EPA/600/R-97/072

Methods for the Determination of Chemical Substances in

Marine and Estuarine Environmental Matrices - 2nd Edition

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
ii

 

DISCLAIMER

This manual has been reviewed by the National Exposure Research Laboratory

- Cincinnati, U.S. Environmental Protection Agency, and approved for publication. 

Mention of trade names or commercial products does not constitute endorsement or

recommendation for use.
iii

 

FOREWORD

Environmental measurements are required to determine the quality of ambient

waters and the character of waste effluents. The National Exposure Research

Laboratory - Cincinnati (NERL-Cincinnati) conducts research to:

# Develop and evaluate analytical methods to identify and measure the

concentration of chemical pollutants in marine and estuarine waters,

drinking waters, surface waters, ground waters, wastewaters, sediments,

sludges, and solid wastes.

# Investigate methods for the identification and measurement of viruses,

bacteria, and other microbiological organisms in aqueous samples and to

determine the responses of aquatic organisms to water quality.

# Develop and operate a quality assurance program to support the

achievement of data quality objectives in measurements of pollutants in

marine and estuarine waters, drinking waters, surface waters, ground

waters, wastewaters, sediments, and solid wastes.

# Develop methods and models to detect and quantify responses in aquatic

and terrestrial organisms exposed to environmental stressors and to

correlate the exposure with effects on chemical and biological indicators.

This NERL-Cincinnati publication, “Methods for the Determination of Chemical

Substances in Marine and Estuarine Environmental Matrices - 2nd Edition” was

prepared as the continuation of an initiative to gather together under a single cover a

compendium of standardized laboratory analytical methods for the determination of

nutrients, metals, chlorophyll and organics in marine matrices. It is the goal of this

initiative that the methods that appear in this manual will be multilaboratory validated. 

We are pleased to provide this manual and believe that it will be of considerable value

to many public and private laboratories involved in marine studies for regulatory or

other reasons.

Alfred P. Dufour, Director

Microbiological and Chemical Exposure

Assessment Research Division,

National Exposure Research Laboratory - Cincinnati
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ABSTRACT

This manual contains eleven methods for determination of nutrients, metals, and

chlorophyll. Since Revision 1.0 appeared in 1992, four new methods have been added, one

deleted and four have been multilaboratory validated. Methods 440.0, 445.0, 446.0 and 447.0

have been multilaboratory validated, and Method 353.4 has been replaced with an improved

method.

The metals methods, Methods 200.10, 200.12 and 200.13 have not changed since the

1992 manual. Method 365.5 has remained the same and Method 440.0, that appeared in 1992,

now contains multilaboratory validation data. Two new chlorophyll methods, Methods 446.0 and

447.0, have been added and all three chlorophyll methods have been multilaboratory validated. 

Since the chlorophyll methods validation study was also a comparison study of the methods, that

data has been added to the methods. Anyone interested in obtaining a copy of the full

chlorophyll study final report should contact the Chemical Exposure Research Branch office of

NERL-Cincinnati.
v

CONTENTS

Page

Disclaimer...................................................................................................ii

Foreword...................................................................................................iii

Abstract.....................................................................................................iv

Acknowledgments....................................................................................vii

Introduction................................................................................................1

Method Multilab

Number Title Revision Validation Status

200.10 Determination of Trace Elements in 1.6 No

Marine Waters by On-line Chelation

Preconcentration and Inductively

Coupled Plasma - Mass Spectrometry

200.12 Determination of Trace Elements in 1.0 No

Marine Waters by Stabilized Temperature

Graphite Furnace Atomic Absorption

200.13 Determination of Trace Elements in 1.0 No

Marine Water by Off-Line Chelation

Preconcentration with Graphite Furnace

Atomic Absorption

349.0 Determination of Ammonia in Estuarine and 1.0 No

Coastal Waters by Gas Segmented Continuous

Flow Colorimetric Analysis

353.4 Determination of Nitrate and Nitrite in 1.0 No

Estuarine and Coastal Waters by Gas 

Segmented Continuous Flow Colorimetric

Analysis

365.5 Determination of Orthophosphate in Estuarine 1.4 Yes

and Coastal Waters by Automated Colorimetric

Analysis

366.0 Determination of Dissolved Silicate in 1.0 No 

Estuarine and Coastal Waters by Gas

Segmented Continuous Flow Colorimetric Analysis
vi

440.0 Determination of Carbon and Nitrogen in 1.4 Yes

Sediments and Particulates of Estuarine/Coastal

Waters Using Elemental Analysis

445.0 In Vitro Determination of Chlorophyll a 1.2 Yes

and Pheophytin a in Marine and Freshwater

Phytoplankton by Fluorescence

446.0 In Vitro Determination of Chlorophylls 1.2 Yes

a, b, c1+c2 and Pheopigments in Marine and

Freshwater Algae by Visible Spectrophotometry

447.0 Determination of Chlorophylls a and b and 1.0 Yes

Identification of Other Pigments of Interest

in Marine and Freshwater Algae Using High

Performance Liquid Chromatography with

Visible Wavelength Detection
vii

 

ACKNOWLEDGMENTS

This manual is dedicated to the memory of Dr. Barbara Metzger, late Director of the

Environmental Services Division of USEPA Region 2. She was the impetus and driving force for

this work.

This manual was prepared by the Chemical Exposure Research Branch of the

Microbiological and Chemical Exposure Assessment Research Division, NERL-Cincinnati. The

metals and chlorophyll methods were authored by in-house scientists and the nutrient methods

were authored under contract by Carl Zimmermann and Carolyn Keefe at the Chesapeake

Biological Laboratory, University of Maryland and under an interagency agreement by Dr. JiaZhong Zhang, National Oceanic and Atmospheric Administration, Atlantic Oceanographic and

Meteorological Laboratory, Ocean Chemistry Division. Dr. Zhang deserves recognition for the

outstanding efforts he put into making these methods both informative and practical.

Special thanks go out to Dr. Margo Hunt of USEPA Region 2 for staying so involved in

the chlorophyll methods study. The need to standardize analytical methods for use in the marine

environment was identified and championed by the USEPA regions. The staff at Regions 2 and 3

were instrumental in identifying resources for this project. They provided insight from the

regional perspective and served as technical advisors. Their input has been valuable.

Diane Shirmann and Helen Brock put a tremendous effort into preparing this manuscript

and we are extremely thankful for their hard work.
1

 

INTRODUCTION 

Since the first edition of this manual was published in 1992, the Environmental

Monitoring Systems Laboratory (EMSL) has been reorganized and its name changed to the

National Exposure Research Laboratory (NERL). The principal aim of this manual is to bring

together under one cover a suite of analytical methods specifically adapted or developed for the

examination of coastal and estuarine environmental samples. Many of the methods presented here

are adaptations of analytical techniques which, for many years, have been used routinely by the

marine community. Hallmarks of the methods which appear in this manual, however, are the

integrated quality control/quality assurance requirements, the use of standardized terminology and

the Environmental Monitoring Management Council (EMMC) format. The mandatory

demonstration of laboratory capability and the continuing checks on method performance ensure

the quality and comparability of data reported by different laboratories and programs. Another

distinction of this manual is the multilaboratory validation data for many of the methods.

Multilaboratory validation studies test the ruggedness of methods, provide single-analyst

and multilaboratory precision and accuracy statements, and method detection limits that are

"typical" of what most laboratories can achieve. Methods that reach this level of evaluation have

been thoroughly investigated to the fullest extent possible by a single laboratory and have usually

been informally adopted as standard methods by the analytical community. When a method does

not perform as expected in a multilaboratory study, it must be returned to the development phase. 

For example, although widely accepted and routinely used in the marine community, Method

353.4 (Determination of Nitrite + Nitrate in Estuarine and coastal Waters by Automated

Colorimetric Analysis) failed the ruggedness test in 1992 when 50% of the participating

laboratories in the multilaboratory study returned unacceptable data. Review of the data

suggested that the cadmium reduction column chemistry and maintenance required further

investigation. The method was subsequently reevaluated by Dr. Jia-Zhong Zhang, under an

Interagency Agreement between the U.S. EPA and NOAA. The new nitrite/nitrate method is

improved in technical detail and QA/QC requirements. 

We are pleased to present this 2 Edition manual to the public and to research and nd

monitoring labs in the hope that it contributes to better protection and preservation of our

estuarine and coastal ecosystems. 

Elizabeth J. Arar, William L. Budde, Thomas D. Behymer

Microbiological and Chemical Exposure Assessment Research Division

September, 1997
200.10-1

Method 200.10

Determination of Trace Elements in Marine Waters by

On-Line Chelation Preconcentration and Inductively

Coupled Plasma - Mass Spectrometry

Stephen E. Long

Technology Applications, Inc.

and

Theodore D. Martin

Chemical Exposure Research Branch

Human Exposure Research Division

Revision 1.6

September 1997

Edited by

John T. Creed

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Revision 1.6 September 1997 200.10-2

Method 200.10

Determination of Trace Elements in Marine Waters by On-Line Chelation

Preconcentration and Inductively Coupled Plasma - Mass Spectrometry

1.0 Scope and Application

1.1 This method describes procedures for

preconcentration and determination of total recoverable

trace elements in marine waters, including estuarine

water, seawater, and brines.

1.2 Acid solubilization is required prior to the

determination of total recoverable elements to facilitate

breakdown of complexes or colloids that might influence

trace element recoveries. This method should only be

used for preconcentration and determination of trace

elements in aqueous samples.

1.3 This method is applicable to the following

elements:

Chemical Abstracts Service

Element Registry Numbers (CASRN)

Cadmium (Cd) 7440-43-9

Cobalt (Co) 7440-48-4

Copper (Cu) 7440-50-8

Lead (Pb) 7439-92-1

Nickel (Ni) 7440-02-0

Uranium (U) 7440-61-1

Vanadium (V) 7440-62-2

1.4 Method detection limits (MDLs) for these elements will be dependent on the specific instrumentation

employed and the selected operating conditions. However, the MDLs should be essentially independent of the

matrix because elimination of the matrix is a feature of

the method. Reagent water MDLs, which were determined using the procedure described in Section 9.2.4,

are listed in Table 1.

1.5 A minimum of 6-months experience in the use of

commercial instrumentation for inductively coupled

plasma mass spectrometry (ICP-MS) is recommended.

2.0 Summary of Method

2.1 This method is used to preconcentrate trace

elements using an iminodiacetate functionalized chelating

resin. Following acid solubilization, the sample is

1,2

buffered prior to the chelating column using an on-line

system. Groups I and II metals, as well as most anions,

are selectively separated from the analytes by elution with

ammonium acetate at pH 5.5. The analytes are

subsequently eluted into a simplified matrix consisting of

dilute nitric acid and are determined by ICP-MS using a

directly coupled on-line configuration.

2.2 The determinative step in this method is ICPMS. Sample material in solution is introduced by

3-5

pneumatic nebulization into a radio frequency plasma

where energy transfer processes cause desolvation,

atomization and ionization. The ions are extracted from

the plasma through a differentially pumped vacuum

interface and separated on the basis of their mass-tocharge ratio by a quadrupole mass spectrometer having

a minimum resolution capability of 1 amu peak width at

5% peak height. The ions transmitted through the

quadrupole are registered by a continuous dynode electron multiplier or Faraday detector and the ion information

is processed by a data handling system. Interferences

relating to the technique (Section 4) must be recognized

and corrected. Such corrections must include

compensation for isobaric elemental interferences and

interferences from polyatomic ions derived from the

plasma gas, reagents or sample matrix. Instrumental drift

must be corrected for by the use of internal standardization.

3.0 Definitions

3.1 Calibration Blank (CB) -- A volume of reagent

water fortified with the same matrix as the calibration

standards but without the analytes, internal standards, or

surrogate analytes.

3.2 Calibration Standard (CAL) -- A solution prepared from the primary dilution standard solution or stock

standard solutions and the internal standards and surrogate analytes. The CAL solutions are used to calibrate the

instrument response with respect to analyte concentration.

3.3 Instrument Detection Limit (IDL) -- The minimum quantity of analyte or the concentration equivalent

that gives an analyte signal equal to three times the

standard deviation of the background signal at the selected wavelength, mass, retention time, absorbance line,

etc.

3.4 Instrument Performance Check Solution (IPC)

-- A solution of one or more method analytes, surrogates,

internal standards, or other test substances used to

evaluate the performance of the instrument system with

respect to a defined set of criteria.

3.5 Internal Standard (IS) -- A pure analyte(s) added

to a sample, extract, or standard solution in known

amount(s) and used to measure the relative responses
200.10-3 Revision 1.6 September 1997

of other method analytes and surrogates that are compo- materials or purchased from a reputable commercial

nents of the same sample or solution. The internal source.

standard must be an analyte that is not a sample component. 3.14 Total Recoverable Analyte (TRA) -- The

3.6 Laboratory Fortified Blank (LFB) -- An aliquot sample or an unfiltered aqueous sample following

of reagent water or other blank matrices to which known treatment by refluxing with hot dilute mineral acid(s) as

quantities of the method analytes are added in the specified in the method.

laboratory. The LFB is analyzed exactly like a sample,

and its purpose is to determine whether the methodology 3.15 Tuning Solution (TS) -- A solution that is used to

is in control and whether the laboratory is capable of adjust instrument performance prior to calibration and

making accurate and precise measurements. sample analyses.

3.7 Laboratory Fortified Sample Matrix (LFM) -- An

aliquot of an environmental sample to which known

quantities of the method analytes are added in the

laboratory. The LFM is analyzed exactly like a sample,

and its purpose is to determine whether the sample matrix

contributes bias to the analytical results. The background

concentrations of the analytes in the sample matrix must

be determined in a separate aliquot and the measured

values in the LFM corrected for background

concentrations.

3.8 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water or other blank matrices that are treated

exactly as a sample including exposure to all glassware,

equipment, solvents, reagents, internal standards, and

surrogates that are used with other samples. The LRB is

used to determine if method analytes or other interferences are present in the laboratory environment, the

reagents, or the apparatus.

3.9 Linear Dynamic Range (LDR) -- The absolute

quantity or concentration range over which the instrument

response to an analyte is linear.

3.10 Material Safety Data Sheet (MSDS) -- Written

information provided by vendors concerning a chemical’s

toxicity, health hazards, physical properties, fire, and

reactivity data including storage, spill, and handling

precautions.

3.11 Method Detection Limit (MDL) -- The minimum

concentration of an analyte that can be identified,

measured, and reported with 99% confidence that the

analyte concentration is greater than zero.

3.12 Quality Control Sample (QCS) -- A solution of

method analytes of known concentrations that is used to

fortify an aliquot of LRB or sample matrix. The QCS is

obtained from a source external to the laboratory and

different from the source of calibration standards. It is

used to check laboratory performance with externally

prepared test materials.

3.13 Stock Standard Solution (SSS) -- A concentrated solution containing one or more method analytes

prepared in the laboratory using assayed reference 

concentration of analyte determined to be in either a solid

4.0 Interferences

4.1 Several interference sources may cause inaccuracies in the determination of trace elements by ICP-MS.

These are:

4.1.1 Isobaric elemental interferences -- Are caused

by isotopes of different elements that form singly or

doubly charged ions of the same nominal mass-tocharge ratio and that cannot be resolved by the mass

spectrometer in use. All elements determined by this

method have, at a minimum, one isotope free of isobaric

elemental interference. The analytical isotopes recommended for use with this method are listed in Table 1.

4.1.2 Abundance sensitivity -- Is a property defining

the degree to which the wings of a mass peak contribute

to adjacent masses. The abundance sensitivity is affected by ion energy and quadrupole operating pressure.

Wing overlap interferences may result when a small ion

peak is being measured adjacent to a large one. The

potential for these interferences should be recognized

and the spectrometer resolution adjusted to minimize

them.

4.1.3 Isobaric polyatomic ion interferences -- Are

caused by ions consisting of more than one atom that

have the same nominal mass-to-charge ratio as the

isotope of interest and that cannot be resolved by the

mass spectrometer in use. These ions are commonly

formed in the plasma or interface system from support

gases or sample components. Such interferences must

be recognized, and when they cannot be avoided by the

selection of alternative analytical isotopes, appropriate

corrections must be made to the data. Equations for the

correction of data should be established at the time of the

analytical run sequence as the polyatomic ion

interferences will be highly dependent on the sample

matrix and chosen instrument conditions.

4.1.4 Physical interferences -- Are associated with the

physical processes that govern the transport of sample

into the plasma, sample conversion processes in the

plasma, and the transmission of ions through the plasma

mass spectrometer interface. These interferences may

result in differences between instrument responses for
Revision 1.6 September 1997 200.10-4

the sample and the calibration standards. Physical as cyanides or sulfides. Acidification of samples should be

interferences may occur in the transfer of solution to the performed in a fume hood.

nebulizer (e.g., viscosity effects), at the point of aerosol

formation and transport to the plasma (e.g., surface 5.4 All personnel handling environmental samples

tension), or during excitation and ionization processes known to contain or to have been in contact with human

within the plasma itself. Internal standardization may be waste should be immunized against known disease

effectively used to compensate for many physical causative agents.

interference effects. Internal standards ideally should

6

have similar analytical behavior to the elements being 5.5 It is the responsibility of the user of this method to

determined. comply with relevant disposal and waste regulations. For

4.1.5 Memory interferences -- Result when isotopes of

elements in a previous sample contribute to the signals

measured in a new sample. Memory effects can result

from sample deposition on the sampler and skimmer

cones and from the buildup of sample material in the

plasma torch and spray chamber. The site where these

effects occur is dependent on the element and can be

minimized by flushing the system with a rinse blank

between samples. Memory interferences from the chelating system may be encountered especially after

analyzing a sample containing high concentrations of the

analytes. A thorough column rinsing sequence following

elution of the analytes is necessary to minimize such

interferences.

4.2 A principal advantage of this method is the

selective elimination of species giving rise to polyatomic

spectral interferences on certain transition metals (e.g.,

removal of the chloride interference on vanadium). As

the majority of the sample matrix is removed, matrix

induced physical interferences are also substantially

reduced.

4.3 Low recoveries may be encountered in the

preconcentration cycle if the trace elements are

complexed by competing chelators in the sample or are

present as colloidal material. Acid solubilization pretreatment is employed to improve analyte recovery and to

minimize adsorption, hydrolysis, and precipitation effects.

5.0 Safety

5.1 Each chemical reagent used in this method

should be regarded as a potential health hazard and

exposure to these reagents should be as low as reasonably achievable. Each laboratory is responsible for

maintaining a current awareness file of OSHA regulations regarding the safe handling of the chemicals

specified in this method. A reference file of material

7,8

data handling sheets should also be available to all

personnel involved in the chemical analysis.

5.2 Analytical plasma sources emit radio frequency

radiation in addition to intense UV radiation. Suitable

precautions should be taken to protect personnel from

such hazards.

5.3 The acidification of samples containing reactive

materials may result in the release of toxic gases, such

guidance see Sections 14.0 and 15.0.

6.0 Equipment and Supplies

6.1 Preconcentration System -- System containing

no metal parts in the analyte flow path, configured as

shown in Figure 1.

6.1.1 Column -- Macroporous iminodiacetate chelating

resin (Dionex Metpac CC-1 or equivalent).

6.1.2 Sample loop -- 10-mL loop constructed from

narrow bore, high-pressure inert tubing, Tefzel ethylene

tetra-fluoroethylene (ETFE) or equivalent.

6.1.3 Eluent pumping system (Pl) -- Programmable

flow, high pressure pumping system, capable of

delivering either one of two eluents at a pressure up to

2000 psi and a flow rate of 1-5 mL/min.

6.1.4 Auxiliary pumps -- On line buffer pump (P2),

piston pump (Dionex QIC pump or equivalent) for

delivering 2M ammonium acetate buffer solution; carrier

pump (P3), peristaltic pump (Gilson Minipuls or equivalent) for delivering 1% nitric acid carrier solution; sample

pump (P4), peristaltic pump for loading sample loop.

6.1.5 Control valves -- Inert double stack, pneumatically operated four-way slider valves with connectors.

6.1.5.1 Argon gas supply regulated at 80-100 psi.

6.1.6 Solution reservoirs -- Inert containers, e.g., high

density polyethylene (HDPE), for holding eluent and

carrier reagents.

6.1.7 Tubing -- High pressure, narrow bore, inert

tubing (e.g., Tefzel ETFE or equivalent) for interconnection of pumps/valve assemblies and a minimum length

for connection of the preconcentration system to the ICPMS instrument.

6.2 Inductively Coupled Plasma - Mass Spectrometer

6.2.1 Instrument capable of scanning the mass range

5-250 amu with a minimum resolution capability of 1 amu

peak width at 5% peak height. Instrument may be fitted

with a conventional or extended dynamic range detection

system.
200.10-5 Revision 1.6 September 1997

6.2.2 Argon gas supply (high-purity grade, 99.99%). 6.4.4 Centrifuge -- Steel cabinet with guard bowl,

6.2.3 A mass-flow controller on the nebulizer gas

supply is recommended. A water-cooled spray chamber

may be of benefit in reducing some types of interferences (e.g., polyatomic oxide species).

6.2.4 Operating conditions -- Because of the diversity electrode system with a resolution of ± 0.1 pH units.

of instrument hardware, no detailed instrument operating

conditions are provided. The analyst is advised to follow

the recommended operating conditions provided by the

manufacturer.

6.2.5 If an electron multiplier detector is being used,

precautions should be taken, where necessary, to

prevent exposure to high ion flux. Otherwise changes in

instrument response or damage to the multiplier may

result. Samples having high concentrations of elements

beyond the linear range of the instrument and with

isotopes falling within scanning windows should be di--

luted prior to analysis.

6.3 Labware -- For the determination of trace

elements, contamination and loss are of critical concern.

Potential contamination sources include improperly

cleaned laboratory apparatus and general contamination

within the laboratory environment. A clean laboratory

work area, designated for trace element sample handling, must be used. Sample containers can introduce

positive and negative errors in the determination of trace

elements by (1) contributing contaminants through

surface desorption or leaching or (2) depleting element

concentrations through adsorption processes. For these

reasons, borosilicate glass is not recommended for use

with this method. All labware in contact with the sample

should be cleaned prior to use. Labware may be soaked

overnight and thoroughly washed with laboratory-grade

detergent and water, rinsed with water, and soaked for 4

hr in a mixture of dilute nitric and hydrochloric acids,

followed by rinsing with ASTM type I water and oven

drying.

6.3.1 Griffin beakers, 250-mL, polytetrafluoroethylene

(PTFE) or quartz.

6.3.2 Storage bottles -- Narrow mouth bottles, Teflon

FEP (fluorinated ethylene propylene), or HDPE, 125-mL

and 250-mL capacities.

6.4 Sample Processing Equipment

6.4.1 Air displacement pipetter -- Digital pipet system

capable of delivering volumes from 10 to 2500 FL with an

assortment of metal-free, disposable pipet tips.

6.4.2 Balances -- Analytical balance, capable of

accurately weighing to ±0.1 mg; top pan balance, accurate to ± 0.01g.

6.4.3 Hot plate -- Corning PC100 or equivalent.

electric timer and brake.

6.4.5 Drying oven -- Gravity convection oven with

thermostatic control capable of maintaining 105EC±5EC.

6.4.6 pH meter -- Bench mounted or hand-held

7.0 Reagents and Standards

7.1 Water -- For all sample preparation and dilutions, ASTM type I water (ASTM D1193) is required.

7.2 Reagents may contain elemental impurities that

might affect the integrity of analytical data. Because of

the high sensitivity of this method, ultra high-purity

reagents must be used unless otherwise specified. To

minimize contamination, reagents should be prepared

directly in their designated containers where possible.

7.2.1 Acetic acid, glacial (sp. gr. 1.05).

7.2.2 Ammonium hydroxide (20%).

7.2.3 Ammonium acetate buffer 1M, pH 5.5 -- Add 58-

mL (60.5 g) of glacial acetic acid to 600-mL of ASTM

type water. Add 65 mL (60 g) of 20% ammonium hydroxide and mix. Check the pH of the resulting solution by

withdrawing a small aliquot and testing with a calibrated

pH meter, adjusting the solution to pH 5.5±0.1 with small

volumes of acetic acid or ammonium hydroxide as necessary. Cool and dilute to 1 L with ASTM type I water.

7.2.4 Ammonium acetate buffer 2M, pH 5.5 -- Prepare

as for Section 7.2.3 using 116 mL (121g) glacial acetic

acid and 130 mL (120 g) 20% ammonium hydroxide,

diluted to 1000 mL with ASTM type I water.

Note: The ammonium acetate buffer solutions may be

further purified by passing them through the

chelating column at a flow rate of 5.0-mL/min.

With reference to Figure 1, pump the buffer

solution through the column using pump P1, with

valves A and B off and valve C on. Collect the

purified solution in a container at the waste

outlet. Following this, elute the collected contaminants from the column using 1.25M nitric acid for

5 min at a flow rate of 4.0 mL/min.

7.2.5 Nitric acid, concentrated (sp.gr. 1.41).

7.2.5.1 Nitric acid 1.25M -- Dilute 79 mL (112 g) conc.

nitric acid to 1000-mL with ASTM type I water.

7.2.5.2 Nitric acid 1% -- Dilute 10 mL conc. nitric acid to

1000 mL with ASTM type I water.
Revision 1.6 September 1997 200.10-6

7.2.5.3 Nitric acid (1+1) -- Dilute 500 mL conc. nitric acid heating to effect solution. Cool and dilute to 100 mL with

to 1000-mL with ASTM type I water. ASTM type I water.

7.2.5.4 Nitric acid (1+9) -- Dilute 100 mL conc. nitric acid 7.3.7 Scandium solution, stock 1 mL = 1000 Fg Sc:

to 1000-mL with ASTM type I water.

