTE: Each filter type has advantages,
disadvantages, and special precautions associated with their use. See Section 4.2.1 for more details on
choosing the appropriate filter.
2. Vigorously shake the polyethylene sample bottle and invert the bottle into the funnel; the bottle
need not be rinsed if vigorously shaken first. If not added previously, add 1 ml of magnesium carbonate
suspension to the last few hundred milliliters of sample as the sample is filtered. NOTE: The
magnesium carbonate is added at this stage to prevent decomposition of the phytoplankton chlorophyll
pigments into phaeophytin pigments. Strickland and Parsons express some doubt as to the efficacy of
such an addition compared with the addition of a completely soluble organic base, but they acknowledge
the established practice and assume the addition has some value as a precautionary measure.
3. Drain the filter completely under suction before removing it from the filtration equipment and, if a
Millipore filter is used, trim the peripheral excess of unstained membrane with clean scissors. NOTE:
The troublesome blank, measured at 7500 A, found with Millipore filters is caused almost entirely by the
salt left in the filter at this stage, which subsequently "salts out" membrane material from the acetone.
The blank can be greatly reduced if filters are very thoroughly sucked dry of sea water at this stage
and the unwanted peripheral filter is removed.
4. If possible, immediately extract the pigment. If the sample must be stored, store according to
procedure described in Section 4.1.
5. Place the filter in a 15-mL stoppered graduated centrifuge tube. If a Millipore filter is used, add
approximately 8 mL of 90 percent acetone, stopper the tube, and dissolve the filter by vigorously
shaking the tube. If a glass paper is used, add approximately 10 mL of 90 percent acetone, stopper the
tube, and disperse and disintegrate the filter by vigorously shaking the tube. NOTE: If poor extraction
is anticipated, use a glass filter. After filtration insert the filter into the bottom of a 20-mL "Potter"-
type grinder. Add approximately 2 mL of acetone and grind for 1 to 2 min in subdued light. Press the
pestle hard against the bottom of the tube and occasionally push the tube up and down the pestle during
the extraction. After use, rinse the pestle into the tube with a few milliliters of 90 percent acetone,
and, using 90 percent acetone, transfer the contents of the grinder tube into a 15-mL centrifuge tube
(the total volume in the centrifuge tube should not exceed 10 mL). Place the centrifuge tube in the
dark for a few hours to ensure complete removal of all extractable pigments.
6. Allow the pigments to be extracted by placing the tube in a refrigerator in complete darkness for
approximately 20 h (vigorously shake the tubes" after they have been in the refrigerator 1 or 2 h).
Pigments are very photosensitive during the extraction process and neither extracts or unextracted
-------
METHOD NO. A-CHLOR-2
filters should be exposed to strong sunlight. Exposure to strong light will reduce chlorophyll values to a
small fraction of their initial level in less than 1 h. NOTE: The period of extraction should be
approximately 15 to 20 h. After this time, the rate of further extraction is very slow and additional
extraction time has little merit. Pigment extracts should preferably be kept chilled but they can be kept
at room temperature for many hours without deterioration. If cells are pretreated in a grinder (see
Section 4.3.5 NOTE), any further extraction is slow and tubes should be stored for a few hours to
complete leaching of cell fragments.
7. Remove the tubes from the refrigerator and let them warm up in the dark to nearly room
temperature. Add 90 percent acetone to make the extracts from the Millipore filters up to exactly 10.0
mL and those from the glass filters to exactly 12.0 ml_ NOTE: The use of 10 mL of solution in a 10-
cm-path-length cell is recommended for maximum sensitivity. Greater sensitivity can be obtained by
using 10-cm cells containing less than 10 mL but this is scarcely great enough to warrant the increased
difficulties in manipulation. The ultimate sensitivity is, in practice, more dependent on the size and
reproducibility of blanks. Glass filters disintegrate to pulp instead of dissolving in acetone, and the pulp
retains at least 1 mL of acetone. To ensure enough extract to fill a 10-cm cell, therefore, 12 mL of
acetone instead of 10 mL should be used.
8. Replace the glass stoppers on the centrifuge tubes with plastic stoppers and centrifuge the contents
of the tubes for 5 to 10 min. NOTE: Centrifugation should be as complete as possible when Millipore
filters are used. In most small centrifuges, centrifugation at 3000 to 400 rpm for 10 min is usually
satisfactory, but the efficiency of each instrument should be tested. Difficulties may be encountered
when centrifuging down the glass pulp from glass filters. Tubes should be centrifuged for 1 to 2 min to
pack most of the fibers to the bottom. The centrifuge is then stopped, the tubes removed, and glass
fibers adhering to the walls of the tubes above the level of the solvent are taken down into the bulk of
the liquid by gently splashing the walls by flicking the tubes. The tubes are then returned to the
centrifuge and spun for approximately 5 min. If this precaution is not taken, some fibers held above the
solvent layer may enter the spectrophotometer cell.