7.2.6 Oxalic acid dihydrate (CASRN 6153-56-6), 0.2M

-- Dissolve 25.2 g reagent grade C2H2O4·2H2O in 250-mL

ASTM type I water and dilute to 1000 mL with ASTM type 7.3.8 Terbium solution, stock 1 mL = 1000 Fg Tb:

I water. Caution - Oxalic acid is toxic; handle with care.

7.3 Standard Stock Solutions -- May be purchased

from a reputable commercial source or prepared from

ultra high-purity grade chemicals or metals (99.99-

99.999% pure). All salts should be dried for 1 h at 105EC,

unless otherwise specified. (Caution- Many metal salts

are extremely toxic if inhaled or swallowed. Wash hands

thoroughly after handling.) Stock solutions should be

stored in plastic bottles. The following procedures may

be used for preparing standard stock solutions:

Note: Some metals, particularly those that form surface oxides require cleaning prior to being

weighed. This may be achieved by pickling the

surface of the metal in acid. An amount in excess of the desired weight should be pickled

repeatedly, rinsed with water, dried, and

weighed until the desired weight is achieved.

7.3.1 Cadmium solution, stock 1 mL = 1000 Fg Cd:

Pickle cadmium metal in (1+9) nitric acid to an exact

weight of 0.100 g. Dissolve in 5-mL (1+1) nitric acid,

heating to effect solution. Cool and dilute to 100-mL with

ASTM type I water.

7.3.2 Cobalt solution, stock 1 mL = 1000 Fg Co:

Pickle cobalt metal in (1+9) nitric acid to an exact weight

of 0.100 g. Dissolve in 5 mL (1+1) nitric acid, heating to

effect solution. Cool and dilute to 100 mL with ASTM type

I water.

7.3.3 Copper solution, stock 1 mL = 1000 Fg Cu:

Pickle copper metal in (1+9) nitric acid to an exact weight

0.100 g. Dissolve in 5 mL (1+1) nitric acid, heating to

effect solution. Cool and dilute to 100 mL with ASTM type

I water.

7.3.4 lndium solution, stock 1 mL = 1000 Fg In: Pickle

indium metal in (1+1) nitric acid to an exact weight 0.100

g. Dissolve in 10 mL (1+1) nitric acid, heating to effect

solution. Cool and dilute to 100 mL with ASTM type I

water.

7.3.5 Lead solution, stock 1 mL = 1000 Fg Pb: Dissolve 0.1599 g PbNO in 5 mL (1+1) nitric acid. Dilute to 7.5 Blanks -- Four types of blanks are required for 3

100 mL with ASTM type I water. this method. A calibration blank is used to establish the

7.3.6 Nickel solution, stock 1 mL = 1000 Fg Ni: blank is used to assess possible contamination from the

Dissolve 0.100 g nickel powder in 5 mL conc. nitric acid,

Dissolve 0.1534 g Sc O in 5 mL (1+1) nitric acid, 2 3

heating to effect solution. Cool and dilute to 100 mL with

ASTM type I water.

Dissolve 0.1176 g Tb4O7 in 5 mL conc. nitric acid, heating

to effect solution. Cool and dilute to 100 mL with ASTM

type I water.

7.3.9 Uranium solution, stock 1 mL = 1000 Fg U:

Dissolve 0.2110 g UO2(NO3)2·6H2O (Do Not Dry) in 20

mL ASTM type I water. Add 2-mL (1+1) nitric acid and

dilute to 100-mL with ASTM type I water.

7.3.10 Vanadium solution, stock 1 mL = 1000 Fg V:

Pickle vanadium metal in (1+9) nitric acid to an exact

weight of 0.100 g. Dissolve in 5-mL (1+1) nitric acid,

heating to effect solution. Cool and dilute to 100 mL with

ASTM type I water.

7.3.11 Yttrium solution, stock 1 mL = 1000 Fg Y:

Dissolve 0.1270 g Y2O3 in 5 mL (1+1) nitric acid, heating

to effect solution. Cool and dilute to 100 mL with ASTM

type I water.

7.4 Multielement Stock Standard Solution -- Care

must be taken in the preparation of multielement stock

standards that the elements are compatible and stable.

Originating element stocks should be checked for

impurities that might influence the accuracy of the

standard. Freshly prepared standards should be transferred to acid cleaned, new FEP or HDPE bottles for

storage and monitored periodically for stability. A

multielement stock standard solution containing the

elements, cadmium, cobalt, copper, lead, nickel, uranium, and vanadium (1 mL = 10 Fg) may be prepared by

diluting 1 mL of each single element stock in the list to

100 mL with ASTM type I water containing 1% (v/v) nitric

acid.

7.4.1 Preparation of calibration standards -- Fresh

multielement calibration standards should be prepared

weekly. Dilute the stock multielement standard solution

in 1% (v/v) nitric acid to levels appropriate to the required

operating range. The element concentrations in the

standards should be sufficiently high to produce good

measurement precision and to accurately define the

slope of the response curve. A suggested mid-range

concentration is 10 Fg /L.

analytical calibration curve, and the laboratory reagent

sample preparation procedure. The laboratory fortified

blank is used to assess the recovery of the method
200.10-7 Revision 1.6 September 1997

analytes and the rinse blank is used between samples to standard using the direct addition method (Meminimize memory from the nebulizer/spray chamber thod A, Section 10.5) as it is not efficiently

surfaces. concentrated on the iminodiacetate column.

7.5.1 Calibration blank -- Consists of 1% (v/v) nitric acid

in ASTM type I water (Section 7.2.5.2).

7.5.2 Laboratory reagent blank (LRB) -- Must contain

all the reagents in the same volumes as used in processing the samples. The LRB must be carried through the

entire sample digestion and preparation scheme.

7.5.3 Laboratory Fortified Blank (LFB) -- To an aliquot

of LRB, add aliquots from the multielement stock standard (Section 7.4) to produce a final concentration of 10

Fg/L for each analyte. The fortified blank must be carried

through the entire sample pretreatment and analytical

scheme.

7.5.4 Rinse Blank (RB) -- Is a 1% (v/v) nitric acid

solution that is delivered to the lCP-MS between samples

(Section 7.2.5.2).

7.6 Tuning Solution -- This solution is used for

instrument tuning and mass calibration prior to analysis

(Section 10.2). The solution is prepared by mixing nickel,

yttrium, indium, terbium, and lead stock solutions (Section 7.3) in 1% (v/v) nitric acid to produce a concentration

of 100 Fg/L of each element.

7.7 Quality Control Sample (QCS) -- A quality

control sample having certified concentrations of the

analytes of interest should be obtained from a source

outside the laboratory. Dilute the QCS if necessary with

1% nitric acid, such that the analyte concentrations fall

within the proposed instrument calibration range.

7.8 Instrument Performance Check (IPC) Solution

-- The IPC solution is used to periodically verify instrument performance during analysis. It should be prepared

by combining method analytes at appropriate concentrations to approximate the midpoint of the calibration curve.

The IPC solution should be prepared from the same

standard stock solutions used to prepare the calibration

standards and stored in a FEP bottle. Agency programs

may specify or request that additional instrument performance check solutions be prepared at specified concentrations in order to meet particular program needs.

7.9 Internal Standards Stock Solution, 1 mL =

100 Fg -- Dilute 10-mL of scandium, yttrium, indium,

terbium, and bismuth stock standards (Section 7.3) to

100-mL with ASTM type I water, and store in a Teflon

bottle. Use this solution concentrate for addition to

blanks, calibration standards and samples (Method A,

Section 10.5), or dilute by an appropriate amount using

1% (v/v) nitric acid, if the internal standards are being

added by peristaltic pump (Method B, Section 10.5).

Note: Bismuth should not be used as an internal

8.0 Sample Collection, Preservation, and

Storage

8.1 Prior to the collection of an aqueous sample,

consideration should be given to the type of data required, so that appropriate preservation and pretreatment

steps can be taken. Acid preservation should be performed at the time of sample collection or as soon

thereafter as practically possible. The pH of all aqueous

samples must be tested immediately prior to aliquoting

for analysis to ensure the sample has been properly

preserved. If properly acid preserved, the sample can be

held up to 6 months before analysis.

8.2 For the determination of total recoverable

elements in aqueous samples, acidify with (1+1) nitric

acid (high purity) at the time of collection to pH<2;

normally, 3 mL of (1+1) acid per liter of sample is

sufficient for most samples. The sample should not be

filtered prior to analysis.

Note: Samples that cannot be acid preserved at the

time of collection because of sampling limitations or transport restrictions, or are >pH2

because of high alkalinity should be acidified

with nitric acid to pH<2 upon receipt in the

laboratory. Following acidification, the sample

should be held for 16 h and the pH verified to be

<2 before withdrawing an aliquot for sample

processing.

8.3 For aqueous samples, a field blank should be

prepared and analyzed as required by the data user. Use

the same container and acid as used in sample collection.

9.0 Quality Control

9.1 Each laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an

initial demonstration of laboratory capability and the

periodic analysis of laboratory reagent blanks, fortified

blanks and other laboratory solutions as a continuing

check on performance. The laboratory is required to

maintain performance records that define the quality of

the data generated.

9.2 Initial Demonstration of Performance (Mandatory)

9.2.1 The initial demonstration of performance is used

to characterize instrument performance (determination of
Revision 1.6 September 1997 200.10-8

linear dynamic ranges and analysis of quality control single laboratory MDL values using this method

samples) and laboratory performance (determination of are given in Table 1.

method detection limits) prior to samples being analyzed

by this method. MDLs should be determined every six months, when a

9.2.2 Linear calibration ranges -- The upper limit of the cant change in the background or instrument response.

linear calibration range should be established for each

analyte. Linear calibration ranges should be determined

every six months or whenever a significant change in

instrument response is expected.

9.2.3 Quality control sample (QCS) -- When beginning must analyze at least one LRB (Section 7.5.2) with each

the use of this method, on a quarterly basis or as required to meet data-quality needs, verify the calibration

standards and acceptable instrument performance with

the preparation and analyses of a QCS (Section 7.7). If

the determined concentrations are not within ± 10% of

the stated values, performance of the determinative step

of the method is unacceptable. The source of the problem must be identified and corrected before either

proceeding with the initial determination of method

detection limits or continuing with ongoing analyses. must analyze at least one LFB (Section 7.5.3) with each

9.2.4 Method detection limit (MDL) -- MDLs must be ery (Section 9.4.3). If the recovery of any analyte falls

established for all analytes, using reagent water (blank)

fortified at a concentration of two to three times the

estimated instrument detection limit. To determine MDL 9

values, take seven replicate aliquots of the fortified

reagent water and process through the entire analytical

method. Perform all calculations defined in the method

and report the concentration values in the appropriate assess laboratory performance against the required conunits. Calculate the MDL as follows: trol limits of 85-115% (Section 9.3.2). When sufficient

MDL = (t) x (S) minimum of 20-30 analyses), optional control limits can

where: t = Student's t value for a 99% confidence level the standard deviation (S) of the mean recovery. These

and a standard deviation estimate with n-1 data can be used to establish the upper and lower control

degrees of freedom [t = 3.14 for seven limits as follows:

replicates].

S = standard deviation of the replicate analyses.

Note: If the relative standard deviation (RSD) from the

analyses of the seven aliquots is <15%, the The optional control limits must be equal to or better than

concentration used to determine the analyte the required control limits of 85-115%. After each five to

MDL may have been inappropriately high for the ten new recovery measurements, new control limits can

determination. If so, this could result in the be calculated using only the most recent 20-30 data

calculation of an unrealistically low MDL. If points. Also, the standard deviation (S) data should be

additional confirmation of the MDL is desired, used to established an ongoing precision statement for

reanalyze the seven replicate aliquots on two the level of concentrations included in the LFB. These

more nonconsecutive days and again calculate data must be kept on file and be available for review.

the MDL values for each day. An average of the

three MDL values for each analyte may provide

for a more appropriate MDL estimate. Concur- For all determinations the laboratory must analyze the

rently, determination of MDL in reagent water IPC solution (Section 7.8) and a calibration blank immerepresents a best case situation and does not diately following daily calibration, after every tenth

reflect possible matrix effects of real world sample (or more frequently, if required) and at the end of

samples. However, successful analyses of LFMs the sample run. Analysis of the IPC solution and calibra-

(Section 9.4) can give confidence to the MDL tion blank immediately following calibration must verify

value determined in reagent water. Typical that the instrument is within ±10% of calibration. Subsenew operator begins work or whenever there is a signifi9.3 Assessing Laboratory Performance (Mandatory)

9.3.1 Laboratory reagent blank (LRB) -- The laboratory

batch of 20 or fewer samples. LRB data are used to

assess contamination from the laboratory environment.

LRB values that exceed the MDL indicate laboratory or

reagent contamination should be suspected. Any determined source of contamination must be corrected and

the samples reanalyzed for the affected analytes after

acceptable LRB values have been obtained.

9.3.2 Laboratory fortified blank (LFB) -- The laboratory

batch of samples. Calculate accuracy as percent recovoutside the required control limits of 85-115%, that

analyte is judged out of control, and the source of the

problem should be identified and resolved before continuing analyses.

9.3.3 The laboratory must use LFB analyses data to

internal performance data become available (usually a

be developed from the percent mean recovery (x) and

Upper Control Limit = x + 3S

Lower Control Limit = x - 3S

9.3.4 Instrument performance check (IPC) solution --
R'

(Cs&C)

S

x 100

200.10-9 Revision 1.6 September 1997

quent analyses of the IPC solution must verify the 9.4.4 If the recovery of any analyte falls outside the

calibration within ±15%. If the calibration cannot be

verified within the specified limits, reanalyze the IPC

solution. If the second analysis of the IPC solution

confirms calibration to be outside the limits, sample

analysis must be discontinued, the cause determined

and/or in the case of drift the instrument recalibrated. All

samples following the last acceptable IPC solution must

be reanalyzed. The analysis data of the calibration blank

and IPC solution must be kept on file with the sample

analyses data.

9.3.5 The overall sensitivity and precision of this

method are strongly influenced by a laboratory's ability to

control the method blank. Therefore, it is recommended

that the calibration blank response be recorded for each

set of samples. This record will aid the laboratory in

assessing both its long- and short-term ability to control

the method blank.

9.4 Assessing Analyte Recovery and Data

Quality

9.4.1 Sample homogeneity and the chemical nature of

the sample matrix can affect analyte recovery and the

quality of the data. Taking separate aliquots from the

sample for replicate and fortified analyses can in some

cases assess these effects. Unless otherwise specified

by the data user, laboratory or program, the following

laboratory fortified matrix (LFM) procedure (Section

9.4.2) is required.

9.4.2 The laboratory must add a known amount of

each analyte to a minimum of 10% of the routine samples. In each case the LFM aliquot must be a duplicate

of the aliquot used for sample analysis and for total

recoverable determinations added prior to sample

preparation. For water samples, the added analyte

concentration must be the same as that used in the

laboratory fortified blank (Section 9.3.2).

9.4.3 Calculate the percent recovery for each analyte,

corrected for concentrations measured in the unfortified

sample, and compare these values to the designated

LFM recovery range of 75-125%. Recovery calculations

are not required if the concentration added is less than

25% of the unfortified sample concentration. Percent

recovery may be calculated in units appropriate to the

matrix, using the following equation:

where, R = percent recovery.

C = fortified sample concentration. S

C = sample background concentration.

S = concentration equivalent of analyte added

to sample.

designated LFM recovery range and the laboratory performance for that analyte is shown to be in control

(Section 9.3), the recovery problem encountered with the

LFM is judged to be either matrix or solution related, not

system related.

9.4.5 If analysis of LFM sample(s) and the test routines above indicate an operative interference and the

LFMs are typical of the other samples in the batch, those

samples that are similar must be analyzed in the same

manner as the LFMs. Also, the data user must be

informed when a matrix interference is so severe that it

prevents the successful analysis of the analyte or when

the heterogeneous nature of the sample precludes the

use of duplicate analyses.

9.4.6 Where reference materials are available, they

should be analyzed to provide additional performance

data. The analysis of reference samples is a valuable

tool for demonstrating the ability to perform the method

acceptably.

10.0 Calibration and Standardization

10.1 Initiate proper operating configuration of ICP-MS

instrument and data system. Allow a period of not less

than 30 min for the instrument to warm up. During this

process conduct mass calibration and resolution checks

using the tuning solution. Resolution at low mass is

indicated by nickel isotopes 60, 61, 62. Resolution at

high mass is indicated by lead isotopes 206, 207, 208.

For good performance adjust spectrometer resolution to

produce a peak width of approximately 0.75 amu at 5%

peak height. Adjust mass calibration if it has shifted by

more than 0.1 amu from unit mass.

10.2 Instrument stability must be demonstrated by

analyzing the tuning solution (Section 7.6) a minimum of

five times with resulting relative standard deviations of

absolute signals for all analytes of less than 5%.

10.3 Prior to initial calibration, setup proper instrument

software routines for quantitative analysis and connect

the ICP-MS instrument to the preconcentration apparatus. The instrument must be calibrated for the analytes

of interest using the calibration blank (Section 7.5.1) and

calibration standard (Section 7.4.1) prepared at one or

more concentration levels. The calibration solutions

should be processed through the preconcentration

system using the procedures described in Section 11.

10.4 Demonstration and documentation of acceptable

initial calibration is required before any samples are

analyzed. After initial calibration is successful, a calibration check is required at the beginning and end of each

period during which analyses are performed and at

requisite intervals.
Revision 1.6 September 1997 200.10-10

10.4.1 After the calibration has been established, it Note: For proper heating, adjust the temperature

must be initially verified for all analytes by analyzing the control of the hot plate such that an uncovered

IPC (Section 7.8). If the initial calibration verification Griffin beaker containing 50 mL of water placed

exceeds ±10% of the established IPC value, the analysis in the center of the hot plate can be maintained

should be terminated, the source of the problem identi- at a temperature approximately but no higher

fied and corrected, the instrument recalibrated, and the than 85EC. (Once the beaker is covered with a

new calibration verified before continuing analyses. watch glass the temperature of the water will rise

10.4.2 To verify that the instrument is properly calibrated

on a continuing basis, analyze the calibration blank

(Section 7.5.1) and IPC (Section 7.8) after every 10 about 20-mL by gentle heating at 85EC. Do Not Boil.

analyses. The results of the analyses of the standards This step takes about 2 h for a 100-mL aliquot with the

will indicate whether the calibration remains valid. If the rate of evaporation rapidly increasing as the sample

indicated concentration of any analyte deviates from the volume approaches 20 mL. (A spare beaker containing

true concentration by more than 15%, reanalyze the 20-mL of water can be used as a gauge.)

standard. If the analyte is again outside the 15% limit, the

instrument must be recalibrated and the previous 10 11.1.3 Cover the lip of the beaker with a watch glass to

samples reanalyzed. The instrument responses from the

calibration check may be used for recalibration purposes.

10.5 Internal Standardization -- Internal standardization must be used in all analyses to correct for instrument

drift and physical interferences. For full mass range

scans, a minimum of three internal standards must be

used. Internal standards must be present in all samples,

standards and blanks at identical levels. This may be

achieved by directly adding an aliquot of the internal

standards to the CAL standard, blank or sample solution

(Method A), or alternatively by mixing with the solution

prior to nebulization using a second channel of the

peristaltic pump and a mixing coil (Method B). The

concentration of the internal standard should be sufficiently high that good precision is obtained in the measurement of the isotope used for data correction and to

minimize the possibility of correction errors if the internal

standard is naturally present in the sample. Internal

standards should be added to blanks, samples and

standards in a like manner, so that dilution effects resulting from the addition may be disregarded.

Note: Bismuth should not be used as an internal

standard using the direct addition method (Method A, Section 10.5) because it is not efficiently

concentrated on the iminodiacetate column.

11.0 Procedure

11.1 Sample Preparation -- Total Recoverable

Elements

11.1.1 Add 2-mL(1+1) nitric acid to the beaker containing 100-mL of sample. Place the beaker on the hot plate

for solution evaporation. The hot plate should be located

in a fume hood and previously adjusted to provide evaporation at a temperature of approximately but no higher

than 85EC. (See the following note.) The beaker should

be covered with an elevated watch glass or other necessary steps should be taken to prevent sample contamination from the fume hood environment.

to approximately 95EC.)

11.1.2 Reduce the volume of the sample aliquot to

reduce additional evaporation and gently reflux the

sample for 30 min. (Slight boiling may occur, but vigorous boiling must be avoided.)

11.1.4 Allow the beaker to cool. Quantitatively transfer

the sample solution to a 100-mL volumetric flask, dilute

to volume with reagent water, stopper and mix.

11.1.5 Allow any undissolved material to settle overnight, or centrifuge a portion of the prepared sample until

clear. (If after centrifuging or standing overnight, the

sample contains suspended solids, a portion of the

sample may be filtered prior to analysis. However, care

should be exercised to avoid potential contamination

from filtration.) The sample is now ready for analysis.

Because the effects of various matrices on the stability of

diluted samples cannot be characterized, all analyses

should be performed as soon as possible after the

completed preparation.

11.2 Prior to first use, the preconcentration system

should be thoroughly cleaned and decontaminated using

0.2M oxalic acid.

11.2.1 Place approximately 500-mL 0.2M oxalic acid in

all the eluent/solution containers and fill the sample loop

with 0.2M oxalic acid using the sample pump (P4) at a

flow rate of 3-5 mL/min. With the preconcentration

system disconnected from the ICP-MS instrument, use

the pump program sequence listed in Table 2 to flush the

complete system with oxalic acid. Repeat the flush sequence three times.

11.2.2 Repeat the sequence described in Section

11.2.1 using 1.25M nitric acid and again using ASTM

type I water in place of the 0.2M oxalic acid.

11.2.3 Rinse the containers thoroughly with ASTM type

I water, fill them with their designated reagents (see

Figure 1) and run through the sequence in Table 2 once

to prime the pump and all eluent lines with the correct

reagents.
200.10-11 Revision 1.6 September 1997

11.3 Initiate ICP-MS instrument operating configuration. Tune the instrument for the analytes of interest

(Section 10).

11.4 Establish instrument software run procedures for

quantitative analysis. Because the analytes are eluted

from the preconcentration column in a transient manner,

it is recommended that the instrument software is configured in a rapid scan/peak hopping mode. The instrument

is now ready to be calibrated.

11.5 Reconnect the preconcentration system to the

ICP-MS instrument. With valves A and B in the off

position and valve C in the on position, load sample

through the sample loop to waste using pump P4 for 4

min at 4 mL/min. Switch on the carrier pump (P3) and

pump 1% nitric acid to the nebulizer of the ICP-MS

instrument at a flow rate of 0.8-1.0-mL/min.

11.6 Switch on the buffer pump (P2), and pump 2M

ammonium acetate at a flow rate of 1.0 mL/min.

11.7 Preconcentration of the sample may be achieved

by running through an eluent pump program (P1) sequence similar to that illustrated in Table 2. The exact

timing of this sequence should be modified according to

the internal volume of the connecting tubing and the

specific hardware configuration used.

11.7.1 Inject sample -- With valves A, B, and C on, load

sample from the loop onto the column using 1M ammonium acetate for 4.5 min at 4.0 mL/min. The analytes are

retained on the column, while the majority of the matrix

is passed through to waste.

11.7.2 Elute analytes -- Turn off valves A and B and

begin eluting the analytes by pumping 1.25M nitric acid

through the column at 4.0 mL/min, then turn off valve C

and pump the eluted analytes into the ICP-MS instrument

at 1.0 mL/min. Initiate ICP-MS software data acquisition

and integrate the eluted analyte profiles.

11.7.3 Column Reconditioning -- Turn on valve C to

direct column effluent to waste, and pump 1.25M nitric

acid, 1M ammonium acetate, 1.25M nitric acid and 1M

ammonium acetate alternately through the column at 4.0

mL/min. During this process, the next sample can be

loaded into the sample loop using the sample pump (P4).

11.8 Repeat the sequence described in Section 11.7

for each sample to be analyzed. At the end of the analytical run leave the column filled with 1M ammonium

acetate buffer until it is next used.

11.9 Samples having concentrations higher than the

established linear dynamic range should be diluted into

range with 1% HNO (v/v) and reanalyzed. 3

12.0 Data Analysis and Calculations

12.1 Analytical isotopes and elemental equations

recommended for sample data calculations are listed in

Table 3. Sample data should be reported in units of μg/L.

Do not report element concentrations below the determined MDL.

12.2 For data values less than 10, two significant

figures should be used for reporting element concentrations. For data values greater than or equal to 10, three

significant figures should be used.

12.3 Reported values should be calibration blank subtracted. If additional dilutions were made to any samples,

the appropriate factor should be applied to the calculated

sample concentrations.

12.4 Data values should be corrected for instrument

drift by the application of internal standardization. Corrections for characterized spectral interferences should be

applied to the data.

12.5 The QC data obtained during the analyses

provide an indication of the quality of the sample data

and should be provided with the sample results.

13.0 Method Performance

13.1 Experimental conditions used for single laboratory testing of the method are summarized in Table 4.

13.2 Data obtained from single laboratory testing of

the method are summarized in Tables 5 and 6 for two

reference water samples consisting of National Research

Council Canada (NRCC) Estuarine Water (SLEW-1) and

Seawater (NASS-2). The samples were prepared using

the procedure described in Section 11.1.1. For each

matrix, three replicates were analyzed and the average

of the replicates was used to determine the sample

concentration for each analyte. Two further sets of three

replicates were fortified at different concentration levels,

one set at 0.5 Fg/L, the other at 10 μg/L. The sample

concentration, mean percent recovery, and the relative

standard deviation of the fortified replicates are listed for

each method analyte. The reference material certificate

values are also listed for comparison.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

EPA has established a preferred hierarchy of environmental management techniques that place pollution pre-
Revision 1.6 September 1997 200.10-12

vention as the management option of first choice. Whenever feasible, laboratory personnel should use pollution

prevention techniques to address their waste generation

(e.g., Section 7.8). When wastes cannot be feasibly

reduced at the source, the Agency recommends recy--

cling as the next best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street N.W.,

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

15.0 Waste Management

15.1 The Environmental Protection Agency requires

that laboratory waste management practices be conducted consistent with all applicable rules and regulations. The Agency urges laboratories to protect the air,

water, and land by minimizing and controlling all releases

from hoods and bench operations, complying with the

letter and spirit of any sewer discharge permits and

regulations, and by complying with all solid and hazardous waste regulations, particularly the hazardous waste

identification rules and land disposal restrictions. For

further information on waste management, consult The

Waste Management Manual for Laboratory Personnel,

available from the American Chemical Society at the

address listed in Section 14.2.