9. Decant the clear supernatant liquid into a 10-cm-path-length spectrophotometer cell designed to hold
10 mL or less of liquid. In the event of extinction values exceeding about 1.3 the measurements
described below should be made with 2.5-cm or 1-cm cells and the extinction values multiplied by 4 or
10, respectively, to normalize them to the values expected with a 10-cm cell. If 12 mL of acetone is
used with glass papers multiply the extinction values by 1.2 to normalize them to the values expected
from 10 mL of extract.
10. Without delay, measure the extinction of the solution against a cell containing 90 percent acetone,
at wavelengths of 7500,6650,6450,6300, and 4800 X. These extracts should not be allowed to
evaporate and should be exposed only to subdued light for the briefest possible time. The measurement
of extinction against acetone (instead of against water) is recommended because acetone has markedly
less absorption in a 10-cm cell at 7500 X than has distilled water. If the Richards equations are to be
used for carotenoids, a further measurement at 5100 X is required. If the SCOR/UNESCO equations are
used the measurement at 6650 X should be replaced with one at 6630 X. Record the extinction values to
the nearest 0.001 unit in the range 0-0.4 and the nearest 0.005 for extinctions exceeding about 0.4.
Correct the extinctions at each wavelength by the procedure described in Section 4.4.
11. If phaeo-pigment concentrations are to be determined, add two drops of dilute hydrochloric acid to
the cuvette, mix thoroughly, and remeasure the extinction at 6650 and 7500 X. All samples are best
mixed by holding a small piece of aluminum foil over the mouths of the cuvettes and inverting them
several times. The destruction of chlorophyll a to phaeophytin is not instantaneous and the sample
-------
METHOD NO. A-CHLOR-2
should be allowed to stand for 4 to 5 min before being measured again. Subtract each 7500 X reading
from the corresponding 6650 X reading and use the equation in Section 6.1.3 to calculate the
concentration of chlorophyll a and phaeo-pigments in the sample. Rinse the cuvette thoroughly with 90 .
percent acetone after each determination to ensure that no acid is carried over to the next sample.
NOTE: If the extraction of pigment was made using acetone-soluble filters, the addition of acid will
cause a transient turbidity which disappears on mixing.
12. Calculate the concentration of pigments in sea water from the equation given in Section 6.1.2.
5. DATA QUAUTY REQUIREMENTS AND ASSESSMENTS
5,1 Cell-to-cell blanks. As the precise values of comparatively small extinctions have to be measured,
corrections for all optical inequalities become important. Fill both spectrophotometer cells with 90
percent and find the "cell-to-cell" blank of the sample cell against the reference cell at all wavelengths
used in the method. Correct all extinction values by this cell-to-cell blank, which may amount to 0.01
or more.
5.2 Turbidity blanks. If glass papers are used there should be only a very small blank value measured
by the spectrophotometer reading at 7500 X where there is known to be no absorption of light from
pigments. Strickland and Parsons sometimes found a small negative blank for reasons which are not
clear. In any case, the value positive or negative should not exceed about 0.002 and may be corrected
for cell-to-cell blank and used for the extinctions at all wavelengths.
A certain amount of colloidal material remains after the solution of an AA Millipore filter, even after
centrifugation. The extinction from this material depends on the wavelength of light used, increasing at
shorter wavelengths because of light scattering effects.
The extinction at 7500 X is corrected for any cell-to-cell blank at this wavelength and the resulting
extinction is multiplied by a factor f to give the turbidity blank extinction to be used with
spectrophotometer readings at other wavelengths (see Section 6.1).
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations.
1. Total blank correction.
Total blank correction = (cell-to-cell blank) + (f x Eb)
where f = 1 at 6650. 6450, and 6300 X; 2 at 5100 X; and 3 at 4800 X. NOTE: These values
for f are very approximate. Extinction values at 4800 A should undoubtedly be
corrected by a greater blank than the one obtained at 7500 X but the value of 3 is
so approximate that there is no substitute for having low Eb values. If a good
correction is required, Eb must not exceed about 0.02.
Eb = corrected value of extinction at 7500 X for any cell-to-cell blank
-------
METHOD NO. A-CHLOR-2
2. Concentration of pigments.
mg (or m-SPU) pigment/m3 = C/V
where C =value obtained from the equations given in Section 6.2.1 through 6.2.4.
V =volume of seawater filtered (L)
3. Chlorophyll a and Phaeo-pigments.
NOTE: Use this calculation only when the procedures in Section 4.3.11 are used to determine ,
extinctions.