16.0 References

1. Siraraks, A., H.M. Kingston, and J.M. Riviello,

Anal Chem. 62,1185 (1990).

2. Heithmar, E.M., T.A. Hinners, J.T. Rowan, and

J.M. Riviello, Anal Chem., 62, 857 (1990).

3. Gray A.L. and A.R. Date, Analyst, 108, 1033

(1983).

4. Houk, R.S., et al. Anal. Chem., 52, 2283 (1980).

5. Houk, R.S., Anal.Chem., 58, 97A (1986).

6. J. J. Thompson and R.S. Houk, Appl. Spec., 41,

801 (1987).

7. OSHA Safety and Health Standards, General

Industry, (29 CFR 1910), Occupational Safety

and Health Administration, OSHA 2206, (Revised, January 1976).

8. Safety in Academic Chemistry Laboratories,

American Chemical Society Publication, Committee on Chemical Safety, 3rd Edition, 1979.

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

Appendix B.
200.10-13 Revision 1.6 September 1997

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Table 1. Total Recoverable Method Detection Limits for Reagent Water

Recommended MDL1

Element Analytical Mass Fg/L

Cadmium 111 0.041

Cobalt 59 0.021

Copper 63 0.023

Lead 206, 207,208 0.074

Nickel 60 0.081

Uranium 238 0.031

Vanadium 51 0.014

 Determined using 10-mL sample loop. 1

Table 2. Eluent Pump Programming Sequence for Preconcentration of Trace Elements

Time Flow Valve Valve

(min) (mL/min) Eluent A,B C

0.0 4.0 1M ammonium acetate ON ON

4.5 4.0 1.25M nitric acid ON ON

5.1 1.0 1.25M nitric acid OFF ON

5.5 1.0 1.25M nitric acid OFF OFF

7.5 4.0 1.25M nitric acid OFF ON

8.0 4.0 1M ammonium acetate OFF ON

10.0 4.0 1.25M nitric acid OFF ON

11.0 4.0 1M ammonium acetate OFF ON

12.5 0.0 OFF ON

Table 3. Recommended Analytical Isotopes and Elemental Equations for Data Calculations

Element Isotope Elemental Equation Note

Cd 106, 108, 111, 114 (1.000)( C)-(1.073)[( C)-(0.712)( C)] (1) 111 108 106

Co 59 (1.000)( C) 59

Cu 63, 65 (1.000)( C) 63

Pb 206, 207, 208 (1.000)( C)+(1.000)( C)+(1.000)( C) (2) 206 207 208

Ni 60 (1.000)( C) 60

U 238 (1.000)( C) 238

V 51 (1.000)( C) 51

C - calibration blank subtracted counts at specified mass.

(1) - correction for MoO interference. An additional isobaric elemental correction should be made if palladium is present.

(2) - allowance for isotopic variability of lead isotopes.

NOTE: As a minimum, all isotopes listed should be monitored. Isotopes recommended for analytical determination are italized.
Revision 1.6 September 1997 200.10-14

Table 4. Experimental Conditions for Single Laboratory Validation

Chromatography

Instrument Dionex chelation system

Preconcentration column Dionex MetPac CC-1

ICP-MS Instrument Conditions

Instrument VG PlasmaQuad Type I

Plasma forward power 1.35 kW

Coolant flow rate 13.5 L/min

Auxiliary flow rate 0.6 L/min

Nebulizer flow rate 0.78 L/min

Internal standards Sc, Y, In, Tb

Data Acquisition

Detector mode Pulse counting

Mass range 45-240 amu

Dwell time 160 Fs

Number of MCA channels 2048

Number of scan sweeps 250

Table 5. Precision and Recovery Data for Estuarine Water (SLEW-1)

Sample Spike Average Spike Average

Certificate Conc. Addition Recovery RSD Addition Recovery RSD

Analyte (Fg/L) (Fg/L) (Fg/L) (%) (%) (Fg/L) (%) (%)

Cd 0.018 <0.041 0.5 94.8 9.8 10 99.6 1.1

Co 0.046 0.078 0.5 102.8 4.0 10 96.6 1.4

Cu 1.76 1.6 0.5 106.0 2.7 10 96.0 4.8

Pb 0.028 <0.074 0.5 100.2 4.0 10 106.9 5.8

Ni 0.743 0.83 0.5 100.0 1.5 10 102.0 2.1

U -- 1.1 0.5 96.7 7.4 10 98.1 3.6

V -- 1.4 0.5 100.0 3.2 10 97.0 4.5

-- No certificate value

Table 6. Precision and Recovery Data for Seawater (NASS-2)

Sample Spike Average Spike Average

Certificate Conc. Addition Recovery RSD Addition Recovery RSD

Analyte (Fg/L) (Fg/L) (Fg/L) (%) (%) (Fg/L) (%) (%)

Cd 0.029 <0.041 0.5 101.8 1.0 10 96.4 3.7

Co 0.004 <0.021 0.5 98.9 3.0 10 99.2 1.7

Cu 0.109 0.12 0.5 95.8 2.3 10 93.1 0.9

Pb 0.039 <0.074 0.5 100.6 8.5 10 92.1 2.6

Ni 0.257 0.23 0.5 102.2 2.3 10 98.2 1.2

U 3.00 3.0 0.5 94.0 0.7 10 98.4 1.7

V -- 1.7 0.5 104.0 3.4 10 109.2 3.7

--No certificate value
Waste Waste Waste

A

B

B

A

C

Chelating

Column

10 mL

Loop

Buffer

Pump

Eluent

Pump

Carrier

Pump

P/STALTIC

Pump

Sample

1MNHOAc 1.25 MNitricAcid

1%Nitric Acid

P3

ICP-MS

Mixing Tee

Off On

P2 P4 P1

4

2MNHOAc

4

Figure 1. Configuration of Preconcentration System.

200.10-15 Revision 1.6 September 1997
200.12 - 1

Method 200.12

Determination of Trace Elements in Marine Waters by Stabilized

Temperature Graphite Furnace Atomic Absorption

John T. Creed and Theodore D. Martin

Chemical Exposure Research Branch

Human Exposure Research Division

Revision 1.0

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Revision 1.0 September 1997 200.12 - 2

Chemical Abstracts

Service Registry

Analyte Numbers (CASRN)

Arsenic (As) 7440-38-2

Cadmium (Cd) 7440-43-9

Chromium (Cr) 7440-47-3

Copper (Cu) 7440-50-8

Lead (Pb) 7439-92-1

Nickel (Ni) 7440-02-0

Selenium (Se) 7782-49-2

Method 200.12

Determination of Trace Elements in Marine Waters by Stabilized 

Temperature Graphite Furnace Atomic Absorption

1.0 Scope and Application

1.1 This method provides procedures for the

determination of total recoverable elements by graphite

furnace atomic absorption (GFAA) in marine waters,

including estuarine, ocean and brines with salinities of up

to 35 ppt. This method is applicable to the following

analytes: 

1.2 For determination of total recoverable analytes in

marine waters, a digestion/extraction is required prior to

analysis.

1.3 Method detection limit and instrumental operating

conditions for the applicable elements are listed in Tables

1 and 2. These are intended as a guide and are typical of

a commercial instrument optimized for the element.

However, actual method detection limits and linear working ranges will be dependent on the sample matrix,

instrumentation and selected operating conditions.

1.4 Users of the method data should state the data

quality objectives prior to analysis. The ultra-trace metal

concentrations typically associated with marine water may

preclude the use of this method based on its sensitivity.

Users of the method must document and have on file the

required initial demonstration performance data described

in Section 9.2 prior to using the method for analysis.

2.0 Summary of Method

2.1 Nitric acid is dispensed into a beaker containing

an accurately weighed or measured, well-mixed, 

homogeneous aqueous sample. Then, for samples with

undissolved material, the beaker is covered with a watch

glass and heated, made up to volume, centrifuged or

allowed to settle, and the sample is then analyzed.

2.2 The analytes listed in this method are determined

by stabilized temperature platform graphite furnace

atomic absorption (STPGFAA). In STPGFAA, the sample

and the matrix modifier are first pipetted onto the platform

or a device which provides delayed atomization. 

The furnace chamber is then purged with a continuous

flow of a premixed gas (95% argon - 5% hydrogen) and

the sample is dried at a relatively low temperature (about

120EC) to avoid spattering. Once dried, the sample is

pretreated in a char or ashing step which is designed to

minimize the interference effects caused by the concomitant sample matrix. After the char step, the furnace is

allowed to cool prior to atomization. The atomization

cycle is characterized by rapid heating of the furnace to a

temperature where the metal (analyte) is atomized from

the pyrolytic graphite surface into a stopped gas flow

atmosphere of argon containing 5% hydrogen. (Only

selenium is determined in an atmosphere of high purity

argon.) The resulting atomic cloud absorbs the elementspecific atomic emission produced by a hollow cathode

lamp (HCL) or an electrodeless discharge lamp (EDL).

Following analysis, the furnace is subjected to a cleanout

period of high temperature and continuous argon flow.

Because the resulting absorbance usually has a nonspecific component associated with the actual analyte absorbance, Zeeman background correction is required to

subtract from the total signal the component which is

nonspecific to the analyte. In the absence of interferences, the background-corrected, absorbance is directly

related to the concentration of the analyte. Interferences

relating to STPGFAA (Section 4.0) must be recognized

and corrected. Suppressions or enhancements of instrument response caused by the sample matrix must be

corrected for by the method of standard addition (Section

11.3).
200.12 - 3 Revision 1.0 September 1997

3.0 Definitions

3.1 Calibration Blank (CB) -- A volume of reagent

water fortified with the same matrix as the calibration

standards, but without the analytes, internal standards, or

surrogate analytes.

3.2 Calibration Standard (CAL) -- A solution prepared from the primary dilution standard solution or stock

standard solutions and the internal standards and surrogate analytes. The CAL solutions are used to calibrate the

instrument response with respect to analyte concentration.

3.3 Field Reagent Blank (FRB) -- An aliquot of

reagent water or other blank matrix that is placed in a

sample container in the laboratory and treated as a

sample in all respects, including shipment to the sampling

site, exposure to sampling site conditions, storage,

preservation, and all analytical procedures. The purpose

of the FRB is to determine it method analytes or other

interferences are present in the field environment.

3.4 Instrument Detection Limit (IDL) -- The minimum quantity of analyte or the concentration equivalent

which gives an analyte signal equal to three times the

standard deviation of the background signal at the selected wavelength, mass, retention time, absorbance line,

etc.

3.5 Instrument Performance Check Solution (IPC)

-- A solution of one or more method analytes, surrogates,

internal standards, or other test substances used to

evaluate the performance of the instrument system with

respect to a defined set of criteria.

3.6 Laboratory Duplicates (LD1 and LD2) -- Two

aliquots of the same sample taken in the laboratory and

analyzed separately with identical procedures. Analyses

of LD1 and LD2 indicate precision associated with

laboratory procedures, but not with sample collection,

preservation, or storage procedures.

3.7 Laboratory Fortified Blank (LFB) -- An aliquot

of reagent water or other blank matrices to which known

quantities of the method analytes are added in the

laboratory. The LFB is analyzed exactly like a sample,

and its purpose is to determine whether the methodology

is in control, and whether the laboratory is capable of

making accurate and precise measurements.

3.8 Laboratory Fortified Sample Matrix (LFM) -- An

aliquot of an environmental sample to which known

quantities of the method analytes are added in the

laboratory. The LFM is analyzed exactly like a sample,

and its purpose is to determine whether the sample matrix

contributes bias to the analytical results. The background

concentrations of the analytes in the sample matrix must

be determined in a separate aliquot and the measured

values in the LFM corrected for background concentrations.

3.9 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water or other blank matrices that are treated

exactly as a sample including exposure to all glassware,

equipment, solvents, reagents, internal standards, and

surrogates that are used with other samples. The LRB is

used to determine if method analytes or other interferences are present in the laboratory environment, the

reagents, or the apparatus.

3.10 Linear Dynamic Range (LDR) -- The absolute

quantity or concentration range over which the instrument

response to an analyte is linear.

3.11 Material Safety Data Sheet (MSDS) -- Written

information provided by vendors concerning a chemical’s

toxicity, health hazards, physical properties, fire, and

reactivity data including storage, spill, and handling precautions.

3.12 Matrix Modifier (MM) -- A substance added to

the instrument along with the sample in order to minimize

the interference effects by selective volatilization of either

analyte or matrix components.

3.13 Matrix Performance Check (MPC) -- A solution

of method analytes used to evaluate the laboratory’s

ongoing capabilities in analyzing high salinity samples.

The reference material NASS-3 or its equivalent is fortified with the same analytes at the same concentration as

the LFB. This provides an ongoing check of furnace

operating conditions to assure the analyte false positives

are not being introduced via elevated backgrounds.

3.14 Method Detection Limit (MDL) -- The minimum

concentration of an analyte that can be identified, measured and reported with 99% confidence that the analyte

concentration is greater than zero.

3.15 Quality Control Sample (QCS) -- A solution of

method analytes of known concentrations which is used

to fortify an aliquot of LRB or sample matrix. The QCS is

obtained from a source external to the laboratory and
Revision 1.0 September 1997 200.12 - 4

different from the source of calibration standards. It is suggested matrix modifiers and the appropriate furnace

used to check laboratory performance with externally charring conditions. Figure 1 is a plot of integrated

prepared test materials. background absorbance vs. char temperature for Ni, Cd,

3.16 Standard Addition -- The addition of a known absorbance in a saline matrix is strongly affected by the

amount of analyte to the sample in order to determine the

relative response of the detector to an analyte within the

sample matrix. The relative response is then used to

assess either an operative matrix effect or the sample

analyte concentration.

3.17 Stock Standard Solution (SSS) -- A concentrated solution containing one or more method analytes

prepared in the laboratory using assayed reference

materials or purchased from a reputable commercial

source.

3.18 Total Recoverable Analyte (TRA) -- The concentration of analyte determined to be in either a solid

sample or an unfiltered aqueous sample following treatment by refluxing with hot dilute mineral acid(s) as

specified in the method.

4.0 Interferences

4.1 Several interference sources may cause

inaccuracies in the determination of trace elements by

GFAA. These interferences can be classified into three

major subdivisions: spectral, matrix, and memory.

4.2 Spectral interferences are caused by absorbance

of light by a molecule or atom which is not the analyte of

interest or emission from black body radiation.

4.2.1 Spectral interferences caused by an element only

occur if there is a spectral overlap between the wavelength of the interfering element and the analyte of

interest. Fortunately, this type of interference is relatively

uncommon in STPGFAA because of the narrow atomic

line widths associated with STPGFAA. In addition, the use

of appropriate furnace temperature programs and high

spectral purity lamps as light sources can minimize the

possibility of this type of interference. However, molecular

absorbances can span several hundred nanometers

producing broadband spectral interferences. This type of

interference is far more common in STPGFAA. The use

of matrix modifiers, selective volatilization, and

background correctors are all attempts to eliminate unwanted nonspecific absorbance. Table 2 contains typical

background absorbances associated with the analysis of

the MPC solution (NASS-3) which has a salinity of 35 ppt.

These background absorbances were obtained using the

Pb, and Se. Figure 1 indicates that the background

char temperature. Therefore, char temperature optimization is a critical part of the successful analysis of metals

in saline water by GFAA. The elevated backgrounds

associated with ocean water can produce false positives.

For this reason, the char temperature profiles shown in

Figure 1 should be constructed for each analyte prior to

using this method for saline water analysis.

Note: False analyte positives can be generated by large

backgrounds. Figure 2 is an atomization profile for Pb

using a 800EC char temperature. The background shown

in the figure has exceeded the capabilities of the Zeeman

corrector. This profile can be used as a guide in screening

other analyses which may have background absorbances

which exceed the Zeeman capability. The background

profile is characterized by a smooth baseline in the

beginning of the atomization cycle followed by a sharp

increase. During this sharp increase the background peak

profile may remain relatively smooth, but when the

background exceeds the Zeeman correction capability,

the background profile will appear extremely erratic. The

atomic profile is also erratic during this part of the atomization cycle. These types of background/atomic profiles

obtained during atomization result in false positives.

Since the nonspecific component of the total absorbance

can vary considerably from sample type to sample type,

to provide effective background correction and eliminate

the elemental spectral interference of palladium on copper and iron on selenium, the exclusive use of Zeeman

background correction is specified in this method.

4.2.2 Spectral interferences are also caused by black

body radiation produced during the atomization furnace

cycle. This black body emission reaches the

photomultiplier tube, producing erroneous results. The

magnitude of this interference can be minimized by

proper furnace tube alignment and monochromator

design. In addition, atomization temperatures which

adequately volatilize the analyte of interest without

producing unnecessary black body radiation can help

reduce unwanted background emission produced during

atomization.

4.3 Matrix interferences are caused by sample components which inhibit the formation of free atomic analyte

atoms during atomization. In this method the use of a

delayed atomization device which provides a warmer gas
200.12 - 5 Revision 1.0 September 1997

phase environment during atomization is required. These response profile is observed in a solution containing

devices provide an environment which is more conducive 10,000 ppm NaCl. Therefore, in well-characterized

to the formation of free analyte atoms and thereby samples of known salinity it is possible to effectively matrix

minimize this type of interference. This type of match the standards with NaCl and perform the analysis

interference can be detected by analyzing the sample directly using matrix matched standards, thereby avoiding

plus a sample aliquot fortified with a known concentration the time consuming method of standard additions. If the

of the analyte. If the determined concentration of the matrix matched standards are going to be used, it is

analyte addition is outside a designated range (Section necessary to document that the use of NaCl is indeed

9.4.3), a possible matrix effect should be suspected. In compensating for the suppression. This documentation

addition, the matrix can produce analyte complexes which should include a response plot of increasing matrix vs.

are lost via volatilization during the char. These losses will relative response similar to Figure 4.

result in poor recovery of the analyte within the matrix and

should be corrected by adjusting the char temperature. 4.5.2 Selenium: The background level associated with

4.4 Memory interferences result from analyzing a

sample containing a high concentration of an element

(typically a high atomization temperature element) which

cannot be removed quantitatively in one complete set of

furnace steps. The analyte which remains in the furnace

can produce false positive signals on subsequent

sample(s). Therefore, the analyst should establish the

analyte concentration which can be injected into the

furnace and adequately removed in one complete set of

furnace cycles. If this concentration is exceeded, the

sample should be diluted and a blank analyzed to assure

the memory effect has been eliminated before reanalyzing the diluted sample.

4.5 Specific Element Interferences. -- The matrix

effects caused by the saline water can be severe. In order

to evaluate the extent of the matrix suppression as a 4.5.3 Arsenic: The elevated char temperatures

function of increasing salinity a plot of normalized integrated absorbance vs. microliters NASS-3 (Reference

Material from the National Research Council of Canada)

is constructed. Figure 3 is a plot of relative response of

As, Se, Cd, Ni, Cu, and Pb in waters containing salinity of

3.5 ppt (1 μL NASS-3) to 35 ppt (10 μL NASS-3). Figure

3 indicates that the matrix effects caused by the increasing salinity are minor for Pb, Cu, and Ni. The relative

responses of Pb, Ni, and Cu shown in Figure 3 are within

± 5% of the 1% HNO standard or zero μL of matrix. 3

Figure 3 indicates that the increasing salinity does cause

a substantial matrix interference for Se and Cd. This

suppression must be compensated for by methods of

standard addition or the use of matrix matched standards

where applicable.

4.5.1 Cadmium: The background level associated with the

direct determination of Cd in NASS-3 exceeds the

Zeeman background correction. Therefore, NH4 NO3 is

used as a matrix removing modifier in addition to the Pd/

Mg(NO3)2. Figure 4 is a plot of the relative Cd response 1 

vs. the amount of seawater on the platform. A similar

the direct determination of Se in NASS-3 exceeds the

Zeeman correction capability. Therefore, HNO3 is used as

a matrix removing modifier in addition to the Pd/ Mg(NO )3 2

for the determination of Se in saline waters. Figure 5 is a

plot of relative response vs. the amount of seawater on

the platform. A similar suppression is observed in a

solution containing 10,000 ppm NaCI. Therefore, in wellcharacterized samples of known salinity it is possible to

effectively matrix match the standards with NaCl and

perform the analysis directly using matrix matched

standards, thereby avoiding the time consuming method

of standard additions. If the matrix matched standards are

going to be used, it is necessary to document that the use

of NaCl is indeed compensating for the suppression. This

documentation should include a response plot of

increasing matrix vs. relative response similar to Figure 5.

possible with the determination of As minimize the

interferences produced by the marine water background

levels. Figure 3 is a plot of relative response vs. the

amount of seawater on the platform. Figure 3 indicates a

matrix suppression on As caused by the seawater.

Although this suppression does cause a slight bias as

shown in the recovery data in Table 3, the suppression

does not warrant the method of standard additions (MSA)

given the recovery criteria of 75-125% for LFMS.

5.0 Safety

5.1 The toxicity or carcinogenicity of each reagent

used in this method has not been fully established. Each

chemical should be regarded as a potential health hazard

and exposure to these compounds should be as low as

reasonably achievable. Each laboratory is responsible for

maintaining a current awareness file of OSHA regulations

regarding the safe handling of the chemicals specified in

this method. A reference file of material data handling 2-5

sheets should also be made available to all personnel
Revision 1.0 September 1997 200.12 - 6

involved in the chemical analysis. Specifically, 6.1.2 Single element hollow cathode lamps or single

concentrated nitric and hydrochloric acids present various

hazards and are moderately toxic and extremely irritating

to skin and mucus membranes. Use these reagents in a

fume hood whenever possible and if eye or skin contact

occurs, flush with large volumes of water. Always wear

safety glasses or a shield for eye protection, protective

clothing, and observe proper mixing when working with

these reagents.

5.2 The acidification of samples containing reactive

materials may result in the release of toxic gases, such as

cyanides or sulfides. Acidification of samples should be

done in a fume hood.

5.3 All personnel handling environmental samples

known to contain or to have been in contact with human

waste should be immunized against known disease

causative agents.

5.4 The graphite tube during atomization emits intense UV radiation. Suitable precautions should be taken

to protect personnel from such a hazard.

5.5 The use of the argon/hydrogen gas mixture

during the dry and char steps may evolve a considerable

amount of HCI gas. Therefore, adequate ventilation is

required.

5.6 It is the responsibility of the user of this method to

comply with relevant disposal and waste regulations. For

guidance see Sections 14.0 and 15.0.

6.0 Equipment and Supplies

6.1 Graphite Furnace Atomic Absorption Spectrometer

6.1.1 The GFAA spectrometer must be capable of

programmed heating of the graphite tube and the

associated delayed atomization device. The instrument

must be equipped with Zeeman background correction

and the furnace device must be capable of utilizing an

alternate gas supply during specified cycles of the

analysis. The capability to record relatively fast (< 1 s)

transient signals and evaluate data on a peak area basis

is preferred. In addition, a recirculating refrigeration unit is

recommended for improved reproducibility of furnace

temperatures.

element electrodeless discharge lamps along with the

associated power supplies.

6.1.3 Argon gas supply (high-purity grade, 99.99%) for

use during the atomization of selenium, for sheathing the

furnace tube when in operation, and during furnace

cleanout. 

6.1.4 Alternate gas mixture (hydrogen 5%-argon 95%)

for use as a continuous gas flow environment during the

dry and char furnace cycles.

6.1.5 Autosampler capable of adding matrix modifier

solutions to the furnace, a single addition of analyte, and

completing methods of standard additions when required.

6.2 Analytical balance, with capability to measure to

0.1 mg, for preparing standards, and for determining

dissolved solids in digests or extracts.

6.3 A temperature adjustable hot plate capable of

maintaining a temperature of 95°C.

6.4 An air displacement pipetter capable of delivering

volumes ranging from 100 to 2500 μL with an assortment

of high quality disposable pipet tips.

6.5 Labware -- All reusable labware (glass, quartz,

polyethylene, PTFE, FEP, etc.) should be sufficiently

clean for the task objectives. Several procedures found to

provide clean labware include washing with a detergent

solution, rinsing with tap water, soaking for 4 h or more in

20% (v/v) nitric acid or a mixture of HCI and HNO3, rinsing

with reagent water and storing clean. Chromic acid

cleaning solutions must be avoided because chromium is

an analyte.

Note: Glassware having ground glass stoppers, etc.

should be avoided because the ground glass surface is

difficult to clean properly and can contain active sites

which adsorb metals.

6.5.1 Glassware -- Volumetric flasks, graduated cylinders, funnels and centrifuge tubes (glass and/or metalfree plastic).

6.5.2 Assorted calibrated pipettes.

6.5.3 Griffin beakers, 250-mL with 75-mm watch

glasses and (optional) 75-mm ribbed watch glasses.
200.12 - 7 Revision 1.0 September 1997

6.5.4 Narrow-mouth storage bottles, FEP (fluorinated that stock solutions be stored in FEP bottles. Replace

ethylene propylene) with screw closure, 125-mL to 1-L

capacities.