Ca = [26.7x (E66500 - E6650a) xv] / (Vx I)
where Ca = concentration of chlorophyll a (mg/m3)
E6650o = extinction at 6650 X before acidification
E6650a = extinction at 6650 X after acidification
v =volume of acetone used for extraction (mL)
V =volume of water filtered (L)
I =path length of the cuvette (cm)
P = [26.7 x (1.7 (E66503) - E6650o) X v] / (V x I)
where P =concentration of phaeo-pigments (mg/m3)
E6650o = extinction at 6650 X before acidification
E6650a =extinction at 6650 X after acidification
v =volume of acetone used for extraction (mL)
V =volume of water filtered (L)
I =path length of the cuvette (cm)
6.2 Formulae (R = Richards (1952,1955); PS = Parsons and Strickland (1963); SU = SCORAJNESCO
(1966)).
1. Chlorophyll a (Ca) in mg/m3.
R : (Ca) = 15.6(E6650) - 2.0(E6450) - 0.8(E6300)
PS: (Ca) = 11 -6(E6650) - 1 -31 (E6450) - 0.14(E6300)
SU: (Ca) = 11.64(E6630)-2.16(E6450) + 0.10(E6300)
2. Chlorophyll b (Cb) in mg/m3.
R : (Cb) = 25.4(E6450) - 4.4(Ee650) -10.3(E*300)
PS: (Cb) = 20.7(E6450) - 4.34(Ee650) - 4.42(^6300)
SU: (Cb) = 20.97(E6450) - 3.94(Ee630) - 3.66(E6300)
3. Chlorophyll c (Cc) in mg/m3 for PS and SU, see Section 6.3.2 for R.
R : (Cc) = 109(E6300) -12.5(Ee650) - 28.7(Ee450)
PS: (Cc) = 55(E6300) - 4.64(E6650) - 16.3(E$450)
SU: (Cc) = 54.22(E6300) -14.81 (E6450) - 5.53(E6630)
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METHOD NO. A-CHLOR-2
4. Plant Carotenoids (Cp).
R (without regard to nature of crop):
(Cp) = 7.6(E4800-1.49(E5100))
PS (if crop is predominantly Chlorophyta or Cyanophyta):
(CP) = 4.0(E4800)
PS (if crop is predominantly Chrysophyta or Pyrrophyta):
(Cp) = 10.0(E4800)
6.3 Reporting units.
1. Parsons-Strickland Equations. When these equations are used, values for chlorophylls a, b, and c will
be in mg/m3 and those for carotenes in a milli-specified plant pigment unit approximating to the
milligram. These equations are described in Section 6.2.
2. Richards Equations. When these equations are used, values are in mg/m3 only for chlorophylls a and
b. The Richards m-SPU is used for chlorophyll c and is considerably greater than the milligram. The
m-SPU is considerably smaller than the milligram if carotenoids are mainly fucoxanthin or peridinin
which are present in Chrysophyta or Pyrrophyta.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
7.2 Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of all equipment. Personnel conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency in the laboratory
techniques described in this method.
8. REFERENCES
Duxbury and Yentsch. 1956. J. Marine Res. 15:92-101.
Parsons and Strickland. 1963. J. Marine Res. 21:155-163.
Richards and Thompson. 1952. J. Marine Res. 11:156.
Richards and Creitz. 1955. J. Marine Res. 14: 211.
SCOR/UNESCO. 1966. Monographs on oceanographic methodology. Published by UNESCO.
Stephens. 1966. J. Fish. Res. Bd. Canada 22:1575.
Strickland, J.D.H. and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries
Research Board of Canada, Ottawa.
8
-------
METHOD NO. A-CHLOR-3
INDEX INFORMATION
Matrices: Seawater, waste
Categories: Estuarine and marine seawater and sea-surface micro-layer, wastewater/effluents
Parameter. Chlorophyll c
1. METHOD TITLE Determination of Chlorophyll c
2. BACKGROUND AND APPLICATION
2.1 Source of method. Determination of Chlorophyll c. Method IV.3.III. In: J.D.H Strickland and T.R.
Parsons. 1972. A Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada,
Ottawa.
2.2 Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. The acetone extract of the phytoplankton in a water sample is treated with a
dilute sodium chloride solution and extracted with n-hexane. The extinction of the aqueous layer
containing the chlorophyll c is then measured by a spectrophotometer at 4500 A before and after
acidification, which converts the chlorophyll c into phaeo-pigment. The chlorophyll c content of
the sample is calculated from the resulting decrease of extinction.
The lower limit of detection, with the filtration of 10 L, has not been statistically determined but
will be approximately 0.05 mg/m3.
2.3.2 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method. The sample is generally 500 mL to 5 L
in volume and filtered through a clean 0.3-mm mesh netting to remove larger zooplankton. For
open ocean samples, filtration of small volumes through a 0.15-mm mesh net will not retain
significant amounts of phytoplankton.