6.5.5 One-piece stem FEP wash bottle with screw closure, 125-mL capacity.

7.0 Reagents and Standards

7.1 Reagents may contain elemental impurities which

might affect analytical data. Only high-purity reagents that

conform to the American Chemical Society specifications6

should be used whenever possible. If the purity of a

reagent is in question, analyze for contamination. All acids

used for this method must be of ultra high-purity grade or

equivalent. Suitable acids are available from a number of

manufacturers. Redistilled acids prepared by sub-boiling

distillation are acceptable.

7.2 Nitric acid, concentrated (sp.gr. 1.41) HNO .3

7.2.1 Nitric acid (1+1) -- Add 500 mL concentrated

HNO to 400 mL reagent water and dilute to 1 L. 3

7.2.2 Nitric acid (1+5) -- Add 50 mL concentrated

HNO to 250 mL reagent water. 3

7.2.3 Nitric acid (1+9) -- Add 10 mL concentrated

HNO to 90 mL reagent water. 3

7.3 Reagent water. All references to water in this

method refer to ASTM Type I grade water.7

7.4 Ammonium hydroxide, concentrated

(sp.gr.0.902).

7.5 Matrix Modifier, dissolve 300 mg palladium (Pd)

powder in concentrated HNO3 (1 mL of HNO3, adding 10

μL of concentrated HCI if necessary). Dissolve 200 mg of

Mg(NO3)2@6H2O in ASTM Type I water. Pour the two

solutions together and dilute to 100 mL with ASTM Type

I water.

Note: It is recommended that the matrix modifier be

analyzed separately in order to assess the contribution of

the modifier to the absorbance of calibration and reagent

blank solutions.

7.6 Standard stock solutions may be purchased or

prepared from ultra-high purity grade chemicals (99.99 to

99.999% pure). All compounds must be dried for 1 h at

105EC, unless otherwise specified. It is recommended

stock standards when succeeding dilutions for preparation of calibration standards cannot be verified.

Caution: Many of these chemicals are extremely toxic if

inhaled or swallowed (Section 5.1). Wash hands thoroughly after handling.

Typical stock solution preparation procedures follow for

1-L quantities, but for the purpose of pollution prevention,

the analyst is encouraged to prepare smaller quantities

when possible. Concentrations are calculated based

upon the weight of the pure element or upon the weight

of the compound multiplied by the fraction of the analyte

in the compound.

From pure element,

Concentration = weight (mg)

volume (L)

From pure compound,

Concentration = weight (mg) x gravimetric factor

volume (L)

where:

gravimetric factor = the weight fraction of the analyte

in the compound.

7.6.1 Arsenic solution, stock, 1 mL = 1000 μg As: Dissolve 1.320 g of As2O3

 (As fraction = 0.7574), weighed

accurately to at least four significant figures, in 100 mL of

reagent water containing 10.0 mL concentrated NH OH. 4

Warm in solution gently to effect dissolution. Acidify the

solution with 20.0 mL concentrated HNO3 and dilute to

volume in a 1-L volumetric flask with reagent water.

7.6.2 Cadmium solution, stock, 1 mL = 1000 μg Cd:

Dissolve 1.000 g Cd metal, acid cleaned with (1+9) HNO ,3

weighed accurately to at least four significant figures, in

50 mL (1+1) HNO with heating to effect dissolution. Let 3

solution cool and dilute with reagent water in a 1-L

volumetric flask.

7.6.3 Chromium solution, stock, 1 mL = 1000 μg Cr:

Dissolve 1.923 g CrO3 (Cr fraction = 0.5200), weighed

accurately to at least four significant figures, in 120 mL (1

+5) HNO3. When solution is complete, dilute to volume in

a 1-L volumetric flask with reagent water.

7.6.4 Copper solution, stock, 1 mL = 1000 μg Cu: Dissolve 1.000 g Cu metal, acid cleaned with (1+9) HNO ,3
Revision 1.0 September 1997 200.12 - 8

weighed accurately to at least four significant figures, in the samples. The preparation blank must be carried

50.0 mL (1+1) HNO3 with heating to effect dissolution. Let through the entire sample digestion and preparation

solution cool and dilute in a 1-L volumetric flask with scheme.

reagent water.

7.6.5 Lead solution, stock, 1 mL = 1000 μg Pb:

Dissolve 1.599 g Pb(NO ) (Pb fraction = 0.6256), all analytes to provide a final concentration which will 3 2

weighed accurately to at least four significant figures, in a produce an absorbance of approximately 0.1 for each

minimum amount of (1+1) HNO3. Add 20.0 mL (1+1) analyte. The LFB must be carried through the complete

HNO and dilute to volume in a 1-L volumetric flask with procedure as used for the samples. 3

reagent water.

7.6.6 Nickel solution, stock, 1 mL = 1000 μg Ni:

Dissolve 1.000 g of nickel metal, weighed accurately to at the appropriate plastic containers.

least four significant figures, in 20.0 mL hot concentrated

HNO , cool, and dilute to volume in a 1-L volumetric flask 7.9 lnstrument Performance Check (IPC) Solution -- 3

with reagent water. The IPC solution is used to periodically verify instrument

7.6.7 Selenium solution, stock, 1 mL = 1000 μg Se: same acid mixture as the calibration standards by comDissolve 1.405 g SeO2

 (Se fraction = 0.7116), weighed

accurately to at least four significant figures, in 200 mL

reagent water and dilute to volume in a 1-L volumetric

flask with reagent water.

7.7 Preparation of Calibration Standards -- Fresh

calibration standards (CAL Solution) should be prepared

weekly, or as needed. Dilute each of the stock standard

solutions to levels appropriate to the operating range of

the instrument using the appropriate acid diluent. The

element concentrations in each CAL solution should be

sufficiently high to produce good measurement precision

and to accurately define the slope of the response curve.

The instrument calibration should be initially verified using

a IPC sample (Section 7.9).

7.8 Blanks -- Four types of blanks are required for

this method. A calibration blank is used to establish the

analytical calibration curve, the laboratory reagent blank

(LRB) is used to assess possible contamination from the

sample preparation procedure and to assess spectral

background, the laboratory fortified blank is used to

assess routine laboratory performance, and a rinse blank

is used to flush the instrument autosampler uptake system. All diluent acids should be made from concentrated

acids (Section 7.2) and ASTM Type I water.

7.8.1 The calibration blank consists of the appropriate

acid diluent in ASTM Type I water. The calibration blank

should be stored in a FEP bottle.

7.8.2 The laboratory reagent blanks must contain all

the reagents in the same volumes as used in processing

7.8.3 The laboratory fortified blank (LFB) is prepared

by fortifying an aliquot of the laboratory reagent blank with

7.8.4 The rinse blank is a 0.1% HCI and 0.1% HNO3

solution used to flush the autosampler tip and is stored in

performance during analysis. It should be prepared in the

bining method analytes at appropriate concentrations to

approximate the midpoint of the calibration curve. The

IPC solution should be prepared from the same standard

stock solutions used to prepare the calibration standards

and stored in a FEP bottle. Agency programs may specify

or request that additional instrument performance check

solutions be prepared at specified concentrations in order

to meet particular program needs.

7.10 Quality Control Sample (QCS) -- For initial and

periodic verification of calibration standards and instrument performance, analysis of a QCS is required. The

QCS must be obtained from an outside source different

from the standard stock solutions and prepared in the

same acid mixture as the calibration standards. The

concentration of the analytes in the QCS solution should

be such that the resulting solution will provide an absorbance reading of approximately 0.1. The QCS solution

should be stored in a FEP bottle and analyzed as needed

to meet data-quality needs. A fresh solution should be

prepared quarterly or as needed.

7.11 Matrix Performance Check (MPC) -- The MPC

solution is used to periodically evaluate the laboratory/

instrument performance in saline samples. It should be

prepared in the same acid mixture as the calibration

standards by combining method analytes at appropriate

concentrations in a seawater matrix (NASS-3, or its

equivalent) to produce an absorbance of 0.1. The MPC

solution should be prepared from the same standard

stock solutions used to prepare the calibration standards

and stored in a FEP bottle. The MPC sample should be

analyzed after every 10 samples to assure saline matrix

is not producing false positives.
200.12 - 9 Revision 1.0 September 1997

8.0 Sample Collection, Preservation and

Storage

8.1 Prior to collection of an aqueous sample,

consideration should be given to the type of data required.

Acid preservation should be performed at the time of

sample collection or as soon thereafter as practically

possible. The pH of all aqueous samples must be tested

immediately prior to aliquoting for analysis to ensure the

sample has been properly preserved. If properly acidpreserved, the sample can be held up to 6 months before

analysis.

8.2 For determination of total recoverable elements

in aqueous samples, acidify with (1+1) nitric acid at the

time of collection to pH<2. Normally, 3 mL of (1+1) nitric

acid (ultra high purity) per liter of sample is sufficient for

most ambient water samples. The sample should not be

filtered prior to analysis.

Note: Samples that cannot be acid-preserved at the

time of collection because of sampling limitations or

transport restrictions, or are > pH 2 because of high

alkalinity should be acidified with nitric acid to pH < 2 upon

receipt in the laboratory. Following acidification, the

sample should be held for 16 h and the pH verified to be

<2 before withdrawing an aliquot for sample processing.

8.3 For aqueous samples, a field blank should be

prepared and analyzed as required by the data user. Use

the same container and acid as used in sample collection.

9.0 Quality Control

9.1 Each laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of laboratory capability, and the periodic

analysis of laboratory reagent blanks, fortified blanks and

other laboratory solutions as a continuing check on

performance. The laboratory is required to maintain

performance records that define the quality of the data

thus generated.

9.2 Initial Demonstration of Performance 

(Mandatory)

9.2.1 The initial demonstration of performance is used

to characterize instrument performance (determination of

linear dynamic ranges and analysis of quality control

samples) and laboratory performance (determination of

method detection limits) prior to samples being analyzed

by this method.

9.2.2 Linear dynamic range (LDR) -- The upper limit of

the LDR must be established for the wavelength utilized

for each analyte by determining the signal responses

from a minimum of six different concentration standards

across the range, two of which are close to the upper limit

of the LDR. Determined LDRs must be documented and

kept on file. The linear calibration range which may be

used for the analysis of samples should be judged by the

analyst from the resulting data. The upper LDR limit

should be an observed signal no more than 10% below

the level extrapolated from the four lower standards. New

LDRs should be determined whenever there is a

significant change in instrument response, a change in

instrument analytical hardware or operating conditions.

Note: Multiple cleanout furnace cycles may be necessary in order to fully define or utilize the LDR for certain

elements such as chromium. For this reason, the upper

limit of the linear calibration range may not correspond to

the upper operational LDR limit.

Measured sample analyte concentrations that exceed the

upper limit of the linear calibration range must either be

diluted and reanalyzed (with concern for memory effects

Section 4.4) or analyzed by another approved method.

9.2.3 Quality control sample (QCS) -- When beginning

the use of this method, on a quarterly basis or as required

to meet data-quality needs, verify the calibration standards and acceptable instrument performance with the

preparation and analyses of a QCS (Section 7.10). If the

determined concentrations are not within ± 10% of the

stated values, performance of the determinative step of

the method is unacceptable. The source of the problem

must be identified and corrected before either proceeding

on with the initial determination of method detection limits

or continuing with ongoing analyses.

9.2.4 Method detection limit (MDL) -- MDLs must be

established for all analytes, using reagent water (blank)

fortified at a concentration of two to three times the

estimated instrument detection limit. To determine MDL 8

values, take seven replicate aliquots of the fortified

reagent water and process through the entire analytical

method. Perform all calculations defined in the method

and report the concentration values in the appropriate

units. Calculate the MDL as follows:
Revision 1.0 September 1997 200.12 - 10

MDL = (t) x (S) outside the required control limits of 85-115%, that

where, t = Student's t value for a 99% confidence level problem should be identified and resolved before

and a standard deviation estimate with n-1 degrees of continuing analyses.

freedom [t = 3.14 for seven replicates].

S = standard deviation of the replicate analyses.

Note: If the percent relative standard deviation (% RSD)

from the analyses of the seven aliquots is < 15%, the

concentration used to determine the analyte MDL may

have been inappropriately high for the determination. If

so, this could result in calculation of an unrealistically low

MDL. If additional confirmation of the MDL is desired,

reanalyze the seven replicate aliquots on two more

nonconsecutive days and again calculate the MDL values

for each day. An average of the three MDL values for

each analyte may provide a more appropriate MDL

estimate. Concurrently, determination of MDL in reagent

water represents a best case situation and does not

reflect possible matrix effects of real world samples.

However, successful analyses of LFMs (Section 9.4) and

the analyte addition test described in Section 9.5.1 can

give confidence to the MDL value determined in reagent

water. Typical single laboratory MDL values using this

method are given in Table 2.

MDLs should be determined every six months, when a

new operator begins work or whenever there is a signifi- 9.3.4 Instrument performance check (IPC) solution --

cant change in the background or instrument response.

The MDLs reported in Table 2 were determined in fortified NASS-3 samples. It is recommended that a certified

saline matrix such as NASS-3 be used to determine

MDLS.

9.3 Assessing Laboratory Performance (Mandatory)

9.3.1 Laboratory reagent blank (LRB) -- The laboratory

must analyze at least one LRB (Section 7.8.2) with each firms calibration to be outside the limits, sample analysis

batch of 20 or fewer samples. LRB data are used to must be discontinued, the cause determined and/or, in

assess contamination from the laboratory environment. the case of drift, the instrument recalibrated. All samples

LRB values that exceed the MDL indicate laboratory or following the last acceptable IPC solution must be reanareagent contamination should be suspected. Any deter- lyzed. Data for the calibration blank and IPC solution

mined source of contamination must be corrected and the must be kept on file with associated sample data.

samples reanalyzed for the affected analytes after

acceptable LRB values have been obtained. 9.3.5 Matrix performance check (MPC) solution -- For

9.3.2 Laboratory fortified blank (LFB) -- The laboratory

must analyze at least one LFB (Section 7.8.3) with each bration, after every tenth sample (or more frequently, if

batch of samples. Calculate accuracy as percent recov- required) and after the last sample in the batch is anaery (Section 9.4.3). If the recovery of any analyte falls lyzed. Analysis of the MPC must verify that the instrument

analyte is judged out of control, and the source of the

9.3.3 The laboratory must use LFB analyses data to

assess laboratory performance against the required

control limits of 85-115%. When sufficient internal performance data become available (usually a minimum of 20-

30 analyses), optional control limits can be developed

from the percent mean recovery (x) and the standard

deviation (S) of the mean recovery. These data can be

used to establish the upper and lower control limits as

follows:

Upper Control Limit = x + 3S

Lower Control Limit = x - 3S

The optional control limits must be equal to or better than

the required control limits of 85-115%. After each five to

ten new recovery measurements, new control limits can

be calculated using only the most recent 20-30 data

points. Also, the standard deviation (S) data should be

used to establish an ongoing precision statement for the

level of concentrations included in the LFB. These data

must be kept on file and be available for review.

For all determinations the laboratory must analyze the

IPC solution (Section 7.9) and a calibration blank

immediately following daily calibration, after every tenth

sample (or more frequently, if required) and after the last

sample in the batch is analyzed. Analysis of the I PC

solution and calibration blank immediately following

calibration must verify that the instrument is within ± 5% of

calibration. Subsequent analyses of the IPC solution must

verify the calibration within ± 10%. If the calibration cannot

be verified within the specified limits, reanalyze the IPC

solution. If the second analysis of the IPC solution conall determinations, the laboratory must analyze the MPC

solution (Section 7.11) immediately following daily cali-
200.12 - 11 Revision 1.0 September 1997

is within ± 15% of calibration and confirm that the matrix 9.4.4 If the recovery of any analyte falls outside the

is not causing matrix/background interferences. If the

MPC is not within ± 15%, reanalyze the MPC solution. If

the second analysis of the MPC solution is outside the

limits, sample analysis must be discontinued the cause

determined and/or, in the case of drift, the instrument

recalibrated. All samples following the last acceptable

MPC solution must be reanalyzed. The analysis data for

the calibration blank and MPC solution must be kept on

file with the sample analyses data.

9.4 Assessing Analyte Recovery and Data Quality

9.4.1 Sample homogeneity and the chemical nature of

the sample matrix can affect analyte recovery and the recovery of the LFM should be calculated. If the fortified

data quality. Taking separate aliquots from the sample for analyte in the diluted LFM is found to be < 25% of the

replicate and fortified analyses can in some cases assess sample concentration or the diluted LFM produces an

these effects. Unless otherwise specified by the data user, atomic signal of <10 times the MDL, the diluted sample

laboratory or program, the following laboratory fortified should be analyzed by methods of standard addition. If

matrix (LFM) procedure (Section 9.4.2) is required. Also, the calculated recovery of the diluted sample is within the

the analyte addition test (Section 9.5.1) can aid in designated range, the sample concentration should be

identifying matrix interferences. However, all samples calculated from the diluted sample. If the calculated

must have a background absorbance < 1.0 before the recovery of the diluted sample is outside the designated

test results obtained can be considered reliable. range, follow the directions given below. If the back9.4.2 The laboratory must add a known amount of dilution, all samples of a similar matrix should be diluted

each analyte to a minimum of 10% of the routine

samples. In each case the LFM aliquot must be a

duplicate of the aliquot used for sample analysis and for

total recoverable determinations added prior to sample

preparation. For water samples, the added analyte

concentration must be the same as that used in the

laboratory fortified blank (Section 9.3.2).

9.4.3 Calculate the percent recovery for each analyte,

corrected for concentrations measured in the unfortified

sample, and compare these values to the designated

LFM recovery range of 75-125%. Recovery calculations

are not required if the concentration added is less than

25% of the unfortified sample concentration. Percent

recovery may be calculated in units appropriate to the

matrix, using the following equation:

R = Cs - C x 100

s

where, R = percent recovery.

C = fortified sample concentration. s

C = sample background concentration.

s = concentration equivalent of analyte

added to sample.

designated LFM recovery range (but is still within the

range of calibration and the background absorbance is <

1.0 abs.) and the laboratory performance for that analyte

is shown to be in control (Section 9.3), the recovery

problem encountered with the LFM is judged to be either

matrix or solution related, not system related. A flowchart

of the remainder of this section can be found in Figure 6.

This flowchart may clarify the verbal discussion given

below.

If the background absorbance is > 1 abs., the sample and

the LFM should be diluted 1:3 and reanalyzed until the

background absorbance is < 1, at which point a percent

ground is reduced and/or the matrix effect is reduced by

and analyzed in a similar fashion. The result should be

flagged indicating the methods sensitivity has been reduced by the dilution. If dilution is unacceptable because

of data quality objectives the sample should be flagged

indicating the analysis is not possible via this analytical

procedure.

If the analyte recovery on the LFM is <75% and the

background absorbance is <1, complete the analyte

addition test (Section 9.5.1) on the original sample (or its

dilution). The results of the test should be evaluated as

follows:

1. If recovery of the analyte addition test (< 85%)

confirms a low recovery for the LFM, a suppressive

matrix interference is indicated and the unfortified

sample aliquot must be analyzed by method of

standard additions (Section 11.3).

2. If the recovery of the analyte addition test is between

85% to 115%, a low recovery of the analyte in the

LFM (< 75%) may be related to the heterogeneity of

the sample, sample preparation or a poor transfer,

etc. Report the sample concentration based on the

unfortified sample aliquot.
Revision 1.0 September 1997 200.12 - 12

3. If the recovery of the analyte addition test is less prevents successful determination of the analyte or when

than recovery calculated for the LFM, matrix sup- the heterogeneous nature of the sample precludes the

pression is confirmed. The unfortified sample use of duplicate analyses.

should be analyzed by MSA (Section 11.3).

Significantly lower recoveries (relative to the LFM) 9.4.6 Where reference materials are available, they

associated with the analyte addition test are

unlikely unless the sample is heterogeneous.

4. If the recovery of the analyte addition test is >115%,

the dramatic change in analyte response should be

verified by fortifying the LFM. The recovery in the

sample and the recovery in the LFM should be

compared. If the recoveries verify the dramatic

response difference, the sample results should be

flagged indicating the sample matrix is not homogeneous.

If the analyte recovery in the LFM is > 125% and the

background absorbance is < 1, complete the analyte

addition test (Section 9.5.1) on the unfortified sample (or

its dilution) aliquot.

1 . If the percent recovery of the analyte addition test is

> 115% and the LFB does not indicate laboratory

contamination, an enhancing matrix interference

(albeit rare) is indicated, and the unfortified sample

aliquot must be analyzed by method of standard

additions (Section 11.3).

2. If the percent recovery of the analyte addition test is

between 85% to 115%, either random sample contamination of the LFM, an incorrect analyte concentration was added to the LFM prior to sample

preparation, or sample heterogeneity should be

suspected. Report analyte data determined from the

analysis of the unfortified sample aliquot.

3. If the percent recovery of the analyte addition test is

< 85%, a heterogeneous sample with matrix interference is suspected. This dramatic change in response should be verified by performing the analyte

addition test to the LFM. The recovery in the sample

and the recovery in the LFM should be compared. If

the recoveries verify the dramatic response difference the sample results should be flagged indicating

the sample matrix is not homogeneous.

9.4.5 If the analysis of a LFM sample(s) and the test

routines above indicate an operative interference and the

LFMs are typical of the other samples in the batch, those

samples that are similar must be analyzed in the same

manner as the LFMS. Also, the data user must be

informed when a matrix interference is so severe that it

should be analyzed to provide additional performance

data. Analysis of reference samples is a valuable tool for

demonstrating the ability to perform the method acceptably. It is recommended that NASS-3 or its equivalent be

fortified and used as an MPC.

9.5 Matrix interference effects and the need for MSA

can be assessed by the following test. Directions for using

MSA are given in Section 11.3.

9.5.1 Analyte addition test: An analyte standard added

to a portion of a prepared sample or its dilution should be

recovered to within 85-115% of the known value. The

analyte addition should occur directly to sample in the

furnace and should produce a minimum absorbance of

0.1. The concentration of the analyte addition plus that in

the sample should not exceed the linear calibration range

of the analyte. If the analyte is not recovered within the

specified limits, a matrix effect should be suspected and

the sample must be analyzed by MSA.

10.0 Calibration and Standardization

10.1 Specific wavelengths and instrument operating

conditions are listed in Table 1. However, because of

differences among makes and models of spectrophotometers and electrothermal furnace devices, the actual

instrument conditions selected may vary from those listed.

10.2 Prior to the use of this method, the instrument

operating conditions must be optimized. The analyst

should follow the instructions provided by the manufacturer while using the conditions listed in Table 1 as a

guide. The appropriate charring condition for each of the

analytes is a critical part of the metal analysis in saline

waters; therefore, the char temperature profiles should be

determined in a saline water matrix. The appropriate

charring temperature should be chosen so as to minimize

background absorbance while providing some furnace

temperature variation without the loss of analyte. For

analytical operation, the charring temperature is usually

set at least 100EC below the point at which analyte begins

to be lost during the char. Because the background

absorbance can be affected by the atomization temperature, care should be taken in the choice of an appropriate

atomization temperature. The optimum conditions se-
200.12 - 13 Revision 1.0 September 1997

lected should provide the lowest reliable MDLs and be 11.1.3 Cover the lip of the beaker with a watch glass to

similar to those listed in Table 2. Once the optimum

operating conditions are determined, they should be

recorded and available for daily reference. The effectiveness of these operating conditions are continually evaluated by analyzing the MPC.

10.3 Prior to an initial calibration the linear dynamic

range of the analyte must be determined (Sect 9.2.2)

using the optimized instrument operating conditions. For

all determinations allow an instrument and hollow cathode lamp warm-up period of not less than 15 min. If an

EDL is to be used, allow 30 min for warm-up.

10.4 Before using the procedure (Section 11.0) to analyze samples, there must be data available documenting

initial demonstration of performance. The required data

and procedure are described in Section 9.2. This data

must be generated using the same instrument operating

conditions and calibration routine to be used for sample

analysis. These documented data must be kept on file

and be available for review by the data user.

11.0 Procedure

11.1 Aqueous Sample Preparation -- Total Recoverable Analytes

11.1.1 Add 2 mL (1+1) nitric acid to the beaker

containing 100 mL of sample. Place the beaker on a hot

plate for solution evaporation. The hot plate should be

located in a fume hood and previously adjusted to provide

evaporation at a temperature of approximately but no

higher than 85EC. (See the following note.) The beaker

should be covered with an elevated watch glass or other

necessary steps should be taken to prevent sample

contamination from the fume hood environment.

Note: For proper heating adjust the temperature control

of the hot plate such that an uncovered Griffin beaker

containing 50 mL of water placed in the center of the hot

plate can be maintained at a temperature approximately

but no higher than 85EC. (Once the beaker is covered

with a watch glass the temperature of the water will rise

to approximately 95EC.)

11.1.2 Reduce the volume of the sample aliquot to

about 20 mL by gentle heating at 85EC. DO NOT BOIL.

This step takes about 2 h for a 100-mL aliquot with the

rate of evaporation rapidly increasing as the sample

volume approaches 20 mL. (A spare beaker containing

20 mL of water can be used as a gauge.)

reduce additional evaporation and gently reflux the

sample for 30 min.

11.1.4 Allow the beaker to cool. Quantitatively transfer

the sample solution to a 100-mL volumetric flask, dilute

to volume with reagent water, stopper and mix.

11.1.5 Allow any undissolved material to settle overnight, or centrifuge a portion of the prepared sample until

clear. (If after centrifuging or standing overnight the

sample contains suspended solids, a portion of the

sample may be filtered prior to analysis. However, care

should be exercised to avoid potential contamination from

filtration.) The sample is now ready for analysis. Because

the effects of various matrices on the stability of diluted

samples cannot be characterized, all analyses should be

performed as soon as possible after the completed

preparation.