2.4 Standardization/validation status of method.
2.4.1 Comparability studies. Comparability studies using this method with a sea water matrix have
not been cited; this method will be updated to include this information as soon as data are
available.
2.4.2 Precision. At the 5 /*g level, the correct value for chlorophyll c is in the range of the mean
of n determinations ± 1.5/n1/2Mg chlorophyll c.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-CHLOFW
3. SPECIFICATIONS
3.1 This procedure requires the following equipment
1. Millipore filtration unit designed to hold 47-mm membrane filters.
2. 300-mL polyethylene wash bottle.
3. 15-mL graduated centrifuge tubes with both glass and polyethylene stoppers.
4. Small volume (< 10 mL) spectrophotometer cells with path length of 10 cm.
5. Spectrophotometer.
6. 60-mL pear-shaped separatory funnels with the stems cut short. Do not grease the taps but grind
them into place with a little fine emery and water to ensure a snug fit.
7. Millipore AA filters, 47-mm diameter.
3.2 This procedure requires the following reagents.
1. Redistilled acetone (Special Reagent). Distill reagent grade acetone over approximately 1 percent of
its weight of both anhydrous sodium carbonate and anhydrous sodium sulphite. Collect the fraction
boiling at a constant temperature near 56.5°C (uncorrected). Pipet 100 mL of water into a 1000-mL
volumetric flask and add acetone to a volume of exactly 1000 mL Store the redistilled acetone in a
tightly stoppered dark glass bottle and store the 90 percent reagent in moderately small amounts
(approximately 1-L at a time) for use. This reagent is conveniently dispensed from a polyethylene wash
bottle which should be kept nearly full. If good quality reagent acetone is available, it should be
shaken with a little granular anhydrous sodium carbonate and decanted directly for use.
2. Magnesium carbonate suspension. Add approximately 1 g of finely powdered magnesium carbonate
(light weight or "Levis" grade) of analytical reagent quality to 100 mL of distilled water in a stoppered
Erlenmeyer flask. Shake vigorously to suspend the powder immediately before use.
3. Concentrated hydrochloric acid.
4. 100 percent acetone. Purify as described in Section 3.2.1.
3.3 Equipment/instrument calibration.
The wavelength setting of the spectrophotometer used should be checked against a standard hydrogen or
neon line source because the precision of the present method depends upon settings being correct to
better than 20 to 30 X. With quartz prisms at wavelength exceeding 6000 X, very slight movements of
the optical system (brought about by vibrations, etc.) can easily result in errors of 50 X or more in
wavelength settings. If a suitable lamp is not available check the extinction of a suitably concentrated
plant extract and adjust the spectrophotometer until a maximum extinction is obtained at 6300 X.
-------
METHOD NO. A-CHLOR-3
4. PROCEDURE
4.1 Sample handling and preservation. The required volume of sample is measured using a polyethylene
measuring cylinder into a polyethylene bottle. Two or three drops (ca. 0.1 to 0.2 mL) of magnesium
carbonate suspension (see Section 3.2.2) are added. The sample may then be stored in a cool dark place
for a maximum of about 8 h. It is desirable, however, that samples be filtered through a membrane
filter at the time of collection. Membrane filters can be stored by folding them in half (with the
plankton innermost) and storing them in the dark in a desiccator frozen to -20° C for no more than a
few weeks. This procedure nearly always leads to low results and makes the extraction of chlorophyll
more difficult; filters should be extracted without delay if at all possible.
4.2 Interferences.
1. Millipore filters dissolve in acetone completely, give no complications at the centrifugation stage, and
require no particular precautions during filtration. However, unless great care is taken, undesirably high
blanks will occur when using Millipore filters, making determination of small concentrations of
carotenoids difficult. These filters are expensive.
2. This technique minimizes interference from chlorophyllides a and b.
4.3 Sample analysis.
1. Place a 47-mm diameter Millipore AA filter onto the base of the Millipore filter apparatus and secure
the funnel. Care should be taken that the Millipore filtration equipment, centrifuge tubes, and
spectrophotometer cells are kept free from acid and that the filter is not touched with acidic fingers.
2. Vigorously shake the polyethylene sample bottle and invert the bottle into the funnel; the bottle
need not be rinsed if vigorously shaken first. If not added previously, add 1 mL of magnesium carbonate
suspension to the last few hundred milliliters of sample as the sample is filtered. NOTE: The
magnesium carbonate is added at this stage to prevent decomposition of the phytoplankton chlorophyll
pigments into phaeophytin pigments. Strickland and Parsons express some doubt as to the efficacy of
such an addition compared with the addition of a completely soluble organic base, but they acknowledge
the established practice and assume the addition has some value as a precautionary measure.
3. Drain the filter completely under suction before removing it from the filtration equipment and trim
the peripheral excess of unstained membrane with clean scissors. NOTE: The troublesome blank,
measured at 7500 A, found with Millipore filters is caused almost entirely by the salt left in the filter at
this stage, which subsequently "salts out" membrane material from the acetone. The blank can be
greatly reduced if filters are very thoroughly sucked dry of sea water at this stage and the unwanted
peripheral filter is removed.
4. If possible, immediately extract the pigment. If the sample must be stored, store according to
procedure described in Section 4.1.
5. Place the filter in a 15-mL stoppered graduated centrifuge tube. Add approximately 8 mL of 90
percent acetone, stopper the tube, and dissolve the filter by vigorously shaking the tube.
6. Allow the pigments to be extracted by placing the tube in a refrigerator in complete darkness for
approximately 20 h (vigorously shake the "ubes after they have been in the refrigerator 1 or 2 h).
Pigments are very photosensitive during the extraction process and neither extracts or unextracted
-------
METHOD NO. A-CHLOR-3
filters should be exposed to strong sunlight. Exposure to strong light will reduce chlorophyll values to a
small fraction of their initial level in less than 1 h. NOTE: The period of extraction should be
approximately 15 to 20 h. After this time, the rate of further extraction is very slow and additional
extraction time has little merit. Pigment extracts should preferably be kept chilled but they can be kept
at room temperature for many hours without deterioration.
7. Remove the tubes from the refrigerator and let them warm up in the dark to nearly room
temperature. Add 90 percent acetone to make the extracts from the Millipore filters up to exactly
10.0 ml_ NOTE: The use of 10 mL of solution in a 10-cm-path-length cell is recommended for maximum
sensitivity. Greater sensitivity can be obtained by using 10-cm cells containing less than 10 mL but this
is scarcely great enough to warrant the increased difficulties in manipulation. The ultimate sensitivity
is, in practice, more dependent on the size and reproducibility of blanks.
8. Replace the glass stoppers on the centrifuge tubes with plastic stoppers and centrifuge the contents
of the tubes for 5 to 10 min. NOTE: Centrifugation should be as complete as possible when Millipore
filters are used. In most small centrifuges, centrifugation at 3000 to 400 rpm for 10 min is usually
satisfactory, but the efficiency of each instrument should be tested. Difficulties may be encountered
when centrifuging down the glass pulp from glass filters. Tubes should be centrifuged for 1 to 2 min to
pack most of the fibers to the bottom. The centrifuge is then stopped, the tubes removed, and glass
fibers adhering to the walls of the tubes above the level of the solvent are taken down into the bulk of
the liquid by gently splashing the walls by flicking the tubes. The tubes are the"n returned to the
centrifuge and spun for approximately 5 min. If this precaution is not taken, some fibers held above the
solvent layer may enter the spectrophotometer cell.
9. Decant the whole 10-mL of clear supernatant liquid into a 10-cm-path-length spectrophotometer cell
designed to hold 10 mL or less of liquid. In the event of extinction values exceeding about 1.3 the
measurements described below should be made with 2.5-cm or 1-cm cells and the extinction values
multiplied by 4 or 10, respectively, to normalize them to the values expected with a 10-cm cell.
10. Transfer the 10 mL of 90 percent acetone from the cell into a clean, dry separatory funnel. Drain
the cell thoroughly but do not rinse.
11. Add 3.5 mL of sodium chloride solution from a 5-mL graduated pipet and 13.5 mL of hexane from a
20- or 25-mL graduated pipet. Shake the funnel gently for 1 min. NOTE: The addition of this saline
solution precipitates and coagulates the Millipore membrane material which should collect at the
interface of the two liquids. All pigments except chlorophyll c are removed from the lower layer.
12. Run off exactly 8.5 mL of the lower aqueous-acetone phase into a 15-mL graduated centrifuge tube.
Add 100 percent acetone to make the volume to exactly 10.0 mL and centrifuge if necessary. NOTE: If
care is taken to avoid particles of precipitated membrane material, 10.0 mL of clear solution should
result. Centrifugation at this stage should rarely be necessary but the 15-mL centrifuge tubes make
convenient measuring vessels.
13. Decant the clear liquid into an acid-free 10-cm-path-length spectrophotometer cell designed to hold
10 mL or less of liquid. Work only in diffuse light. NOTE: The use of this technique of converting
chlorophyll c to phaeophytin c and measuring the corresponding decrease in extinction at 4500 A alone
gives a method having less sensitivity than could be obtained if the extinction at 4500 A alone were
measured but the procedure is more specific for chlorophyll c and removes the possibility of interference
from traces of carotenoids, etc.
-------
METHOD NO. A-CHLOR-3
14. Without delay measure the extinction of the solution against a cell containing 90 percent acetone at
4500 X. These extracts should not be allowed to evaporate and should be exposed only to subdued light
for the briefest possible time.