11.2 Sample Analysis

11.2.1 Prior to daily calibration of the instrument, inspect the graphite tube and contact rings for salt

buildup, etc. Generally, it will be necessary to clean the

contact rings and replace the graphite tube daily. The

contact rings are a cooler environment in which salts

can deposit after atomization. A cotton swab dipped in a

50/50 mixture of isopropyl alcohol (IPA) and H2O (such

that it is damp but not dripping) can be used to remove

the majority of the salt buildup. A second cotton swab is

dipped in IPA and the contact rings are wiped down to

assure they are clean. The rings are then allowed to

thoroughly dry and then a new tube is placed in the

furnace and conditioned according to instrument

manufacturer's specifications.

11.2.2 Configure the instrument system to the selected

optimized operating conditions as determined in Sections

10.1 and 10.2.

11.2.3 Before beginning daily calibration the instrument

should be reconfigured to the optimized conditions. Initiate the data system and allow a period of not less than

15 min for instrument and hollow cathode lamp warm up.

If an EDL is to be used, allow 30 min for warm up.

11.2.4 After the warm up period but before calibration,

instrument stability must be demonstrated by analyzing a

standard solution with a concentration 20 times the IDL a

minimum of five times. The resulting relative standard

deviation of absorbance signals must be < 5%. If the
Cx'

SBVSCS

(SA&SB)VX

Revision 1.0 September 1997 200.12 - 14

relative standard deviation is > 5%, determine and correct 11.2.11 When it is necessary to assess an operative

the cause before calibrating the instrument.

11.2.5 For initial and daily operation, calibrate the instrument according to the instrument manufacturer's

recommended procedures using the calibration blank 11.2.12 Report data as directed in Section 12.

(Section 7.8.1) and calibration standards (Section 7.7)

prepared at three or more concentrations within the

usable linear dynamic range of the analyte (Sections 4.4

and 9.2.2).

11.2.6 An autosampler must be used to introduce all

solutions into the graphite furnace. Once the sample and

the matrix modifier are injected, the furnace controller

completes a set of furnace cycles and a cleanout period

as programmed. Analyte signals must be reported on an

integrated absorbance basis. Background absorbances,

background heights and the corresponding peak profiles

should be displayed to the CRT for review by the analyst

and be available as hard copy for documentation to be

kept on file. Flush the autosampler solution uptake system with the rinse blank (Section 7.8.4) between each

solution injected.

11.2.7 After completion of the initial requirements of

this method (Section 9.2), samples should be analyzed

in the same operational manner used in the calibration

routine.

11.2.8 During sample analyses, the laboratory must

comply with the required quality control described in

Sections 9.3 and 9.4.

11.2.9 For every new or unusual matrix, when practical, where, S and S are the analytical signals (corrected

it is highly recommended that an inductively coupled

plasma atomic emission spectrometer be used to screen

for high element concentration. Information gained from

this may be used to prevent potential damage to the

instrument and to better estimate which elements may

require analysis by graphite furnace.

11.2.10 Determined sample analyte concentrations that

are > 90% of the upper limit of calibration must either

be diluted with acidified reagent water and reanalyzed

with concern for memory effects (Section 4.4), or

determined by another approved but less sensitive

procedure. Samples with background absorbances > 1

must be diluted with appropriate acidified reagent water

such that the background absorbance is < 1 (Section

9.4.4). If the method of standard additions is required,

follow the instructions described in Section 11.3.

matrix interference (e.g., signal reduction due to high

dissolved solids), the test described in Section 9.5 is

recommended.

11.3 Standard Additions -- If the method of standard

addition is required, the following procedure is recommended:

11.3.1 The standard addition technique involves pre9

paring new standards in the sample matrix by adding

known amounts of standard to one or more aliquots of the

processed sample solution. This technique compensates

for a sample constituent that enhances or depresses the

analyte signal, thus producing a different slope from that

of the calibration standards. It will not correct for additive

interference, which causes a baseline shift. The simplest

version of this technique is the single-addition method.

The procedure is as follows: Two identical aliquots of the

sample solution, each of volume V , are taken. To the first x

(labeled A) is added a small volume VS of a standard

analyte solution of concentration CS. To the second

(labeled B) is added the same volume VS of the solvent.

The analytical signals of A and B are measured and

corrected for nonanalyte signals. The unknown sample

concentration CX is calculated:

A B

for the blank) of solutions A and B, respectively. VS and

CS should be chosen so that SA is roughly twice SB on

the average. It is best if VS is made much less than VX,

and thus CS is much greater than CX, to avoid excess

dilution of the sample matrix. If a separation or

concentration step is used, the additions are best made

first and carried through the entire procedure. For the

results from this technique to be valid, the following

limitations must be taken into consideration:

1. The analytical curve must be linear.

2. The chemical form of the analyte added must respond in the same manner as the analyte in the

sample.

3. The interference effect must be constant over the

working range of concern.
200.12 - 15 Revision 1.0 September 1997

4. The signal must be corrected for any additive inter- EPA has established a preferred hierarchy of environference. mental management techniques that places pollution

12.0 Data Analysis and Calculations

12.1 Sample data should be reported in units of Fg/L

for aqueous samples.

12.2 For total recoverable aqueous analytes (Section

11.1), when 100-mL aliquot is used to produce the 100

mL final solution, round the data to the tenths place and

report the data in Fg/L up to three significant figures. If a

different aliquot volume other than 100 mL is used for

sample preparation, adjust the dilution factor accordingly.

Also, account for any additional dilution of the prepared

sample solution needed to complete the determination of

analytes exceeding the upper limit of the calibration curve.

Do not report data below the determined analyte MDL

concentration or below an adjusted detection limit

reflecting smaller sample aliquots used in processing or 15.1 The Environmental Protection Agency requires

additional dilutions required to complete the analysis. that laboratory waste management practices be con12.3 The QC data obtained during the analyses tions. The Agency urges laboratories to protect the air,

provide an indication of the quality of the sample data and water, and land by minimizing and controlling all releases

should be provided with the sample results. from hoods and bench operations, complying with the

13.0 Method Performance

13.1 Instrument operating conditions used for single

laboratory testing of the method and MDLs are listed in

Tables 1 & 2.

13.2 Table 3 contains precision and recovery data obtained from a single laboratory analysis of four fortified

sample replicates of NASS-3. Five unfortified replicates

were analyzed, and their average concentration was used

to determine the sample concentration. Samples were

prepared using the procedure described in Section 11.1.

Four samples were fortified at the levels reported in Table

3. Average percent recovery and percent relative

standard deviation are reported in Table 3 for the fortified

samples.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

prevention as the management option of first choice.

Whenever feasible, laboratory personnel should use

pollution prevention techniques to address their waste

generation. When wastes cannot be feasibly reduced at

the source, the Agency recommends recycling as the

next best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society's Department of Government

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

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

15.0 Waste Management

ducted consistent with all applicable rules and regulaletter and spirit of any sewer discharge permits and

regulations, and by complying with all solid and hazardous waste regulations, particularly the hazardous waste

identification rules and land disposal restrictions. For

further information on waste management consult The

Waste Management Manual for Laboratory Personnel,

available from the American Chemical Society at the

address listed in the Section 14.2.

16.0 References

1. Pruszkowska, E., G. Carnrick, and W. Slavin. Anal.

Chem. 55,182-186,1983.

2. Carcinogens - Working With Carcinogens,

Department of Health, Education, and Welfare,

Public Health Service, Centers for Disease Control,

National Institute for Occupational Safety and

Health, Publication No. 77-206, Aug. 1977.

3. OSHA Safety and Health Standards, General

Industry, (29 CFR 1910), Occupational Safety and

Health Administration, OSHA 2206, (Revised,

January 1976).
Revision 1.0 September 1997 200.12 - 16

4. Safety in Academic Chemistry Laboratories,

American Chemical Society Publication,

Committee on Chemical Safety, 3rd Edition, 1979.

5. Proposed OSHA Safety and Health Standards,

Laboratories, Occupational Safety and Health

Administration, Federal Register, July 24,1986.

6. Rohrbough, W.G. et al. Reagent Chemicals,

American Chemical Society Specifications, 7th

edition. American Chemical Society, Washington,

DC, 1986.

7. American Society for Testing and Materials.

Standard Specification for Reagent Water, D1193-

77. Annual Book of ASTM Standards, Vol. 11.01.

Philadelphia, PA, 1991.

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

Appendix B.

9. Winefordner, J.D., Trace Analysis: Spectroscopic

Methods for Elements, Chemical Analysis, Vol. 46,

pp. 41-42, 1976.
200.12 - 17 Revision 1.0 September 1997

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Table 1. Furnace Conditions for Determination of Metals in Seawater 1

Element Wavelength (nm) Method of Furnaces Temp Temp 5

Slit Width (nm) Analysis Modifier Cycle EC Ramp Hold Time (sec) 2,3

As 193.7 Direct Pd/Mg Dry 130 1 60

0.7 Char 1400 10 60 4

Atomization 2200 0 5

Cd 228.8 Matrix Match Pd/Mg Dry 130 1 60

0.7 Standard + Char 1 350 45 30

or 600 Fg Char 2 850 1 30

 Std. Addition NH4NO3 Atomization 1500 0 5

Cr 357.9 Direct Pd/Mg Dry 130 1 60

0.7 Char 1500 5 30

 Atomization 2600 0 5

Cu 324.8 Direct Pd/Mg Dry 130 1 60

0.7 Char 1300 10 30

Atomization 2600 0 5

Ni 232.4 Direct Pd/Mg Dry 130 1 60

0.2 Char 1400 10 30 4

Atomization 2600 0 7

Pb 283.3 Direct Pd/Mg Dry 130 1 60

0.7 Char 1200 10 45

Atomization 2200 0 5

 Se 196.0 Matrix Match Pd/Mg Dry 130 1 60

2.0 Standard 9% HNO3 on Char 1000 5 60

or Platform Atomization 2100 0 5

Std. Addition

 10-FL sample size. 1

 5FL of (30 mg Pd Powder and 20 mg Mg(NO ) @6H O to 10 mL). 2

3 2 2

 A gas mixture of 5% H in 95% Ar is used during the dry and char. 3

2

 Sodium emission is visibly exiting from the sample inlet port. 4

 The furnace program has a cool down step of 20E between char and atomization and a clean out step of 2600E C after atomization. 5

Table 2. MDLs and Background Absorbances Associated with a Fortified NASS-31-3

Typical

Integrated

MDL Background 5

Element Fg/L Absorbances6

Cd 0.1 1.2

Cr - 0.2

Cu 2.8 0.2

Ni 1.8 0.1

Pb 2.4 0.4

Se 9.5 1.4 4

As 2.6 0.3 4

 Matrix Modifier = 0.015 mg Pd + 0.01 mg Mg(NO ) .

1

3 2

 A 5% H in Ar gas mix is used during the dry and char steps at 300 mL/min for all elements. 2

2

 10-FL sample size. 3

 An electrodeless discharge lamp was used for this element. 4

 MDL calculated based on fortifying NASS-3 with metal analytes. 5

 Background absorbances are affected by the atomization temperature for analysis, therefore, lowering atomization temperatures may be 6

advantageous if large backgrounds are observed.

- Not Determined.
Revision 1.0 September 1997 200.12 - 18

Table 3. Precision and Recovery Data for Fortified NASS-3

Certified Observed Fortified Fortified

Value Value Conc. Avg. Conc. Avg.

Element Fg/L Fg/L Fg/L Recovery, % %RSD Fg/L Recovery, % % RSD 2

As 1.65 ± 0.19 < MDL 15 89 3.6 37.5 85 1.6

Cd 0.029 ± 0.004 < MDL 1.0 107 4.5 2.5 104 3.8 1

Cr 0.175 ± 0.010 < MDL 5 88 0.7 12.5 85 1.6

Cu 0.109 ± 0.011 < MDL 15 95 4.4 37.5 91 0.9

Pb 0.039 ± 0.006 < MDL 15 103 2.3 37.5 99 3.4

Ni 0.257 ± 0.027 < MDL 15 92 10.1 37.5 93 7.1

Se 0.024 ± 0.004 < MDL 25 101 2.9 62.5 99 3.9 1

 Standards were made in 10,000 ppm NaCl for this analysis. 1

 Determined from four sample replicates. 2
200.12 - 19 Revision 1.0 September 1997
Revision 1.0 September 1997 200.12 - 20
200.12 - 21 Revision 1.0 September 1997
200.13-1

Method 200.13

Determination of Trace Elements in Marine Waters by Off-Line

Chelation Preconcentration with Graphite Furnace Atomic Absorption

John T. Creed and Theodore D. Martin

Chemical Expsoure Research Branch

Human Exposure Research Division

Revision 1.0

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Revision 1.0 September 1997 200.13 - 2

Chemical Abstracts Service

Element Registry Numbers (CASRN)

Cadmium (Cd) 7440-43-9

Cobalt (Co) 7440-48-4

Copper (Cu) 7440-50-8

Lead (Pb) 7439-92-1

Nickel (Ni) 7440-02-0

Method 200.13

Determination of Trace Elements in Marine Waters by Off-Line Chelation

Preconcentration with Graphite Furnace Atomic Absorption

1.0 Scope and Application

1.1 This method describes procedures for preconcentration and determination of total recoverable trace

elements in marine waters, including estuarine water,

seawater and brines.

1.2 Acid solubilization is required prior to determination of total recoverable elements to facilitate breakdown

of complexes or colloids which might influence trace

element recoveries. This method should only be used for

preconcentration and determination of trace elements in

aqueous samples. 3.1 Calibration Blank (CB) -- A volume of reagent

1.3 This method is applicable to the following standards, but without the analytes, internal standards, or

elements: surrogate analytes.

1.4 Method detection limits (MDLs) for these reagent water or other blank matrix that is placed in a

elements will be dependent on the specific sample container in the laboratory and treated as a

instrumentation employed and the selected operating sample in all respects, including shipment to the sampling

conditions. MDLs in NASS-3 (Reference Material, site, exposure to sampling site conditions, storage,

National Research Council of Canada) were determined preservation, and all analytical procedures. The purpose

using the procedure described in Section 9.2.4 and are of the FRB is to determine if method analytes or other

listed in Table 1. interferences are present in the field environment.

1.5 A minimum of 6-months experience in graphite 3.4 Instrument Performance Check Solution (IPC)

furnace atomic absorption (GFAA) is recommended. -- A solution of one or more method analytes, surrogates,

2.0 Summary of Method

2.1 Nitric acid is dispensed into a beaker containing

an accurately weighed or measured, well-mixed,

homogeneous aqueous sample. The sample volume is

reduced to approximately 20 mL and then covered and

allowed to reflux. The resulting solution is diluted to

volume and is ready for analysis.

2.2 This method is used to preconcentrate trace elements using an iminodiacetate functionalized chelating

resin. Following acid solubilization, the sample is buff- 1,2

ered using an on-line system prior to entering the chelating column. Group I and II metals, as well as most

anions, are selectively separated from the analytes by

elution with ammonium acetate at pH 5.5. The analytes

are subsequently eluted into a simplified matrix consisting

of 0.75 M nitric acid and are determined by GFAA.

3.0 Definitions

water fortified with the same matrix as the calibration

3.2 Calibration Standard (CAL) -- A solution prepared from the primary dilution standard solution or stock

standard solutions and the internal standards and surrogate analytes. The CAL solutions are used to calibrate

the instrument response with respect to analyte concentration.

3.3 Field Reagent Blank (FRB) -- An aliquot of

internal standards, or other test substances used to

evaluate the performance of the instrument system with

respect to a defined set of criteria.

3.5 Laboratory Fortified Blank (LFB) -- An aliquot

of reagent water or other blank matrices to which known

quantities of the method analytes are added in the

laboratory. The LFB is analyzed exactly like a sample,

and its purpose is to determine whether the methodology
200.13 - 3 Revision 1.0 September 1997

is in control, and whether the laboratory is capable of trated solution containing one or more method analytes

making accurate and precise measurements. prepared in the laboratory using assayed reference ma3.6 Laboratory Fortified Sample Matrix (LFM) -- -

An aliquot of an environmental sample to which known 3.14 Total Recoverable Analyte (TRA) -- The conquantities of the method analytes are added in the centration of analyte determined to be in either a solid

laboratory. The LFM is analyzed exactly like a sample, sample or an unfiltered aqueous sample following treatand its purpose is to determine whether the sample matrix ment by refluxing with hot dilute mineral acid(s) as

contributes bias to the analytical results. The background specified in the method.

concentrations of the analytes in the sample matrix must

be determined in a separate aliquot and the measured

values in the LFM corrected for background

concentrations.

3.7 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water or other blank matrices that are treated

exactly as a sample including exposure to all glassware,

equipment, solvents, reagents, internal standards, and

surrogates that are used with other samples. The LRB is

used to determine if method analytes or other

interferences are present in the laboratory environment,

the reagents, or the apparatus.

3.8 Linear Dynamic Range (LDR) -- The absolute

quantity or concentration range over which the instrument

response to an analyte is linear.

3.9 Matrix Modifier (MM) -- A substance added to

the instrument along with the sample in order to minimize

the interference effects by selective volatilization of either

analyte or matrix components.

3.10 Method Detection Limit (MDL) -- The minimum

concentration of an analyte that can be identified, measured and reported with 99% confidence that the analyte

concentration is greater than zero.

3.11 Quality Control Sample -- A solution of method

analytes of known concentrations which is used to fortify

an aliquot of LRB or sample matrix. The QCS is obtained

from a source external to the laboratory and different

from the source of calibration standards. It is used to

check laboratory performance with externally prepared

test materials.

3.12 Standard Addition -- The addition of a known

amount of analyte to the sample in order to determine the

relative response of the detector to an analyte within the

sample matrix. The relative response is then used to

assess either an operative matrix effect or the sample

analyte concentration.

3.13 Stock Standard Solution (SSS) -- A concenterials or purchased from a reputable commercial source.

4.0 Interferences

4.1 Several interference sources may cause

inaccuracies in the determination of trace elements by

GFAA. These interferences can be classified into three

major subdivisions: spectral, matrix, and memory. Some

of these interferences can be minimized via the preconcentration step, which reduces the Ca, Mg, Na and Cl

concentration in the sample prior to GFAA analysis.

4.2 Spectral interferences are caused by absorbance

of light by a molecule or atom which is not the analyte of

interest or emission from black body radiation.

4.2.1 Spectral interferences caused by an element only

occur if there is a spectral overlap between the wavelength of the interfering element and the analyte of

interest. Fortunately, this type of interference is relatively

uncommon in STPGFAA (Stabilized Temperature Platform Graphite Furnace Atomic Absorption) because of

the narrow atomic line widths associated with STPGFAA.

In addition, the use of appropriate furnace temperature

programs and high spectral purity lamps as light sources

can minimize the possibility of this type of interference.

However, molecular absorbances can span several hundred manometers, producing broadband spectral interferences. This type of interference is far more common

in STPGFAA. The use of matrix modifiers, selective

volatilization, and background correctors are all attempts

to eliminate unwanted nonspecific absorbance. Because

the nonspecific component of the total absorbance can

vary considerably from sample type to sample type, to

provide effective background correction and eliminate the

elemental spectral interference of palladium on copper

and iron on selenium, the exclusive use of Zeeman

background correction is specified in this method.

4.2.2 Spectral interferences are also caused by emissions from black body radiation produced during the

atomization furnace cycle. This black body emission 
Revision 1.0 September 1997 200.13 - 4

reaches the photomultiplier tube, producing erroneous

results. The magnitude of this interference can be minimized by proper furnace tube alignment and monochromator design. In addition, atomization temperatures

which adequately volatilize the analyte of interest without

producing unnecessary black body radiation can help reduce unwanted background emission produced during

atomization.

4.3 Matrix interferences are caused by sample cornponents which inhibit formation of free atomic analyte

atoms during the atomization cycle. In this method the

use of a delayed atomization device which provides

warmer gas phase temperatures is required. These

devices provide an environment which is more conducive

to the formation of free analyte atoms and thereby

minimize this type of interference. This type of interference can be detected by analyzing the sample plus a

sample aliquot fortified with a known concentration of the

analyte. If the determined concentration of the analyte

addition is outside a designated range, a possible matrix

effect should be suspected (Section 9.4).

4.4 Memory interferences result from analyzing a

sample containing a high concentration of an element

(typically a high atomization temperature element) which

cannot be removed quantitatively in one complete set of

furnace steps. The analyte which remains in the furnace

can produce false positive signals on subsequent

sample(s). Therefore, the analyst should establish the

analyte concentration which can be injected into the

furnace and adequately removed in one complete set of

furnace cycles. If this concentration is exceeded, the

sample should be diluted and a blank analyzed to assure

the memory effect has been eliminated before reanalyzing the diluted sample.

4.5 Low recoveries may be encountered in the

preconcentration cycle if the trace elements are

complexed by competing chelators (humic/fulvic) in the

sample or are present as colloidal material. Acid solubilization pretreatment is employed to improve analyte

recovery and to minimize adsorption, hydrolysis and

precipitation effects.

4.6 Memory interferences from the chelating system

may be encountered, especially after analyzing a sample

containing high analyte concentrations. A thorough column rinsing sequence following elution of the analytes is

necessary to minimize such interferences.

5.0 Safety

5.1 The toxicity or carcinogenicity of each reagent

used in this method has not been fully established. Each

chemical should be regarded as a potential health hazard

and exposure to these compounds should be as low as

reasonably achievable. Each laboratory is responsible for

maintaining a current awareness file of OSHA regulations

regarding the safe handling of the chemicals specified in

this method. A reference file of material data handling 3-6

sheets should also be made available to all personnel

involved in the chemical analysis. Specifically,

concentrated nitric and hydrochloric acids present various

hazards and are moderately toxic and extremely irritating

to skin and mucus membranes. Use these reagents in a

fume hood whenever possible and if eye or skin contact

occurs, flush with large volumes of water. Always wear

safety glasses or a shield for eye protection, protective

clothing and observe proper mixing when working with

these reagents.

5.2 Acidification of samples containing reactive materials may result in release of toxic gases, such as cyanides or sulfides. Samples should be acidified in a fume

hood.

5.3 All personnel handling environmental samples

known to contain or to have been in contact with human

waste should be immunized against known disease

causative agents.

5.4 The graphite tube during atomization emits intense UV radiation. Suitable precautions should be taken

to protect personnel from such a hazard.

5.5 The use of the argon/hydrogen gas mixture

during the dry and char steps may evolve a considerable

amount of HCI gas. Therefore, adequate ventilation is

required.

5.6 It is the responsibility of the user of this method to

comply with relevant disposal and waste regulations. For

guidance see Sections 14.0 and 15.0.

6.0 Equipment and Supplies

6.1 Graphite Furnace Atomic Absorption 

Spectrometer

6.1.1 The GFAA spectrometer must be capable of

programmed heating of the graphite tube and the
200.13 - 5 Revision 1.0 September 1997

associated delayed atomization device. The instrument 6.2.5 Eluent pumping system (Gradient Pump) -- Proshould be equipped with an adequate background

correction device capable of removing undesirable nonspecific absorbance over the spectral region of interest.

The capability to record relatively fast (< 1 sec) transient

signals and evaluate data on a peak area basis is 6.2.6 System setup, including sample loop (See

preferred. In addition, a recirculating refrigeration unit is Figure 1).

recommended for improved reproducibility of furnace

temperatures. The data shown in the tables were

obtained using the stabilized temperature platform and

Zeeman background correction.

6.1.2 Single element hollow cathode lamps or single

element electrodeless discharge lamps along with the pump (Dionex QIC pump or equivalent) for delivering 2M

associated power supplies. ammonium acetate buffer solution; carrier pump, peri6.1.3 Argon gas supply (high-purity grade, 99.99%). 1% nitric acid carrier solution; sample pump, peristaltic

6.1.4 A 5% hydrogen in argon gas mix and the

necessary hardware to use this gas mixture during

specific furnace cycles.

6.1.5 Autosampler-- Although not specifically required,

the use of an autosampler is highly recommended.

6.1.6 Graphite Furnace 0perating Conditions -- A

guide to experimental conditions for the applicable

elements is provided in Table 1.

6.2 Preconcentration System -- System containing

no metal parts in the analyte flow path, configured as

shown with a sample loop in Figure 1 and without a

sample loop in Figure 2.

6.2.1 Column -- Macroporous iminodiacetate chelating

resin (Dionex Metpac CC-1 or equivalent).

6.2.2 Control valves -- Inert double stack, pneumatically operated four-way slider valves with connectors.

6.2.2.1 Argon gas supply regulated at 80-100 psi.

6.2.3 Solution reservoirs -- Inert containers, e.g., high

density polyethylene (HDPE), for holding eluent and

carrier reagents.

6.2.4 Tubing -- High pressure, narrow bore, inert tubing

such as Tefzel ETFE (ethylene tetra-fluoro ethylene) or

equivalent for interconnection of pumps/ valve assemblies

and a minimum length for connection of the preconcentration system with the sample collection vessel.

grammable flow, high-pressure pumping system, capable

of delivering either one of three eluents at a pressure up

to 2000 psi and a flow rate of 1-5 mL/min.

6.2.6.1 Sample loop -- 10-mL loop constructed from

narrow bore, high-pressure inert tubing, Tefzel ETFE or

equivalent.

6.2.6.2 Auxiliary pumps -- On-line buffer pump, piston

staltic pump (Gilson Minipuls or equivalent) for delivering

pump for loading sample loop.

6.2.7 System setup without sample loop (See

Figure 2).

6.2.7.1 Auxiliary Pumps - Sample pump (Dionex QIC

Pump or equivalent) for loading sample on the column.

Carrier pump (Dionex QIC Pump or equivalent) used to

flush collection line between samples.

6.3 Labware -- For determination of trace elements,

contamination and loss are of critical consideration.