15. Add one small drop (ca. 0.02 ml) of concentrated hydrochloric acid to the extract, stopper the cell,
and invert several times to mix the acid and the acetone. Immediately remeasure the extinction at 4500
X. It should be noted that the spectrophotometer cell must be completely free from the acid used in
one determination before the non-acidified reading of the next determination is attempted. Cells should
be cleaned by a generous washing with 100 percent acetone between determinations.
16. Calculate the concentration of chlorophyll c in sea water from the equation given in Section 6.1..
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
There is no blank determination as such in this method. The method is difficult to calibrate in the absence
of pure chlorophyll c and the factor given here, obtained by taking known weights of chlorophyll c
throughout the procedure, is probably applicable directly with all correctly aligned spectrophotometers. The
only compounds known to give interference are chlorophyllides a and b, as some would remain with the
chlorophyll c in the acetone layer and be converted on acidification tot he corresponding phaeophorbides.
with a spectral shift. The present technique minimizes the interference from such compounds and
fortunately it does not appear likely that they will often be present in significant amounts in samples from
the open ocean.
6. RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations.
mg chlorophyll c/m3 = [17.5 (Ei - £2)] / V
where EI = extinction at 4500 X before acidification
E2 = extinction at 4500 X after acidification
V =volume of sea water filtered (L)
6.2 Reporting units. Concentration of chlorophyll c will be reported in mg/m3.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
72. Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of all equipment. Personnel conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency in the laboratory
techniques described in this method.
-------
METHOD NO. A-CHLOR-3
8. REFERENCES
Strickland, J.D.H. and T.R. Parsons. 1972. A Practical Handbook of Seawater Analysis, 2nd ed. Fisheries
Research Board of Canada, Ottawa.
-------
METHOD NO. A-CHLOR-4
INDEX INFORMATION
Matrix: Seawater
Categories: Estuarine and marine seawater and sea-surface microlayer
Parameter Chlorophyll a
1. METHOD TTTLE
Spectrophotometric Determination of Chlorophyll a
2. BACKGROUND AND APPUCAT1ON
2.1 Source of method. Fluorometric Determination of Chlorophyll. Method 1002.Q.1. In: Standard
Methods for the Examination of Water and Wastewater, 16th Edition. 1985. APHA, AWWA. WPCF. pp.
1071-1072.
22. Regulatory status. This method is not cited by any regulation.
2.3 Principle and application.
2.3.1 Description. The pigments are extracted from the plankton concentrates with aqueous
acetone and the optical density (absorbance) of the extract is determined with a spectrophotometer.
The ease with which the chlorophylls are removed from the cells varies considerably with different
algae. To achieve consistently the complete extraction of pigments, disrupt the cells mechanically
with a tissue grinder. Glass fiber filters are preferred for removing algae from water. The glass
filters assist in breaking the dells during grinding, larger volumes of water can be filtered, and no
precipitate forms after acidification. Membrane filters may be used where these factors are
irrelevant.
2.32 Reference to compatible sampling procedures. Any conventional water sampling procedures
can be used to obtain samples for analysis by this method.
2.4 Standardization/validation status of method. Comparability studies using this method with a sea
water matrix have not been cited; this method will be updated to include this information as soon as
data are available.
3. SPECIFICATIONS.
3.1 This procedure requires the following equipment
1. Spectrophotometer, with a narrow band (pass) width (0.5 to 2.0 nm) because the chlorophyll
absorption peak is relatively narrow. At a spectral band width of 20 nm the chlorophyll a concentration
may be underestimated by as much as 40 percent.
The registered trademarks and materials suppliers are referenced for reader convenience in replicating
experiments and do not represent endorsement by the U.S. Environmental Protection Agency.
-------
METHOD NO. A-CHLOR-4
2. Cuvettes, with 1-, 4-, and 10-cm path lengths.
3. Clinical centrifuge.
4. Pipets, 0.1 and 5.0 mL
5. Tissue grinder (Kontes Glass Co., Glass/glass grinder model 8855; glass/TFE grinder model 886000; or
equivalent). Successfully macerating glass fiber filters in tissue grinders with grinding tube and pestle
of conical design may be difficult. Preferably use round-bottom grinding tubes with a matching pestle
having grooves in the TFE tip.
6. Centrifuge tubes, 15-mL graduated, screw cap.
7. Filtration equipment, filters, glass fiber (GF/C or GF/A 4.5-cm diameter, or equivalent) membrane
(0.45-Mm porosity, 47-mm diameter); vacuum pump; solvent-resistant disposable filter assembly, 1.0-/«m
pore size (Gelman Acrodisc or equivalent); 10-mL solvent-resistant syringe.