Potential contamination sources include improperly

cleaned laboratory apparatus and general contamination

within the laboratory environment. A clean laboratory

work area, designated for trace element sample handling

must be used. Sample containers can introduce positive

and negative errors in determination of trace elements by

(1) contributing contaminants through surface desorption

or leaching and (2) depleting element concentrations

through adsorption processes. For these reasons, borosilicate glass is not recommended for use with this

method. All labware in contact with the sample should be

cleaned prior to use. Labware may be soaked overnight

and thoroughly washed with laboratory-grade detergent

and water, rinsed with water, and soaked for 4 h in a

mixture of dilute nitric and hydrochloric acids, followed by

rinsing with ASTM type I water and oven drying.

6.3.1 Griffin beakers, 250 mL, polytetrafluoroethylene

(PTFE) or quartz.

6.3.2 Storage bottles -- Narrow mouth bottles, Teflon

FEP (fluorinated ethylene propylene), or HDPE, 125-mL

and 250-mL capacities.
Revision 1.0 September 1997 200.13 - 6

6.4 Sample Processing Equipment Note: It is recommended that the matrix modifier be

6.4.1 Air displacement pipetter -- Digital pipet system the modifier to the overall laboratory blank.

capable of delivering volumes from 100 to 2500 μL with

an assortment of metal-free, disposable pipet tips.

6.4.2 Balances -- Analytical balance, capable of

accurately weighing to ± 0.1 mg; top pan balance,

accurate to ± 0.01 g. nium hydroxide is recommended.

6.4.3 Hot plate -- Corning PC100 or equivalent. 7.1.5 Ammonium acetate buffer 1M, pH 5.5 -- Add 58

6.4.4 Centrifuge -- Steel cabinet with guard bowl,

electric timer and brake.

6.4.5 Drying oven -- Gravity convection oven with thermostatic control capable of maintaining 105EC ± 5EC.

6.4.6 pH meter -- Bench mounted or hand-held electrode system with a resolution of ± 0.1 pH units.

6.4.7 Class 100 hoods are recommended for all

sample handling.

7.0 Reagents and Standards

7.1 Reagents may contain elemental impurities which

might affect analytical data. Only high-purity reagents

that conform to the American Chemical Society specifications should be used whenever possible. If the purity 7

of a reagent is in question, analyze for contamination. All

acids used for this method must be of ultra high-purity

grade or equivalent. Suitable acids are available from a

number of manufacturers. Redistilled acids prepared by

sub-boiling distillation are acceptable.

7.1.1 Nitric acid, concentrated (sp.gr. 1.41).

7.1.1.1 Nitric acid 0.75M -- Dilute 47.7 mL (67.3g) conc.

nitric acid to 1000 mL with ASTM type I water.

7.1.1.2 Nitric acid (1+1) -- Dilute 500 mL conc. nitric acid

to 1000 mL with ASTM type I water.

7.1.1.3 Nitric acid (1+9) -- Dilute 100 mL conc. nitric acid

to 1000 mL with ASTM type I water.

7.1.2 Matrix Modifier, dissolve 300 mg Palladium (Pd)

powder in a minimum amount of concentrated HN03 (1

mL of HNO , adding concentrated HCl only if necessary). 3

Dissolve 200 mg of Mg(NO ) C6H O in ASTM type I water. 3 2 2

Pour the two solutions together and dilute to 100 mL with

ASTM type I water.

analyzed separately in order to assess the contribution of

7.1.3 Acetic acid, glacial (sp.gr. 1.05). High purity acetic

acid is recommended.

7.1.4 Ammonium hydroxide (20%). High purity ammomL (60.5 g) of glacial acetic acid to 600 mL of ASTM type

I water. Add 65 mL (60 g) of 20% ammonium hydroxide

and mix. Check the pH of the resulting solution by

withdrawing a small aliquot and testing with a calibrated

pH meter, adjusting the solution to pH 5.5 ± 0.1 with small

volumes of acetic acid or ammonium hydroxide as necessary. Cool and dilute to 1 L with ASTM type I water.

7.1.6 Ammonium acetate buffer 2M, pH 5.5 -- Prepare

as for Section 7.1.5 using 116 mL (121 g) glacial acetic

acid and 130 mL (120 g) 20% ammonium hydroxide,

diluted to 1000 mL with ASTM type I water.

Note: If the system is configured as shown in Figure 1,

the ammonium acetate buffer solutions may be further

purified by passing them through the chelating column at

a flow rate of 5.0 mL/min. Collect the purified solution in

a container. Following this, elute the collected contaminants from the column using 0.75M nitric acid for 5 min at

a flow rate of 4.0 mL/min. If the system is configured as

shown in Figure 2, the majority of the buffer is being

purified in an on-line configuration via the clean-up column.

7.1.7 Oxalic acid dihydrate (CASRN 6153-56-6),

0.2M -- Dissolve 25.2 g reagent grade C2H2O4C2H2O in

250 mL ASTM type I water and dilute to 1000 mL with

ASTM type I water. CAUTION - Oxalic acid is toxic;

handle with care.

7.2 Water -- For all sample preparation and dilutions,

ASTM type I water (ASTM D1193) is required.

7.3 Standard Stock Solutions -- May be purchased

from a reputable commercial source or prepared from

ultra high-purity grade chemicals or metals (99.99 -

99.999% pure). All salts should be dried for one hour at

105EC, unless otherwise specified. (CAUTION - Many

metal salts are extremely toxic if inhaled or swallowed.

Wash hands thoroughly after handling.) Stock solutions

should be stored in plastic bottles. The following procedures may be used for preparing standard stock solutions:
200.13 - 7 Revision 1.0 September 1997

Note: Some metals, particularly those which form sur- dards should be sufficiently high to produce good meaface oxides require cleaning prior to being weighed. This surement precision and to accurately define the slope of

may be achieved by pickling the surface of the metal in the response curve.

acid. An amount in excess of the desired weight should

be pickled repeatedly, rinsed with water, dried and 7.5 Blanks -- Four types of blanks are required for

weighed until the desired weight is achieved. this method. A calibration blank is used to establish the

7.3.1 Cadmium solution, stock 1 mL = 1000 μg Cd -- (LRB) is used to assess possible contamination from the

Pickle cadmium metal in (1+9) nitric acid to an exact

weight of 0.100 g. Dissolve in 5 mL (1+1) nitric acid,

heating to effect solution. Cool and dilute to 100 mL with

ASTM type I water.

7.3.2 Cobalt solution, stock 1 mL = 1000 μg Co -- acids (Section 7.1) and ASTM type I water.

Pickle cobalt metal in (1+9) nitric acid to an exact weight

of 0.100 g. Dissolve in 5 mL (1+1) nitric acid, heating to

effect solution. Cool and dilute to 100 mL with ASTM type acid diluent in ASTM type I water. The calibration blank

I water. should be stored in a FEP bottle.

7.3.3 Copper solution, stock 1 mL = 1000 μg Cu --

Pickle copper metal in (1+9) nitric acid to an exact weight

of 0.100 g. Dissolve in 5 mL (1+1) nitric acid, heating to

effect solution. Cool and dilute to 100 mL with ASTM type

I water.

7.3.4 Lead solution, stock 1 mL = 1000 μg Pb --

Dissolve 0.1599 g PbNO3 in 5 mL (1+1) nitric acid. Dilute

to 100 mL with ASTM type I water.

7.3.5 Nickel solution, stock 1 mL = 1000 μg Ni --

Dissolve 0.100 g nickel powder in 5 mL conc. nitric acid,

heating to effect solution. Cool and dilute to 100 mL with

ASTM type I water.

7.4 Multielement Stock Standard Solution -- Care

must be taken in the preparation of multielement stock

standards that the elements are compatible and stable.

Originating element stocks should be checked for the

presence of impurities which might influence the accuracy

of the standard. Freshly prepared standards should be

transferred to acid cleaned, new FEP or HDPE bottles for

storage and monitored periodically for stability. A

multielement stock standard solution containing cadmium, cobalt, copper, lead, and nickel may be prepared

by diluting an appropriate aliquot of each single element

stock in the list to 100 mL with ASTM type I water

containing 1% (v/v) nitric acid.

7.4.1 Preparation of calibration standards -- Fresh

multielement calibration standards should be prepared

weekly. Dilute the stock multielement standard solution

in 1% (v/v) nitric acid to levels appropriate to the required

operating range. The element concentrations in the stananalytical calibration curve, the laboratory reagent blank

sample preparation procedure and to assess spectral

background. The laboratory fortified blank is used to

assess routine laboratory performance, and a rinse blank

is used to flush the instrument autosampler uptake system. All diluent acids should be made from concentrated

7.5.1 The calibration blank consists of the appropriate

7.5.2 The laboratory reagent blanks must contain all

the reagents in the same volumes as used in processing

the samples. The preparation blank must be carried

through the entire sample digestion and preparation

scheme.

7.5.3 The laboratory fortified blank (LFB) is prepared

by fortifying an aliquot of the laboratory reagent blank with

all analytes to provide a final concentration which will

produce an absorbance of approximately 0.1 for each

analyte. The LFB must be carried through the complete

procedure as used for the samples.

7.5.4 The rinse blank is prepared as needed by adding

1.0 mL of conc. HNO and 1.0 mL conc. HCI to 1 L of 3

ASTM Type I water and stored in a convenient manner.

7.6 Instrument Performance Check (IPC) Solution

-- The IPC solution is used to periodically verify instrument

performance during analysis. The IPC solution should be

a fortified seawater prepared in the same acid mixture as

the calibration standards and should contain method

analytes such that the resulting absorbances are near the

midpoint of the calibration curve. The IPC solution should

be prepared from the same standard stock solutions used

to prepare the calibration standards and stored in a FEP

bottle. Agency programs may specify or request that

additional instrument performance check solutions be

prepared at specified concentrations in order to meet

particular program needs.

7.7 Quality Control Sample (QCS) -- A quality control sample having certified concentrations of the analytes

of interest should be obtained from a source outside the
Revision 1.0 September 1997 200.13 - 8

laboratory. Dilute the QCS if necessary with 1% nitric 9.2 Initial Demonstration of Performance 

acid, such that the analyte concentrations fall within the (Mandatory)

proposed instrument calibration range.

8.0 Sample Collection, Preservation and

Storage

8.1 Prior to collection of an aqueous sample,

consideration should be given to the type of data required,

so that appropriate preservation and pretreatment steps

can be taken. Acid preservation, etc., should be

performed at the time of sample collection or as soon

thereafter as practically possible. The pH of all aqueous

samples must be tested immediately prior to aliquoting for

analysis to ensure the sample has been properly

preserved. If properly acid-preserved, the sample can be

held up to 6 months before analysis.

8.2 For determination of total recoverable elements

in aqueous samples, acidify with (1+1) nitric acid at the

time of collection to pH < 2. Normally 3 mL of (1+1) acid

per liter of sample is sufficient. The sample should not be

filtered prior to analysis.

Note: Samples that cannot be acid-preserved at the

time of collection because of sampling limitations or

transport restrictions, or have pH > 2 because of high

alkalinity should be acidified with nitric acid to pH < 2 upon

receipt in the laboratory. Following acidification, the

sample should be held for 16 h and the pH verified to be

<2 before withdrawing an aliquot for sample processing.

8.3 For aqueous samples, a field blank should be

prepared and analyzed as required by the data user. Use

the same container type and acid as used in sample

collection.

9.0 Quality Control

9.1 Each laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of laboratory capability and periodic

analysis of laboratory reagent blanks, fortified blanks and

other laboratory solutions as a continuing check on

performance. The laboratory is required to maintain

performance records that define the quality of the data

generated.

9.2.1 The initial demonstration of performance is used to

characterize instrument performance (determination of

linear dynamic ranges and analysis of quality control

samples) and laboratory performance (determination of

method detection limits) prior to samples being analyzed

by this method.

9.2.2 Linear dynamic range (LDR) -- The upper limit of

the LDR. must be established for the wavelength utilized

for each analyte by determining the signal responses

from a minimum of 6 different concentration standards

across the range, two of which are close to the upper limit

of the LDR. Determined LDRs must be documented and

kept on file. The linear calibration range which may be

used for analysis of samples should be judged by the

analyst from the resulting data. The upper LDR. limit

should be an observed signal no more than 10% below

the level extrapolated from the four lower standards. New

LDRs should be determined whenever there is a

significant change in instrument response, a change in

instrument analytical hardware or operating conditions.

Note: Multiple cleanout furnace cycles may be necessary

in order to fully define or utilize the LDR. for certain

elements such as nickel. For this reason, the upper limit

of the linear calibration range may not correspond to the

upper LDR limit.

Measured sample analyte concentrations that exceed the

upper limit of the linear calibration range must either be

diluted and reanalyzed with concern for memory effects

(Section 4.4) or analyzed by another approved method.

9.2.3 Quality control sample (QCS) -- When beginning

the use of this method, on a quarterly basis or as required

to meet data-quality needs, verify the calibration standards and acceptable instrument performance with the

preparation and analyses of a QCS (Section 7.7). If the

determined concentrations are not within ± 10% of the

stated values, performance of the determinative step of

the method is unacceptable. The source of the problem

must be identified and corrected before either proceeding

on with the initial determination of method detection limits

or continuing with ongoing analyses.

9.2.4 Method detection limit (MDL) -- MDLs must be

established for all analytes, using reagent water (blank)

fortified at a concentration of two to three times the

estimated instrument detection limit. To determine MDL 8

values, take seven replicate aliquots of the fortified
200.13 - 9 Revision 1.0 September 1997

reagent water and process through the entire analytical problem should be identified and resolved before

method. Perform all calculations defined in the method continuing analyses.

and report the concentration values in the appropriate

units. Calculate the MDL as follows: 9.3.3 The laboratory must use LFB analyses data to

MDL = (t) x (S)

where, t = Student's t value for a 99% confidence level

and a standard deviation estimate with n-1

degrees of freedom [t = 3.14 for seven

replicates].

 S= standard deviation of the replicate

analyses.

Note: If the relative standard deviation (RSD) from the

analyses of the seven aliquots is < 15%, the concentration

used to determine the analyte MDL may have been in

appropriately high for the determination. lf so, this could

result in the calculation of an unrealistically low MDL. If

additional confirmation of the MDL is desired, reanalyze

the seven replicate aliquots on two more nonconsecutive

days and again calculate the MDL values for each day.

An average of the three MDL values for each analyte may

provide for a more appropriate MDL estimate. Determination of MDL in reagent water represents a best case

situation and does not reflect possible matrix effects of

real world samples. However, successful analyses of 9.3.4 Instrument Performance Check (IPC) Solution --

LFMs (Section 9.4) can give confidence to the MDL value

determined in reagent water. Typical single laboratory

MDL values using this method are given in Table 1. MDLs

should be determined every 6 months, when a new

operator begins work, or whenever there is a significant

change in the background or instrument response.

9.3 Assessing Laboratory Performance 

(Mandatory)

9.3.1 Laboratory reagent blank (LRB) - The laboratory

must analyze at least one LRB (Section 7.5.2) with each the second analysis of the IPC solution confirms

batch of 20 or fewer samples. LRB data are used to calibration to be outside the limits, sample analysis must

assess contamination from the laboratory environment. be discontinued, the cause determined and/or in the case

LRB values that exceed the MDL indicate laboratory or of drift the instrument recalibrated. All samples following

reagent contamination should be suspected. Any deter- the last acceptable IPC solution must be reanalyzed. The

mined source of contamination must be corrected and the analysis data of the calibration blank and IPC solution

samples reanalyzed for the affected analytes after must be kept on file with the sample analyses data.

acceptable LRB values have been obtained.

9.3.2 Laboratory fortified blank (LFB) -- The laboratory

must analyze at least one LFB (Section 7.5.3) with each control the method blank. Therefore, it is recommended

batch of samples. Calculate accuracy as percent recov- that the calibration blank response be recorded for each

ery (Section 9.4.3). If the recovery of any analyte falls set of samples. This record will aid the laboratory in

outside the required control limits of 85-115%, that assessing both its long- and short-term ability to control

analyte is judged out of control, and the source of the the method blank.

assess laboratory performance against the required control limits of 85-115% (Section 9.3.2). When sufficient

internal performance data become available (usually a

minimum of 20-30 analyses), optional control limits can

be developed from the percent mean recovery (x) and the

standard deviation (S) of the mean recovery. These data

can be used to establish the upper and lower control

limits as follows:

Upper Control Limit = x + 3S

Lower Control Limit = x - 3S

The optional control limits must be equal to or better than

the required control limits of 85-115%. After each 5-10

new recovery measurements, new control limits can be

calculated using only the most recent 20-30 data points.

Also, the standard deviation (S) data should be used to

establish an ongoing precision statement for the level of

concentrations included in the LFB. These data must be

kept on file and be available for review.

For all determinations the laboratory must analyze the

IPC solution (Section 7.6) and a calibration blank immediately following each calibration, after every tenth sample

(or more frequently, if required) and at the end of the

sample run. The IPC solution should be a fortified

seawater matrix. Analysis of the IPC solution and

calibration blank immediately following calibration must

verify that the instrument is within ±10% of calibration.

Subsequent analyses of the IPC solution must be within

±10% of calibration. If the calibration cannot be verified

within the specified limits, reanalyze the IPC solution. If

9.3.5 The overall sensitivity and precision of this

method are strongly influenced by a laboratory’s ability to
Revision 1.0 September 1997 200.13 - 10

9.4 Assessing Analyte Recovery and Data influences the column's ability to preconcentrate the

Quality metals; therefore, a low recovery may be caused by a low

9.4.1 Sample homogeneity and the chemical nature of analyst is advised to make an in furnace analyte addition

the sample matrix can affect analyte recovery and data

quality. Taking separate aliquots from the sample for

replicate and fortified analyses can, in some cases,

assess these effects. Unless otherwise specified by the

data user, laboratory or program, the following laboratory

fortified matrix (LFM) procedure (Section 9.4.2) is

required.

9.4.2 The laboratory must add a known amount of as NASS-3 (from the Research Council of Canada) be

each analyte to a minimum of 10% of routine samples.

In each case, the LFM aliquot must be a duplicate of the

aliquot used for sample analysis and for total recoverable

determinations added prior to sample preparation. For

water samples, the added analyte concentration must be

the same as that used in the laboratory fortified blank

(Section 7.5.3). Over time, samples from all routine

sample sources should be fortified.

9.4.3 Calculate the percent recovery for each analyte,

corrected for concentrations measured in the unfortified

sample, and compare these values to the designated

LFM recovery range of 75-125%. Recovery calculations

are not required if the concentration added is <25% of the

unfortified sample concentration. Percent recovery may

be calculated in units appropriate to the matrix, using the

following equation:

R = Cs - C x 100

s

where, R = percent recovery.

C = fortified sample concentration. s

C = sample background concentration.

s = concentration equivalent of analyte 

added to sample.

9.4.4 If the recovery of any analyte falls outside the

designated LFM recovery range (but is still within the

range of calibration and the background absorbance is <

1 abs.) and the laboratory performance for that analyte is

shown to be in control (Section 9.3), the recovery problem

encountered with the LFM is judged to be either matrix or

solution related, not system related. This situation should

be rare given the matrix elimination preconcentration step

prior to analysis. If a low recovery is found, check the pH

of the sample plus the buffer mixture. The resulting pH

should be about 5.5. The pH of the sample strongly

pH. If the pH for the LFM/buffer mixture is about 5.5, the

to the LFM using the preconcentrated standard solution.

If recovery of the in furnace analyte addition is shown to

be out of control, a matrix interference is confirmed and

the sample must be analyzed by MSA.

9.5 Utilizing Reference Materials

9.5.1 It is recommended that a reference material such

fortified and used as an Instrument Performance Check

Solution.

10.0 Calibration and Standardization

10.1 The preconcentration system can be configured

utilizing a sample loop to define the sample volume

(Figure 1) or the system can be configured such that a

sample pump rate and a pumping time defines the

sample volume (Figure 2). The system illustrated in

Figure 1 is recommended for sample sizes of <10 mL. A

thorough rinsing of the sample loop between samples

with HNO3 is required. This rinsing will minimize the

cross-contamination which may be caused by the sample

loop. The system in Figure 2 should be used for sample

volumes of >10 mL. The sample pump used in Figure 2

must be calibrated to assure that a reproducible/defined

volume is being delivered.

10.2 Specific wavelengths and instrument operating

conditions are listed in Table 1. However, because of

differences among makes and models of spectrophotometers and electrothermal furnace devices, the actual

instrument conditions selected may vary from those listed.

10.3 Prior to the use of this method, instrument operating conditions must be optimized. The analyst should

follow the instructions provided by the manufacturer while

using the conditions listed in Table 1 as a guide. Of

particular importance is the determination of the charring

temperature limit for each analyte. This limit is the furnace temperature setting where a loss in analyte will

occur prior to atomization. This limit should be determined by conducting char temperature profiles for each

analyte and when necessary, in the matrix of question.

The charring temperature selected should minimize background absorbance while providing some furnace temperature variation without loss of analyte. For routine

analytical operation the charring temperature is usually
200.13 - 11 Revision 1.0 September 1997

set at least 100EC below this limit. The optimum condi- 11.1.3 Cover the lip of the beaker with a watch glass to

tions selected should provide the lowest reliable MDLs

and be similar to those listed in Table 1. Once the

optimum operating conditions are determined, they

should be recorded and available for daily reference.

10.4 Prior to an initial calibration, the linear dynamic

range of the analyte must be determined (Section 9.2.2)

using the optimized instrument operating conditions. For

all determinations allow an instrument and hollow cathode lamp warm-up period of not less than 15 min. If an

EDL is to be used, allow 30 min for warm-up.

10.5 Before using the procedure (Section 11.0) to analyze samples, data must be available to document initial

demonstration of performance. The required data and

procedure are described in Section 9.2. This data must be

generated using the same instrument operating

conditions and calibration routine (Section 11.4) to be

used for sample analysis. These documented data must

be kept on file and be available for review by the data

user.

11.0 Procedure

11.1 Sample Preparation -- Total Recoverable

Elements

11.1.1 Add 2 mL (1+1) nitric acid to the beaker

containing 100 mL of sample. Place the beaker on the

hot plate for solution evaporation. The hot plate should

be located in a fume hood and previously adjusted to

provide evaporation at a temperature of approximately but

no higher than 85EC. (See the following note.) The

beaker should be covered with an elevated (ribbed) watch

glass or other necessary steps should be taken to prevent

sample contamination from the fume hood environment.

Note: For proper heating adjust the temperature control

of the hot plate such that an uncovered Griffin beaker

containing 50 mL of water placed in the center of the hot

plate can be maintained at a temperature approximately

but no higher than 85EC. (Once the beaker is covered

with a watch glass the temperature of the water will rise

to approximately 95EC.)

11.1.2 Reduce the volume of the sample aliquot to

about 20 mL by gentle heating at 85EC. DO NOT BOIL.

This step takes about 2 hr for a 100-mL aliquot with the

rate of evaporation rapidly increasing as the sample

volume approaches 20 mL. (A spare beaker containing

20 mL of water can be used as a gauge.)

reduce additional evaporation and gently reflux the

sample for 30 min. Slight boiling may occur, but vigorous

boiling must be avoided.

11.1.4 Allow the beaker to cool. Quantitatively transfer

the sample solution to a 100-mL volumetric flask, dilute

to volume with reagent water, stopper and mix.

11.1.5 Allow any undissolved material to settle overnight, or centrifuge a portion of the prepared sample until

clear. (If after centrifuging or standing overnight the

sample contains suspended solids that would clog or

affect the sample introduction system, a portion of the

sample may be filtered prior to analysis. However, care

should be exercised to avoid potential contamination from

filtration.) The sample is now ready for analysis. Because

the effects of various matrices on the stability of diluted

samples cannot be characterized, all analyses should be

performed as soon as possible after the completed

preparation.

11.2 Prior to first use, the preconcentration system

should be thoroughly cleaned and decontaminated using

0.2M - oxalic acid.

11.2.1 Precleaning the Preconcentration System

11.2.1.1 Place approximately 500 mL 0.2M - oxalic acid

in each of the sample/eluent containers. Flush the entire

system by running the program used for sample analysis

3 times.

11.2.1.2 Rinse the containers with ASTM type I water

and repeat the sequence described in Section 11.2.1.1

using 0.75M nitric acid and again using ASTM type I water

in place of the 0.2M - oxalic acid.

11.2.1.3 Rinse the containers thoroughly with ASTM type

I water, fill them with their designated reagents and run

through the program used for sample analysis in order to

prime the pump and all eluent lines with the correct

reagents.

11.2.2 Peak Profile Determination

11.2.2.1 The peak elution time or the collection window

should be determined using an ICP-AES (Inductively

Coupled Plasma Atomic Emission Spectrometer) or

Flame AA. Figure 3 is a plot of time vs. emission intensity

for Cd, Pb, Ni, and Cu. The collection window is marked

in Figure 3 and should provide about 30 sec buffer on
Revision 1.0 September 1997 200.13 - 12

either side of the peak. If an ICP-AES is not available, it 11.3.2.1 Sample Loading -- With the valves 1 and 2 on

is recommended that the peak profile be determined by and the sample pump on, load the sample on the column

collecting 200-μL samples during the elution part of the buffering the sample utilizing the gradient pump and the

preconcentration cycle and then reconstructing the peak 2M buffer. The actual sample volume is determined by

profile from the analysis of the 200-μL samples. knowing the sample pump rate and the time. While the

11.3 Sample Preconcentration flush the collection line.

11.3.1 Preconcentration utilizing a sample loop. 11.3.2.2 Elution Matrix -- With valve 1 in the off position

11.3.1.1 Loading Sample Loop -- With valve 1 in the off 1M ammonium acetate. During which time the carrier,

position and valve 2 in the on position, load sample buffer and the sample pumps are all off.

through the sample loop to waste using the sample pump

for 4 min at 4 mL/min. Switch on the carrier pump and 11.3.2.3 Elution of Analytes -- With valves l and 2 in the

pump 1 % nitric acid to flush the sample collection line. off position the gradient pump is switched to 0.75M HNO

11.3.1.2 Column Loading -- With valve 1 in the on The analytes should be eluted into a 2 mL sample

position, load sample from the loop onto the column volume.

using 1 M ammonium acetate for 4.5 min at 4.0 mL/min.