3.2 This procedure requires the following reagents.
1. Saturated magnesium carbonate solution. Add 1.0 g finely powdered MgCOs to 100 mL distilled
water.
2. Aqueous acetone solution. Mix 90 parts acetone (reagent grade BP 56° C) with 10 parts saturated
magnesium carbonate solution.
3. Hydrochloric acid. 0.1 N.
3.3 Equipment/instrumentation calibration.
1. Calibrate the fluorometer spectrophotometrically with a sample from the same source to achieve
acceptable results. Optimum sensitivity for chlorophyll a extract measurements is obtained at an
excitation wavelength of 430 nm and an emission wavelength of 663 nm. (A method for the continuous
measurement of chlorophyll a is available, but is reported to be less efficient than the in-vitro method
given here, yielding about one-tenth as much fluorescence per unit weight as the same amount in
solution.)
2. Calibrate the fluorometer with a chlorophyll solution of known concentration prepared as follows.
Prepare chlorophyll extract and analyze spectrophotometrically. Prepare serial dilutions of the extract to
provide concentrations of approximately 2,6, 20, and 60 /*g chlorophyll a/L Make fluorometric readings
for each solution at each sensitivity setting (sliding window orifice): 1X, 3X, 10X, and 30X. Using the
values obtained, derive calibration factors to convert fluorometric readings in each sensitivity level to
concentrations of chlorophyll a, presented in Section 6.1.1
3. Determine extract fluorescence at each sensitivity setting before and after acidification.
4. PROCEDURE
NOTE: Conduct all work with chlorophyll extracts in subdues light to avoid degradation. Use opaque
containers or wrap with aluminum foil.
-------
METHOD NO. A-CHLOR-4
4.1 Sample handling and preservation. Whole water samples may be stored up to 2 weeks in the dark
at 4°C. Use opaque bottles because even brief exposure to light during storage will alter chlorophyll
values. Samples on filters collected from water having pH 7 or higher may be placed in airtight plastic
bags and stored frozen for 3 weeks. Samples from acidic water must be processed promptly to prevent
chlorophyll degradation. Use glassware and cuvettes that are clean and acid-free.
42 Interferences. The determination of chlorophyll a by the trichromatic method is of questionable
value because it tends to overestimate chlorophyll a when no correction for phaeophytin a is made.
4.3 Sample extraction.
1. Concentrate samples by centrifuging or filtering (see Section 4.1 for handling procedures.
2. Place the sample in a glass tissue grinder, cover with 2 to 3 mL 90 percent aqueous acetone solution,
and macerate at 500 rpm for 1 min. Use TFE/glass grinder for glass-fiber filters and glass/glass grinder
for membrane filter.
3. Transfer sample to a screw-cap centrifuge tube, rinse grinder with a few milliliters 90 percent
aqueous acetone, and add the rinse to the extraction slurry. Adjust total volume to a constant level, 5
to 10 mL, with 90 percent aqueous acetone. Use solvent sparingly and avoid excessive dilution of
pigments. Steep samples at least 2 h at 4° C in the dark.
4. Clarify by filtering through a solvent-resistent disposable filter (to minimize retention of extract in
filter and filter holder, force 1 to 2 mL air through the filter after the extract) or by centrifuging in
closed tubes for 20 min at 500 g. Decant clarified extract into a clean, calibrated, 15-mL screw-cap
centrifuge tube and measure total volume.
4.4 Sample analysis-Determination of chlorophyll a in the presence of phaeophytin a
NOTE: Chlorophyll a may be overestimated by including phaeo-pigments that absorb near the same
wavelength as chlorophyll a. Addition of acid to chlorophyll a results in loss of the magnesium atom,
converting it to phaeophytin a.
1. Acidify carefully to a final molartty of not more than 3 x 1Q-3M to prevent certain accessory
pigments from changing to absorb at the same wavelength as phaeophytin a. When a solution of pure
chlorophyll a is converted to phaeophytin a by acidification, the absorption-peak-ratio (OD664/OD665) of
1.70 is used in correcting the apparent chlorophyll a concentration for phaeophytin a.
Samples with an OD664 before/00665 after acidification ratio (664b/665a) of 1.70 are considered to
contain no phaeophytin a and to be in excellent physiological condition. Solutions of pure phaeophytin
show no reduction in OD665 upon acidification and have a 664b/665a ratio of 1.0. Thus, mixtures of
chlorophyll a and phaeophytin a have absorption peak ratios ranging between 1.0 and 1.7. These ratios
are based on the use of 90 percent acetone as a solvent. Using 100 percent acetone as solvent results
in a chlorophyll a before-to-after acidification ratio of about 2.0.
2. Transfer 3 mL clarified extract to a 1-cm cuvette and read optical density (OD) at 750 and 664 nm.
3. Acidify extract in the cuvette with 0.1 mL of 0.1 N HCI. Gently agitate the acidified extract and
read OD at 750 and at 665 nm., 90 s after acidification. The volumes of extract and acid and the time
after acidification are critical for accurate, consistent results.