Switch on the buffer pump, and pump 2M ammonium 11.3.2.4 Column Reconditioning -- Turn on valve 2 to

acetate at a flow rate of 1 mL/min. The analytes are direct column effluent to waste, and pump 0.75M nitric

retained on the column, while the majority of the matrix is acid, 1M ammonium acetate, 0.75M nitric acid and 1M

passed through to waste. ammonium acetate alternately through the column at 4.0

11.3.1.3 Elution Matrix -- With valve 1 in the on position

the gradient pump is allowed to elute the matrix using the Note: When switching the gradient pump from nitric

1M ammonium acetate. During which time the carrier, acid back to the ammonium acetate it is necessary to

buffer and the sample pumps are all off. flush the line connecting the gradient pump to valve 2 with

11.3.1.4 Elute Analytes -- Turn off valve 1 and begin line contains nitric acid it will elute the metals from the

eluting the analytes by pumping 0.75M nitric acid through cleanup column.

the column and turn off valve 2 and pump the eluted

analytes into the collection flask. The analytes should be 11.3.2.5 Preconcentration of the sample may be aeluted into a 2-mL sample volume. chieved by running through an eluent pump program.

11.3.1.5 Column Reconditioning -- Turn on valve 2 to according to the internal volume of the connecting tubing

direct column effluent to waste, and pump 0.75M nitric and the specific hardware configuration used.

acid, 1M ammonium acetate, 0.75M nitric acid and 1M

ammonium acetate alternately through the column at 4.0 11.4 Repeat the sequence described in Section 11.3.1

mL/min. Each solvent should be pumped through the or 11.3.2 for each sample to be analyzed. At the end of

column for 2 min. During this process, the next sample the analytical run leave the column filled with 1M ammocan be loaded into the sample loop using the sample nium acetate buffer until it is next used.

pump.

11.3.1.6 Preconcentration of the sample may be a- established linear dynamic range should be diluted into

chieved by running through an eluent pump program. range and reanalyzed.

The exact timing of this sequence should be modified

according to the internal volume of the connecting tubing 11.6 Sample Analysis

and the specific hardware configuration used.

11.3.2 Preconcentration utilizing an auxiliary pump to

determine sample volume. sampler injector for any change that would affect

sample is being loaded the carrier pump can be used to

the gradient pump is allowed to elute the matrix using the

3

and the analytes are eluted into the collection vessel.

mL/min.

the ammonium acetate prior to switching the valve. If the

The exact timing of this sequence should be modified

11.5 Samples having concentrations higher than the

11.6.1 Prior to daily instrument calibration, inspect the

graphite furnace, the sample uptake system and autoinstrument performance. Clean the system and replace
200.13 - 13 Revision 1.0 September 1997

the graphite tube and/or platform when needed or on a 11.6.8 During sample analyses, the laboratory must

daily basis. A cotton swab dipped in a 50/50 mixture of

isopropyl alcohol (IPA) and H O (such that it is damp but 2

not dripping) can be used to remove the majority of the

salt buildup. A second cotton swab is dipped in IPA and

the contact rings are wiped down to assure they are

clean. The rings are then allowed to thoroughly dry and

then a new tube is placed in the furnace and conditioned

according to instrument manufacturers specifications.

11.6.2 Configure the instrument system to the selected

optimized operating conditions as determined in Sections

10.1 and 10.2.

11.6.3 Before beginning daily calibration the instrument

should be reconfigured to the optimized conditions. Ini- 11.6.10 Report data as directed in Section 12.

tiate data system and allow a period of not less than 15

min for instrument and hollow cathode lamp warm-up. If

an EDL is to be used, allow 30 min for warm-up.

11.6.4 After the warm-up period but before calibration,

instrument stability must be demonstrated by analyzing a

standard solution with a concentration 20 times the IDL a

minimum of five times. The resulting relative standard

deviation of absorbance signals must be <5%. If the

relative standard deviation is >5%, determine and correct

the cause before calibrating the instrument.

11.6.5 For initial and daily operation calibrate the instrument according to the instrument manufacturer’s recommended procedures using the calibration blank (Section

7.5.1) and calibration standards (Section 7.4) prepared at

three or more concentrations within the usable linear

dynamic range of the analyte (Sections 4.4 & 9.2.2).

11.6.6 An autosampler must be used to introduce all

solutions into the graphite furnace. Once the standard, corrected for nonanalyte signals. The unknown sample

sample or QC solution plus the matrix modifier is injected, concentration C is calculated:

the furnace controller completes furnace cycles and

cleanout period as programmed. Analyte signals must be C = S V C

integrated and collected as peak area measurements. (S -S )V

Background absorbances, background corrected analyte

signals, and determined analyte concentrations on all where, S and S are the analytical signals (corrected for

solutions must be able to be displayed on a CRT for the blank) of solutions A and B, respectively. V and C

immediate review by the analyst and be available as hard should be chosen so that S is roughly twice S on the

copy for documentation to be kept on file. Flush the average. It is best if V is made much less than V , and

autosampler solution uptake system with the rinse blank thus C is much greater than C , to avoid excess dilution

(Section 7.5.4) between each solution injected. of the sample matrix. If a separation or concentration

11.6.7 After completion of the initial requirements of this through the entire procedure. For the results from this

method (Section 9.2), samples should be analyzed in the

same operational manner used in the calibration routine.

comply with the required quality control described in

Sections 9.3 and 9.4.

11.6.9 Determined sample analyte concentrations that

are >90% of the upper limit of calibration must either be

diluted with acidified reagent water and reanalyzed with

concern for memory effects (Section 4.4), or determined

by another approved test procedure that is less sensitive.

Samples with a background absorbance > 1.0 must be

appropriately diluted with acidified reagent water and

reanalyzed (Section 9.4.6). If the method of standard

additions is required, follow the instructions described in

Section 11.5.

11.7 Standard Additions -- If the method of standard

addition is required, the following procedure is recommended:

11.7.1 The standard addition technique involves pre9

paring new standards in the sample matrix by adding

known amounts of standard to one or more aliquots of the

processed sample solution. This technique compensates

for a sample constituent that enhances or depresses the

analyte signal, thus producing a different slope from that

of the calibration standards. It will not correct for additive

interference, which causes a baseline shift. The simplest

version of this technique is the single addition method.

The procedure is as follows: Two identical aliquots of the

sample solution, each of volume Vx, are taken. To the

first (labeled A) is added a small volume VS of a standard

analyte solution of concentration Cs. To the second

(labeled B) is added the same volume V of the solvent. s

The analytical signals of A and B are measured and

x

X B S S

A B X

A B

S S

A B

S X

S X

step is used, the additions are best made first and carried

technique to be valid, the following limitations must be

taken into consideration:
Revision 1.0 September 1997 200.13 - 14

1. The analytical curve must be linear.

2. The chemical form of the analyte added must respond in the same manner as the analyte in the

sample.

3. The interference effect must be constant over the

working range of concern.

4. The signal must be corrected for any additive interference.

12.0 Data Analysis and Calculations

12.1 Sample data should be reported in units of μg/L

for aqueous samples. 14.2 For information about pollution prevention that

12.2 For total recoverable aqueous analytes (Section tions, consult Less is Better: Laboratory Chemical Man11.1), when 100-mL aliquot is used to produce the 100 agement for Waste Reduction, available from the AmerimL final solution, round the data to the tenths place and can Chemical Society’s Department of Government Rereport the data in μg/L up to three significant figures. If an lations and Science Policy, 1155 16th Street N.W., Washaliquot volume other than 100 mL is used for sample ington D.C. 20036, (202)872-4477.

preparation, adjust the dilution factor accordingly. Also,

account for any additional dilution of the prepared sample

solution needed to complete the determination of analytes exceeding the upper limit of the calibration curve.

Do not report data below the determined analyte MDL

concentration or below an adjusted detection limit

reflecting smaller sample aliquots used in processing or

additional dilutions required to complete the analysis.

12.3 The QC data obtained during the analyses

provide an indication of the quality of the sample data and

should be provided with the sample results.

13.0 Method Performance

13.1 Experimental conditions used for single available from the American Chemical Society at the

laboratory testing of the method are summarized in Table address listed in the Section 14.2.

1.

13.2 Table 2 contains precision and recovery data obtained from a single laboratory analysis of a fortified and

a non-fortified sample of NASS-3. The samples were

prepared using the procedure described in Section 11.1.

Four replicates of the non-fortified samples were

analyzed and the average of the replicates was used for

determining the sample analyte concentration. The fortified samples of NASS-3 were also analyzed and the

average percent recovery and the percent relative standard deviation is reported. The reference material certified values are also listed for comparison.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

EPA has established a preferred hierarchy of environmental management techniques that places pollution

prevention as the management option of first choice.

Whenever feasible, laboratory personnel should use

pollution prevention techniques to address their waste

generation (e.g., Section 7.8). When wastes cannot be

feasibly reduced at the source, the Agency recommends

recycling as the next best option.

may be applicable to laboratories and research institu15.0 Waste Management

15.1 The Environmental Protection Agency requires

that laboratory waste management practices be conducted consistent with all applicable rules and regulations. The Agency urges laboratories to protect the air,

water, and land by minimizing and controlling all releases

from hoods and bench operations, complying with the

letter and spirit of any sewer discharge permits and

regulations, and by complying with all solid and hazardous waste regulations, particularly the hazardous waste

identification rules and land disposal restrictions. For

further information on waste management consult The

Waste Management Manual for Laboratory Personnel,

16.0 References

1. A. Siraraks, H.M. Kingston and J.M. Riviello, Anal

Chem. 62 1185 (1990).

2. E.M. Heithmar, T.A. Hinners, J.T. Rowan and J.M.

Riviello, Anal Chem. 62 857 (1990).

3. OSHA Safety and Health Standards, General

Industry, (29 CFR 1910), Occupational Safety and

Health Administration, OSHA 2206, (Revised,

January 1976).
200.13 - 15 Revision 1.0 September 1997

4. Carcinogens - Working With Carcinogens, Department of Health, Education, and Welfare, Public

Health Service, Centers for Disease Control, National Institute for Occupational Safety and Health,

Publication No. 77-206, Aug. 1977.

5. Proposed OSHA Safety and Health

Standards,Laboratories, Occupational Safety and

Health Administration, Federal Register, July 24,

1986.

6. Safety in Academic Chemistry Laboratories, American Chemical Society Publication, Committee on

Chemical Safety, 3rd Edition, 1979.

7. Rohrbough, W.G. et al. Reagent Chemicals,

American Chemical Society Specifications, 7th

edition. American Chemical Society, Washington,

DC, 1986.

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

Appendix B.

9. Winefordner, J.D., Trace Analysis: Spectroscopic

Methods for Elements, Chemical Analysis, Vol. 46,

pp. 41-42,1976.
Revision 1.0 September 1997 200.13 - 16

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Table 1. Method Detection Limits for Total Recoverable Analytes in Reagent Water1

Recommended

Slit, analytical Char Atomization MDL ,

2

Element nm Wavelengths, nm Temp, EC Temp, EC μg/L

Cadmium 0.7 228.8 800 1600 0.016

Cobalt 0.2 242.5 1400 2500 -

Copper 0.7 324.8 1300 2600 0.36

Lead 0.7 283.3 1250 2000 0.28

Nickel 0.2 232.4 1400 2500 *

 MDLs were calculated using NASS-3 as the matrix. 1

 MDLs were calculated based on a 10-mL sample loop. 2

* MDL was not calculated because the concentration in the matrix exceeds the MDL spike level.

- Not Determined.

Table 2. Precision and Recovery Data for NASS-3 Using System Illustrated in Figure 11,2

Certified Sample Fortified

Value, Conc., Conc., Avg.

Analyte μg/L μg/L μg/L Recovery, % % RSD 3 3

Cd 0.029 ± 0.004 0.026 ± 0.012 0.25 93 3.3

Co 0.004 ± 0.001 - - - -

Cu 0.109 ± 0.011 <0.36 5.0 87 1.4

Pb 0.039 ± 0.006 <0.28 5.0 90 3.7

Ni 0.257 ± 0.027 0.260 ± 0.04 5.0 117 8.3

 Data collected using 10-mL sample loop. 1

 Matrix modifier is Pd/Mg(NO ) /H .

2

3 2 2

 Uncertainties based on 95% confidence limits. 3

- Not determined.
200.13 - 17 Revision 1.0 September 1997

Valves Buffer Carrier Sample

1 2 Pump Pump Pump

Sample Loop 

Loading Off On Off On On

Column

Loading On On On Off Off

Elution of

Matrix On On Off Off Off

Elution of

Analytes Off Off Off Off Off

Column

Recondition Off On Off Off Off
Revision 1.0 September 1997 200.13 - 18

Valves Carrier Sample

Event 1 2 Pump Pump

Sample

Loading On On On On

Elution of

Matrix Off On Off Off

Elution of

Analytes Off Off Off Off

Column

Recondition Off On Off On
200.13 - 19 Revision 1.0 September 1997
349.0-1

Method 349.0

 Determination of Ammonia in Estuarine and Coastal Waters

 by Gas Segmented Continuous Flow Colorimetric Analysis

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

Rosenstiel School of Marine and Atmospheric Science/AOML, NOAA

University of Miami, Miami, FL 33149

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

Atmospheric Administration, Atlantic Oceanographic and Meteorological Laboratory,

Ocean Chemistry Division, Miami, FL 33149

Project Officer

Elizabeth J. Arar

Version 1.0

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Version 1.0 September 1997 349.0-2

Method 349.0

 Determination of Ammonia in Estuarine and Coastal Waters

 by Gas Segmented Continuous Flow Colorimetric Analysis

1.0 Scope and Application

1.1 This method provides a procedure for the

determination of ammonia in estuarine and coastal

waters. The method is based upon the indophenol

reaction, here adapted to automated gas-segmented

1-5

continuous flow analysis.

The term ammonia as used in this method denotes total

concentration of ammonia, including both chemical

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

+ + 

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

+ 

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

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

other aquatic organisms.

The concentration of ammonia in estuarine and coastal

water shows considerable temporal and spatial variability.

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

estuarine and coastal water, but in anoxic water, the

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

Although other forms of nitrogen contribute to primary

productivity and nutrient cycling in marine and estuarine

waters, ammonia is particularly important. Because

ammonia represents the most reduced form of inorganic

nitrogen available, it is preferentially assimilated by

phytoplankton. Whereas nitrate is the source of nitrogen,

it must first be reduced to ammonia before it can be

assimilated and incorporated into amino acids and other

compounds. Ammonia is released during the

decomposition of organic nitrogen compounds by

proteolytic bacteria, but also excreted directly by

invertebrates along with urea and peptides. In regions of 7

coastal upwelling, ammonia released by zooplankton can

play a significant role in supplying the nitrogen that

supports phytoplankton production.8

Chemical Abstracts Service

Analyte Registry Numbers (CASRN)

___________________________________________

Ammonia 7664-41-7

___________________________________________

1.2 A statistically determined method detection limit

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

9

laboratory from seawaters of four different salinities. The

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

System (Alpkem, Wilsonville, Oregon).

1.3 Approximately 60 samples per hour can be

analyzed.

1.4 This method should be used by analysts both

experienced in the use of automated gas segmented

continuous flow colorimetric analyses, and also familiar

with matrix interferences and the procedures used in their

correction. A minimum of 6-months experience under the

close supervision of a qualified analyst is recommended.

2.0 Summary of Method 

2.1 The automated gas segmented continuous flow

colorimetric method is used for the analysis of ammonia

concentration. Ammonia in solution reacts with alkaline

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

indophenol blue in the presence of sodium

nitroferricyanide as a catalyst. The absorbance of

indophenol blue at 640 nm is linearly proportional to the

concentration of ammonia in the sample. A small

systematic negative error caused by differences in the

refractive index of seawater and reagent water, and a

positive error caused by the matrix effect on the color

formation, may be corrected for during data processing.

3.0 Definitions

3.1 Calibration Standard (CAL) -- A solution

prepared from the primary dilution standard solution or

stock standard solution containing analytes. The CAL

solutions are used to calibrate the instrument response

with respect to analyte concentration.

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

reagent water to which known quantities of the method

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

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

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

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

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

prepared in reagent water that is analyzed as a sample.

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

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

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

laboratory. The LFM is analyzed exactly like a sample,

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

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

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

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

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

concentrations. used to check laboratory performance with externally

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

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

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

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

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

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

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

quantity or concentration range over which the instrument

response to an analyte is linear.

3.6 Method Detection Limit (MDL) -- The minimum

concentration of an analyte that can be identified,

measured and reported with 99% confidence that the

analyte concentration is greater than zero.9

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

water equal to or exceeding the standards established by

the American Society for Testing and Materials (ASTM).

Reverse osmosis systems or distilling units followed by

Super-Q Plus Water System that produce water with 18

megohm resistance are examples of acceptable water

sources. To avoid contamination of ammonia from the air,

the reagent water should be stored in a sealed or a

collapsible container and used the day of preparation.

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

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

relative refractive index is the ratio of the velocity of light

in two different media, such as estuarine or sea water

versus reagent water. The correction for this difference is

referred to as refractive index correction in this method.

3.10 Primary Dilution Standard Solution (PDS) -- A

prepared test materials.

concentration standard, but can be any sample that

3.13 Color SYNC Peak Solution -- A colored solution

used to produce a synchronization peak in the refractive

index measurement in which no color reagent is pumped

through system. 

3.14 Sensitivity Drift -- The change in absorbance for

a given concentration of analyte due to instrumental or

chemical drift during the course of measurement.

3.15 Matrix Effect -- The change of absorbance in

different matrices due to the effect of ionic strength and

composition on the kinetics of color forming reactions.

4.0 Interferences

4.1 Hydrogen sulfide at concentrations greater than 2

mg S/L can negatively interfere with ammonia analysis.

Hydrogen sulfide in samples should be removed by

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

stripping with gaseous nitrogen.

4.2 The addition of sodium citrate and EDTA

complexing reagent eliminates the precipitation of

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

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

13) reagent solution.4

4.3 Sample turbidity is eliminated by filtration or

centrifugation after sample collection.

4.4 As noted in Section 2.1 refractive index and salt

error interferences occur when sampler wash solution

and calibration standards are not matched with samples

in salinity, but are correctable. For low concentration

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

with salinity matched to samples, sampler wash solutions

and calibration standards is recommended to eliminate

matrix interferences.

5.0 Safety

5.1 Water samples collected from the estuarine and

coastal environment are rarely hazardous. However, the

individual who collects samples should use proper

technique.

5.2 Good laboratory technique should be used when

preparing reagents. Laboratory personnel should obtain

material safety data sheets (MSDS) for al chemicals used

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

should be worn when handling the concentrated acid.

5.3 Chloroform is used as a preservative in this

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

hood.

6.0 Equipment and Supplies

6.1 Gas Segmented Continuous Flow Autoanalyzer

Consisting of:

6.1.1 Automatic sampler.

6.1.2 Analytical cartridge with reaction coils and heater.

6.1.3 Proportioning pump.

6.1.4 Spectrophotometer equipped with a tungsten lamp

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

filter (maximum 2 nm bandwidth).

6.1.5 Strip chart recorder or computer based data

acquisition system.

6.2 Glassware and Supplies

6.2.1 Gaseous ammonia concentration in the laboratory

air should be minimal to avoid sample or reagent

contamination. Remove any NH4OH solution stored in the

laboratory. Smoking should be strictly forbidden. An air

filtration unit might also be used to obtain ammonia-free

lab air.

6.2.2 All labware used in the analysis must be free of

residual ammonia to avoid sample or reagent

contamination. Soaking with laboratory grade detergent,

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

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

found to be sufficient when working at moderate and high

concentration of ammonia. Ammonia is known for its high

surface reactivity. When working at low levels of

10

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

mandatory. Plastic bottles and glass volumetric flasks

should be cleaned in an ultrasonic bath with reagent

water for 60 minutes. Bottles and sample tubes made of

glass can be easily cleaned by boiling in reagent water.

Repeat the cleaning process with fresh reagent water

prior to use if necessary.

6.2.3 Automatic pipetters with disposable pipet tips

capable of delivering volumes ranging from 100 μL to

1000 μL and 1 mL to 10 mL.

6.2.4 Analytical balance, with accuracy to 0.1 mg, for

preparing standards.

6.2.5 60-mL glass or high density polyethylene sample

bottles, glass volumetric flasks and glass sample tubes.

6.2.6 Drying oven.

6.2.7 Desiccator.

6.2.8 Membrane filters with 0.45 μm nominal pore size.

Plastic syringes with syringe filters.

6.2.9 Centrifuge.

6.2.10 Ultrasonic water bath cleaner. 

7.0 Reagents and Standards

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

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

7.1.1 Complexing Reagent - Dissolve 140 g of sodium

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

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

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

approximately 800 mL of reagent water, mix and dilute to

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

approximately 13. This solution is stable for 2 months.

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

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

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

1000 mL glass volumetric flask containing approximately

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

drops of chloroform as a preservative. Dilute the solution

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

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

7.1.3 Low Nutrient Sea Water (LNSW) - Obtain natural

low nutrient seawater from surface water of the Gulf

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

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

this is not available, commercial low nutrient sea water

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

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

Don’t use artificial seawater in this method.

7.2 Working Reagents

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

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

and mix gently.

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

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

9002-92-0).

7.2.2 Working Complexing Reagent - Add 1 mL Brij-35

to 200 mL of stock complexing reagent, mix gently.

Prepare this solution daily. This volume of solution is

sufficient for an 8-hour run.

7.2.3 Sodium Nitroferricyanide Solution - Dissolve 0.25

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

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

reagent water. Store in an amber bottle at room

temperature.

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

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

hydroxide (NaOH, FW 40) and 0.2 g dichloroisocyanuric

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

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

7.2.6 Colored SYNC Peak Solution - Add 50 μL of blue

food coloring solution to 1000 mL reagent water and mix

thoroughly. Further dilute this solution to obtain a peak of

between 25 to 100 percent full scale according to the

AUFS setting used for refractive index measurement.

7.2.7 Primary Dilution Standard Solution - Prepare a

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

5.0 mL of stock standard solution to 100 mL with reagent

water. Prepare this solution daily.

Note: This solution should be prepared to give an

intermediate concentration appropriate for further dilution

in preparing the calibration solutions. Therefore, the

concentration of a primary dilution standard solution must

be adjusted according to the desired concentration range

of calibration solutions. 

7.2.8 Calibration Standards - Prepare a series of

calibration standards (CAL) by diluting suitable volumes

of a primary dilution standard solution (Section 7.2.7) to

100 mL with reagent water or low nutrient seawater.

Prepare these standards daily. The concentration range

of calibration standards should bracket the expected

concentrations of samples and not span more than two

orders of magnitude. At least five calibration standards

with equal increments in concentration should be used to

construct the calibration curve.

When working with samples of a narrow range of

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

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

CAL solutions be prepared in Low Nutrient Seawater

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

Sampler Wash Solution also be Low Nutrient Seawater

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

procedure is employed, it is not necessary to perform the

matrix effect and refractive index corrections outlined in

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

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

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

calibration standard solutions and sampler wash

solutions can be prepared in reagent water. The

corrections for matrix effect and refractive index should be

subsequently applied (Sections 12.2 and 12.3).

7.2.9 Saline Ammonia Standards - If CAL solutions are

not prepared to match sample salinity, then saline centrifugation.

ammonia standards must be prepared in a series of

salinities in order to quantify the matrix effect (the change

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

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

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

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

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

Salinity Volume of Volume of Conc.

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

_______________________________________

0 0 2 .10

9 25 2 .10

18 50 2 .10

27 75 2 .10

35 98 2 .10

_______________________________________

8.0 Sample Collection, Preservation and

Storage

8.1 Sample Collection - Samples collected for

nutrient analyses from estuarine and coastal waters are

normally collected using one of two methods: hydrocast

or submersible pump systems.

8.1.1 A hydrocast uses a series of sampling bottles

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

a hydro wire. These bottles are sent through the water

column open and are closed either electronically or via a

mechanical messenger when the bottles have reached

the desired depth.

8.1.2 In a submersible pump system, a weighted hose

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

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

sample processing.

8.1.3 For collecting surface samples, an acid - cleaned

plastic bucket or a large plastic bottle can be used as 

convenient samplers. Wash the sampler three times with

sample water before collecting samples.

μm membrane filter as soon as possible after collection.

Wash the filter with reagent water before use. Pass at

least 100 mL of sample through the filter and discard

before taking the final sample. Care must be taken to

avoid the contamination of ammonia especially handling

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

An alternative technique to remove particulate is

8.1.5 60-mL glass or high density polyethylene bottles

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

8.2 Sample Preservation - After collection and

filtration or centrifugation, samples should be analyzed as

soon as possible. If samples will be analyzed within 3

hours then keep refrigerated in tightly sealed, glass or

high density polyethylene bottles in the dark at 4 C until

o

the analysis can be performed.