-------
METHOD NO. A-CHLOR-4
4. The OD 664 before acidification should be between 0.1 and 1.0. For very dilute extracts use cuvettes
having longer path length. If a larger cell is used, add a proportionately larger volume of acid.
Correct OD obtained with larger cuvettes to 1 cm before making calculations.
5. Subtract the 750 nm OD value from the readings before (OD 664 nm) and after acidification (OD 665
nm).
6. Calculate chlorophyll a and phaeophytin a according to Section 6.1.1.
4.5 Determination of chlorophyll a. b, and c (trichromatic method).
1. Transfer extract to a 1-cm cuvette and measure optical density (OD) at 750,664,647, and 630 nm.
Choose a path length or dilution to give an OD 664 between 0.1 and 1.0.
2. Use the optical density readings at 664,647,and 630 nm to determine chlorophyll a, b, and c,
respectively. The OD reading at 750 nm is a correction for turbidity. Subtract this reading from each
of the pigment OD values of the other wavelengths before using them in the calculations given in
Section 6.1.2. Because the OD of the extract at 750 nm is very sensitive to changes in the acetone-to-
water proportions, adhere closely to the 90 parts acetone: 10 parts water (v/v) formula for pigment
extraction. Turbidity can be removed easily by filtration through a disposable, solvent-resistant filter
attached to a syringe or by centrifugation for 20 min at 500 g.
3. Calculate the concentrations of chlorophyll a, b, and c in the extract by inserting the corrected
optical densities in the equations given in Section 6.1.2.
4. After determining the concentration of pigment in the extract, calculate the amount of pigment per
unit volume according to the equation given in Section 6.1.2.
5. DATA QUALITY REQUIREMENTS AND ASSESSMENTS
Precision and accuracy. No requirements for measurement of precision and accuracy are cited by this
method, however, analysts should demonstrate the ability to generate acceptable precision with this method
using replicate sample analyses, and demonstrate acceptable accuracy using blank samples.
& RECORDKEEPING AND DATA REPORTING REQUIREMENTS
6.1 Calculations.
1. Concentration of chlorophyll a and phaeophytin a (see Section 4.4).
Chi a = [26.7 (664b -6663) x V1] / (V2 x L)
Ph a = 26.7 [1.7(665a - 664b) x V1] / (V2 x L)
where Chi a = concentration of chlorophyll a (mg/m3)
Ph a = concentration of phaeophytin a (mg/m3)
664b =OD of 90 percent acetone extract before acidification
664a =OD of 90 percent acetone extract after acidification
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METHOD NO. A-CHLOR-4
V-| =volume of extract (L)
V£ =volume of sample (m)3
L = light path length or width of cuvette (cm)
2. Concentration of chlorophylls a, b, and c (see Section 4.5)
Concentration in extract.
Chi a = 1 1 .83(OD664) - 1 .54(OD647) - 0.08(OD630)
Chi b = 21.03(OD647) - 5.43(OD664) - 2.66(OD630)
Chi c = 24.52(OD630) - 7.60(647) - 1.67(OD664)
where Chi a = concentration of chlorophyll a (mg/L)
Chi b = concentration of chlorophyll b (mg/L)
Chi c = concentration of chlorophyll c (mg/L)
OD630 =corrected optical densities (1-cm light path) at 630 nm.
OD630 =corrected optical densities (1 -cm light path) at 630 nm.
OD630 =corrected optical densities (1-cm light path) at 630 nm.
3. Concentration per unit volume.
Chi a = Ca x Vi / V2
where Chi a =concentration of chlorophyll a (mg/m3)
V-| =volume of extract (L)
V2 =volume of sample (m3)
62 Reporting units. If determined according to Section 4.4, chlorophyll a and phaeophytin a
concentrations will be reported in mg/m3. if determined by the trichromatic method, chlorophyll a, b,
and c will be reported in mg/m3.
7. SPECIAL PRECAUTIONS
7.1 Health and safety considerations. Analysts using this method should observe routine laboratory
precautions: wear protective eyewear and clothing, be familiar with laboratory safety devices,
understand the proper handling of acids, solvents, and other reagents.
72. Training/level of expertise. Analysts using this method should be proficient in the operation and
maintenance of all equipment. Personnel conducting this analysis should initially work under the
guidance of an experienced supervisor until he/she can demonstrate proficiency in the laboratory
techniques described in this method.
8. REFERENCES
APHA, AWWA, and WPCF. 1985. Standard Methods for the Examination of Water and Wastewater, 16th
Edition. APHA. Washington, D.C. pp. 1071-1072.
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Sediment
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Tissue
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Air
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Water
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