8.3 Sample Storage - At low concentrations of

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

satisfactory. Samples must be analyzed within 3 hours of

collection. At moderate and high concentrations of

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

addition of 2 mL of chloroform per liter of sample and

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

either glass or high density polyethylene bottles. A

maximum holding time for preserved estuarine and

coastal water samples with moderate to high

concentrations of ammonia is two weeks.12

9.0 Quality Control

9.1 Each laboratory using this method is required to

implement a formal quality control (QC) program. The

minimum requirements of this program consists of an

initial demonstration of performance, continued analysis

of Laboratory Reagent Blanks (LRB), laboratory

duplicates and Laboratory Fortified Blanks (LFB) with

each set of samples as a continuing check on

performance.
 349.0-7 Version 1.0 September 1997

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

(Mandatory)

9.2.1 The initial demonstration of performance is used (Mandatory)

to characterize instrument performance by determining

the MDL and LDR and laboratory performance by

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

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

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

established for the method analyte, using a low level

seawater sample containing, or fortified at, approximately

5 times the estimated detection limit. To determine MDL

values, analyze at least seven replicate aliquots of water

which have been processed through the entire analytical

method. Perform all calculations defined in the method

and report concentration in appropriate units. Calculate

the MDL as follows:

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

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

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

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

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

freedom at the 99% confidence 

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

of freedom. 

MDLs should be determined every 6 months or whenever

a significant change in background or instrument

response occurs or a new matrix is encountered.

 

9.2.3 The LDR should be determined by analyzing a

minimum of eight calibration standards ranging from

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

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

detector. Standards and sampler wash solutions should

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

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

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

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

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

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

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

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

concentration until the measured absorbance response,

R, of a standard no longer yields a calculated

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

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

defines the upper limit of the LDR for the instrument.

Should samples be encountered that have a

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

9.3 Assessing Laboratory Performance

9.3.1 Laboratory Reagent Blank (LRB) - A laboratory

from the laboratory environment. Should an analyte value

contamination should be suspected. When the LRB value

constitutes 10% or more of the analyte concentration

determined for a sample, duplicates of the sample must

be prepared and analyzed again after the source of

contamination has been corrected and acceptable LRB

values have been obtained.

9.3.2 Laboratory Fortified Blank (LFB) - A laboratory

should analyze at least one LFB with each set of

daily calibration range. The LFB data are used to

laboratory performance against the required control limits

of 90 -110%. When sufficient internal performance data

become available (usually a minimum of 20 to 30

analyses), optional control limits can be developed from

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

of the mean recovery. These data can be used to

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

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

Fortified Sample Matrix (LFM) before the actual samples.

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

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

samples or one LFM per sample set, whichever is

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

ambient concentration and should be at least four times

greater than the MDL. 

9.4.2 Calculate percent recovery of analyte, corrected

for background concentration measured in a separate

unfortified sample. These values should be compared

with the values obtained from the LFBs. Percent

recoveries may be calculated using the following

equation:

 (C - C) s

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

S

where, R = percent recovery

C = measured fortified sample s 

 addition in mg N/L

C = sample background 

 concentration (mg N/L)

S = concentration in mg N/L added 

to the environmental sample.

9.4.3 If the recovery of the analyte falls outside the

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

performance for that analyte is within the control limits,

the fortified sample should be prepared again and

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

recovery problem encountered with the fortified sample is

judged to be matrix related and the sample data should

be flagged accordingly.

10.0 Calibration and Standardization

10.1 At least five calibration standards should be

prepared fresh daily for system calibration. 

10.2 A calibration curve should be constructed for

each sample set by analyzing a series of calibration

standard solutions. A sample set should contain no more

than 60 samples. For a large number of samples make

several sample sets with individual calibration curves. 

or more that bracket the conentrations of samples should

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

range should not be greater than two orders of

magnitude. 

10.5 Use a high CAL solution followed by two blank

cups to quantify system carryover. The difference in peak

heights between two blank cups is due to the carryover

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

is calculated as follows:

 Pb1 - Pb2

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

Phigh 

where, P = the peak height of the high high

ammonia standard

P = the peak height of the b1 

first blank sample

P = the peak height of the b2 

second blank sample

The carryover coefficient, k, should be measured in seven

replicates to obtain a statistically significant number. The

carryover coefficient should be remeasured with any

change in manifold plumbing or upon replacement of

pump tubes.

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

proportional to the peak height of the preceding sample,

P .

i-1

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

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

carryover correction.13,14

Pi,c = Pi - CO

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

carryover should be applied to all the peak heights

throughout a run. The carryover coefficient should be less

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

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

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

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

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

After the reagent baseline has stabilized, reset the

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

acquisition software supplied with autoanalyzers. It is

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

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

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

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

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

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

prevent precipitation of Mg(OH) when seawater is

11.0 Procedure

11.1 If samples are stored in a refrigerator, remove

samples and equilibrate to room temperature prior to

analysis.

11.2 Turn on the continuous flow analyzer and data

acquisition components and warm up at least 30 minutes.

11.3 Set up cartridge and pump tubes as shown in

Figure 1.

11.4 Set spectrophotometer wavelength to 640 nm,

and turn on lamp.

11.5 Set the Absorbance Unit Full Scale (AUFS) range

on the spectrophotometer at an appropriate setting

according to the highest concentration of ammonia in the

samples. The highest setting appropriate for this method

is 0.2 AUFS for 6 mg N/L.

11.6 Prepare all reagents and standards.

11.7 Choose an appropriate wash solution for sampler

wash. For analysis of samples with a narrow range of

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

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

CAL solutions be prepared in Low Nutrient Seawater

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

the Sampler Wash Solution also be Low Nutrient

Seawater diluted to the same salinity. For samples with

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

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

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

standards and procedures in Section 12.2 and 12.3 be

employed.

2

introduced into the system. Keep the reagents and

samples free of particulate. Filter the reagents and

samples if necessary.

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

seconds to increase back pressure. If absorbance drops

down rapidly when back pressure increases, this indicates

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

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

in the flowcell can often be eliminated by simply attaching

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

pumping the syringe piston. Alternatively, flushing the

flowcell with alcohhol was found to be effective in

removing air bubbles from the flowcell.

11.9 The sampling rate is approximately 60 samples

per hour with 30 seconds of sample time and 30 seconds

of wash time.

11.10 Use cleaned sample cups or tubes (follow the

procedures outlined in Section 6.2.2). Place CAL

solutions and saline standards (optional) in sampler.

Complete filling the sampler tray with samples, laboratory

reagent blanks, laboratory fortified blanks, laboratory

fortified sample matrices, and QC samples. Place a blank

after every ten samples.

11.11 Commence analysis.

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

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

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

dependent variable. apparent absorbance due to the change in refractive

12.2 Refractive Index Correction for Estuarine and reagent water baseline.

Coastal Samples

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

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

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

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

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

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

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

reagent water with color reagent using reagent water as

the wash and obtain a linear regression of peak height

versus concentration.

12.2.3 Second, replace reagent water wash solution with

Low Nutrient Seawater wash solution.

Note: In ammonia analysis absorbance of the reagent

water is higher than that of the LNSW. When using

reagent water as a wash solution, the change in refractive

index causes the absorbance of seawater to become

negative. To measure the absorbance due to refractive

index change in different salinity samples, Low Nutrient

Seawater must be used as the wash solution to bring the

baseline down.

12.2.4 Third, replace the phenol solution (Section 7.2.4)

and NaDTT solution (Section 7.2.5) with reagent water.

All other reagents remain the same. Replace the

synchronization sample with the colored SYNC peak

solution (Section 7.2.6).

12.2.5 Prepare a series of different salinity samples by

diluting the LNSW. Commence analysis and obtain peak

heights for different salinity samples. The peak heights for

the refractive index correction must be obtained at the

same AUFS range setting and on the same

spectrophotometer as the corresponding standards

(Section 12.2.2).

12.2.6 Using LNSW as the wash water, a maximum

absorbance will be observed for reagent water. No

change in refractive index will be observed in the

seawater sample. Assuming the absolute absorbance for

reagent water ( relative to the seawater baseline ) is equal

to the absorbance for seawater ( relative to reagent water

index between samples of various salinities relative to the

12.2.7 For each sample of varying salinity, calculate the

apparent ammonia concentration due to refractive index

concentration due to refractive index is entered as the

operator to calculate apparent ammonia concentration

due to refractive index when the sample salinity is known.

Thus, the operator would not be required to obtain

refractive index peak heights for all samples.

12.2.8 The magnitude of refractive index correction can

be minimized by using a low refractive index flowcell. An

example of a typical result using a low refractive index

flowcell follows:

_______________________________________

Salinity Apparent ammonia conc. due 

(% ) to refractive index (μg N/L) o

_______________________________________ 

0.0 0.00

4.5 0.18

9.1 0.45

13.9 0.66

17.9 0.86

27.6 1.30

36.2 1.63

_______________________________________

Note: You must calculate the refractive index correction

for your particular detector. The refractive index must be

redetermined whenever a significant change in the design

of the flowcell or a new matrix is encountered.

12.2.9 An example of a typical equation is:

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

where S is sample salinity in parts per thousand. The

apparent ammonia concentration due to refractive index

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

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

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

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

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

for refractive index becomes negligible.

12.3 Correction for Matrix Effect in Estuarine and

Coastal Samples 

12.3.1 When calculating concentrations of samples of

varying salinities from standards and wash solution

prepared in reagent water, it is necessary to first correct

for refractive index errors, then correct for the change in

color development due to the differences in composition

between samples and standards (matrix effect). Even

where the refractive index correction may be small, the

correction for matrix effect can be appreciable.

12.3.2 Plot the salinity of the saline standards (Section

7.2.9) as the independent variable, and the apparent

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

(corrected for refractive index) calculated from the

regression of standards in reagent water, as the

dependent variable for all saline standards. The resulting

regression equation allows the operator to correct the

concentrations of samples of known salinity for the color

enhancement due to matrix effect. An example of a

typical result follows:

_______________________________________

Peak height of UncorrectedNH3

Salinity 0.140 mg N/L conc. calculated

 (% ) from standards o

in reagent water

 (mg N/L)

_______________________________________

0 2420 0.1400

4.5 2856 0.1649

9.1 2852 0.1649

13.9 2823 0.1635

17.9 2887 0.1673

27.6 2861 0.1663

36.2 2801 0.1633

_______________________________________

12.3.3 Using the reagent described in Section 7.0, as

shown above, matrix effect becomes a single factor

independent of sample salinity. An example of a typical

equation to correct for matrix effect is:

in mg N/L or in μg N/L.

mg N/L = ppm (parts per million)

μg N/L = ppb (part per billion)

13.0 Method Performance

13.1 Single Laboratory Validation

13.1.1 Method Detection Limit- A method detection limit

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

laboratory from spiked LNSW of three different salinities

as follows:

_____________________________________

Salinity [NH3] SD Recovery MDL

(% ) (μg N/L) (μg N/L) (%) (μg N/L) o

_____________________________________

36.2 0.7 0.0252 95.4 0.0792

36.2 0.7 0.0784 100.8 0.2463

36.2 1.4 0.0826 104.7 0.2595

36.2 1.4 0.0966 105.6 0.3035

17.9 0.7 0.0322 106.5 0.1012

17.9 0.7 0.0182 92.2 0.0572

17.9 1.4 0.0938 109.1 0.2947

17.9 1.4 0.0882 100 0.2771

4.5 0.7 0.0672 95.1 0.2111

4.5 1.4 0.1008 94.1 0.3167

4.5 1.4 0.126 106.7 0.3959

0.0 0.7 0.077 98.2 0.2419

0.0 0.7 0.0784 100.8 0.2463

0.0 1.4 0.0854 101.9 0.2683

_____________________________________

13.1.2 Single Analyst Precision - A single laboratory

analyzed three samples collected from the Miami River

and Biscayne Bay, Florida. Seven replicates of each

sample were processed and analyzed with salinity

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

______________________________________ acidification is not suitable preservation technique.

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

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

______________________________________ although samples preserved with phenol were stable as

1 35.5 6.3 7.19 those preserved by chloroform.

2 20.0 72.1 1.57

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

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

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

fortified sample matrices were processed in three

different salinities ranging from 4.8 to 35.0 and ambient

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

replicates of each sample were analyzed and the results

were as follows:

_______________________________________

Salinity Concentration RSD Recovery

 ambient fortified

(% ) (μg N/L) (%) (%) o

______________________________________

35.5 6.3 70 5.01 98.3

20.0 72.1 140 1.71 98.3

4.8 0.0 280 1.81 98.1

______________________________________

13.1.4 Linear Dynamic Range - A linear dynamic range

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

laboratory from spiked LNSW using a Flow Solution

System (Alpkem, Wilsonville, Oregon).

13.1.5 Sample Preservation Study - Natural samples

have been preserved by freezing, acidification and

addition of chloroform and phenol as preservatives to the

samples stored in glass and high density polyethylene

bottles. Table 1 summarized the results of preservation

study.

There is no significant difference in recovery of ammonia

from samples stored in glass and high density

polyethylene bottles, suggesting either glass or high

density polyethylene bottles can be used for storage of

ammonia samples.

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

sample 1 in table 1), no preservation technique is

satisfactory. Samples must be analyzed within 3 hours of

collection. 

Freezing cannot preserve ammonia in samples for more

than one week. Acidified samples must be neutralized

with NaOH solution prior to analysis. Addition of NaOH to

acidified samples induces the precipitation of Mg(OH)2

and Ca(OH) . Centrifuging the samples cannot 2

completely eliminate the interference, therefore,

12

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

o

time for preserved estuarine and coastal water samples

with moderate to high concentrations of ammonia is two

weeks.10

13.2 Multi-Laboratory Validation

Multi-laboratory data is unavailable at this time.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

USEPA has established a preferred hierarchy of

environmental management techniques that places

pollution prevention as the management option of first

choice. Whenever feasible, laboratory personnel should

use pollution prevention techniques to address their waste

generation. When wastes cannot be feasibly reduced at

the source, the agency recommends recycling as the next

best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research

institutions, consult Less is Better: Laboratory Chemical

Management for Waste Reduction, available from the

American Chemical Society, Department of Government

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

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

15.0 Waste Management

15.1 The U.S. Environmental Protection Agency

requires that laboratory waste management practices be

conducted consistent with all applicable rules and

regulations. The Agency urges laboratories to protect the

air, water, and land by minimizing and controlling all

releases from hoods and bench operations, complying

with the letter and spirit of any sewer discharge permits

and regulations, and by complying with all solid and

hazardous waste regulations, particularly the hazardous

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

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

The Waste Management Manual for Laboratory

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

at the address listed in Section 14.2.

16.0 References

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

natural waters by the phenylhypochlorite method.

Limnol. Oceanogr., 14:799-801.

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

phenolhypochlorite method for the determination of

ammonia in sea water. Deep-Sea Research,

18:531-532.

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

of two automated ammonia methods in a region of

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

524.

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

Chemical Analysis, In Methods of Seawater

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

Kremling, Eds) Weinheim, Verlag Chemie,

Germany. pp363-365. 

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

Optimization of the indophenol blue method for the

automated determination of ammonia in estuarine

waters. Estuarine, Coastal and Shelf Science,

17:219-224.

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

anoxic waters in the Cariaco Trench. Deep-Sea

Res., 40:1023-1041.

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

the oceans. Pergamon Press, Oxford, England. 

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

zooplankton in the regeneration of nitrogen in a

coastal upwelling off northwest Africa. Deep-Sea

Res. 24: 49-56. 

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

Appendix B. Definition and Procedure for the

Determination of Method Detection Limit. Revision

1.11.

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

ammonium by glass frits and filters: implications for

analyses of blakish and freshwater. Limnol.

Oceanogr. 24:397-399.

preservation of primary calibration solutions of

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

samples for ammonia determination. Limnol.

Oceanogr., 18:146-150.

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

as a function of system performance for continuous

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

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

carry-over and sample interaction in gas

segmented continuous flow analysis. Journal of

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

2

3

4

5

6

7

9

10

0.41

0.25

1.57

Pump

mL/min

Debubbler

Detector

Manifold

Sample

Reagent Water 

or Low Nutrient Seawater

Wash To Sampler

To Waste

To Waste

Sample:Wash = 30":30"

640nm

0.32 Complexing Reagent

Nitrogen

1.01

0.10 Phenol

0.10 NaDTT

0.10 Nitroferricyanide

 C 8

0.41

Coil

o

o

60 C

60

Heater

Version 1.0 September 1997 349.0-14

17.0 Tables, Diagrams, Flowcharts, and Validation Data

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

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

A 

Addition of Chloroform and Phenol.

______________________________________________________________________________

sample method bottle time(day) B C D

0 7 14 21 28

_______________________________________________________________________________

1 none HDPE 100 349 345 18 91

freezing glass 100 100 0 0 0

HDPE 100 102 0 0 0

H2SO4 glass 200 564 285 73 55 E

HDPE 200 113 64 45 36

CHCl3 glass 193 135 29 47 36

HDPE 193 193 18 44 36

phenol glass 153 36 44 0 0

 F

HDPE 153 36 0 0 0

1+ freezing glass 100 101 82 77 102

HDPE 100 97 76 61 81

H2SO4 glass 95 105 69 54 37 E

HDPE 95 91 91 88 116

CHCl3 glass 96 105 85 78 89

HDPE 96 102 85 78 92

phenol glass 130 133 110 148 123 F

HDPE 130 128 102 103 118

2 none HDPE 100 32 0 0 0

freezing glass 100 109 93 77 88

HDPE 100 107 82 67 91

H2SO4 glass 252 162 66 62 50 E

HDPE 252 193 45 41 27

CHCl3 glass 99 114 83 75 96

HDPE 99 98 80 70 83

phenol glass 108 107 88 74 93 F

HDPE 108 101 83 74 86

2+ freezing glass 99 108 109 111 106

HDPE 99 106 95 78 91

H2SO4 glass 100 107 51 86 88 E

HDPE 100 102 39 98 107

CHCl3 glass 99 106 116 94 105

HDPE 99 107 98 95 103

phenol glass 117 121 106 105 116 F

HDPE 117 124 107 106 117

3 none HDPE 100 104 14 1 0

freezing glass 100 - 116 64 106

HDPE 100 108 105 65 75

H2SO4 glass 101 106 44 74 61 E

HDPE 101 108 111 106 109

CHCl3 glass 100 96 98 96 94

HDPE 100 93 97 95 95

phenol glass 112 106 107 112 125 F

HDPE 112 112 108 110 112

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

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

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

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

respectively.

Methods of preservation: C

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

preservative added.

Freezing: Frozen and stored at -20 C. o

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

NaOH solution before analysis.

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

o

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

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

bottle type on the preservation.

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

E

2 2

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

not suitable preservation technique. 

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

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

preservation technique. 
353.4-1

Method 353.4

 Determination of Nitrate and Nitrite in Estuarine and Coastal Waters

 by Gas Segmented Continuous Flow Colorimetric Analysis

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

School of Marine and Atmospheric Science/AOML, NOAA, University of Miami, Miami,

FL 33149

Peter B. Ortner and Charles J. Fischer, Ocean Chemistry Division, Atlantic

Oceanographic and Meteorological Laboratory, National Oceanic and Atmospheric

Administration, Miami, FL 33149

Project Officer

Elizabeth J. Arar

Revision 2.0

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Revision 2.0 September 1997 353.4-2

Method 353.4

 Determination of Nitrate and Nitrite in Estuarine and Coastal Waters

 by Gas Segmented Continuous Flow Colorimetric Analysis

1.0 Scope and Application

1.1 This method provides a procedure for determining

nitrate and nitrite concentrations in estuarine and coastal

waters. Nitrate is reduced to nitrite by cadmium, and the 1-3 

resulting nitrite determined by formation of an azo dye.4-6

In most estuarine and coastal waters nitrogen is thought

to be a limiting nutrient. Nitrate is the final oxidation

product of the nitrogen cycle in natural waters and is

considered to be the only thermodynamically stable

nitrogen compound in aerobic waters. Nitrate in

7

estuarine and coastal water is derived from rock

weathering, sewage effluent and fertilizer run-off. The

concentration of nitrate usually is high in estuarine waters

and lower in surface coastal waters.

Nitrite is an intermediate product in the microbial

reduction of nitrate or in the oxidation of ammonia. It may

also be excreted by phytoplankton as a result of excess

assimilatory reduction. Unlike nitrate, nitrite is usually

present at a concentration lower than 0.01mg N/L except

in high productivity waters and polluted waters in the

vicinity of sewer outfalls. 

Chemical Abstracts Service

Analyte Registry Numbers (CASRN)

_______________________________________

Nitrate 14797-55-8

Nitrite 14797-65-0

_______________________________________

1.2 A statistically determined method detection limit

(MDL) of 0.075 μg N/L has been determined by one

8

laboratory in seawaters of five different salinities. The

method is linear to 5.0 mg N/L using a Flow Solution

System (Alpkem, Wilsonville, Oregon).

1.3 Approximately 40 samples per hour can be

analyzed.

1.4 This method requires experience in the use of

automated gas segmented continuous flow colorimetric

analyses, and familiarity with the techniques of

preparation, activation and maintenance of the cadmium

reduction column. A minimum of six-months training is

recommended.

2.0 Summary of Method 

2.1 An automated gas segmented continuous flow

colorimetric method for the analysis of nitrate

concentration is described. In the method, samples are

passed through a copper-coated cadmium reduction

column. Nitrate in the sample is reduced to nitrite in a

buffer solution. The nitrite is then determined by

diazotizing with sulfanilamide and coupling with N-1-

naphthylethylenediamine dihydrochloride to form a color

azo dye. The absorbance measured at 540 nm is linearly

proportional to the concentration of nitrite + nitrate in the

sample. Nitrate concentrations are obtained by

subtracting nitrite values, which have been separately

determined without the cadmium reduction procedure,

from the nitrite + nitrate values. There is no significant salt

error in this method. The small negative error caused by

differences in the refractive index of seawater and

reagent water is readily corrected for during data

processing.

3.0 Definitions

3.1 Calibration Standard (CAL) - A solution

prepared from the primary dilution standard solution or

stock standard solution containing analytes. The CAL

solutions are used to calibrate the instrument response

with respect to analyte concentration.

3.2 Laboratory Fortified Blank (LFB) - An aliquot of

reagent water to which known quantities of the method

analytes are added in the laboratory. The LFB is analyzed

exactly like a sample, and its purpose is to determine

whether method performance is within acceptable control

limits, and whether the laboratory is capable of making

accurate and precise measurements. This is a standard

prepared in reagent water that is analyzed as a sample.
 353.4-3 Revision 2.0 September 1997

3.3 Laboratory Fortified Sample Matrix (LFM) - An 3.10 Primary Dilution Standard Solution (PDS) - A

aliquot of an environmental sample to which known solution prepared in the laboratory from stock standard 

quantities of the method analytes are added in the solutions and diluted as needed to prepare calibration

laboratory. The LFM is analyzed exactly like a sample, solutions and other needed analyte solutions.

and its purpose is to determine whether the sample matrix

contributes bias to the analytical results. The background 3.11 Quality Control Sample (QCS) - A solution of

concentrations of the analytes in the sample matrix must method analytes of known concentrations which is used

be determined in a separate aliquot and the measured to fortify an aliquot of LRB or sample matrix. The QCS is

values in the LFM corrected for background obtained from a source external to the laboratory and

concentrations. different from the source of calibration standards. It is

3.4 Laboratory Reagent Blank (LRB) - An aliquot prepared test materials.

of reagent water that is treated exactly as a sample

including exposure to all labware, equipment, and 3.12 SYNC Peak Solution - A colored solution used 

reagents that are used with other samples. The LRB is to produce a synchronization peak in the refractive index

used to determine if method analytes or other measurement. A synchronization peak is required by the

interferences are present in the laboratory environment, data acquisition programs to initialize the peak finding

the reagents, or apparatus. parameters. The first cup in every run must always be

3.5 Linear Dynamic Range (LDR) - The absolute usually a high standard, but can be any sample that

quantity or concentration range over which the instrument generates a peak at least 25% of full scale. 

response to an analyte is linear.

3.6 Method Detection Limit (MDL) - The minimum

concentration of an analyte that can be identified,

measured and reported with 99% confidence that the

analyte concentration is greater than zero.8

3.7 Reagent Water (RW) - Type 1 reagent grade

water equal to or exceeding standards established by

American Society for Testing and Materials (ASTM).

Reverse osmosis systems or distilling units followed by

Super-Q Plus Water System that produce water with 18

megohm resistance are examples of acceptable water

sources. To avoid contamination, the reagent water

should be used the day of preparation.

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

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

refractive index is the ratio of the velocity of light in two

different media, such as estuarine or sea water versus

reagent water. The correction for this difference is

referred to as the refractive index correction in this

method.

3.9 Stock Standard Solution (SSS) -A concentrated

solution of method analyte prepared in the laboratory

using assayed reference compounds or purchased from

a reputable commercial source.

used to check laboratory performance with externally

identified as a SYNC sample. The SYNC sample is

4.0 Interferences

4.1 Hydrogen sulfide at concentrations greater than

0.1 mg S/L can interfere with nitrite analysis by

precipitating on the cadmium column . Hydrogen sulfide 9

in samples must be removed by precipitation with

cadmium or copper salt.

4.2 Iron, copper and other heavy metals at

concentrations larger than 1 mg/L alter the reduction

efficiency of the cadmium column. The addition of EDTA

will complex these metal ions.10

4.3 Phosphate at a concentration larger than 0.1

mg/L decreases the reduction efficiency of cadmium .

11

Dilute samples if possible or remove phosphate with ferric

hydroxide prior to analysis. 12

4.4 Particulates inducing turbidity should be removed

by filtration after sample collection.

4.5 This method corrects for small refractive index

interference which occurs if the calibration standard

solution is not matched with samples in salinity.

5.0 Safety

5.1 Water samples collected from the estuarine and

coastal environment are generally not hazardous.
Revision 2.0 September 1997 353.4-4

However, the individual who collects samples should use 6.2.4 60 mL high density polyethylene sample bottles,

proper technique.

5.2 Good laboratory technique should be used when

preparing reagents. Laboratory personnel should obtain

material safety data sheets (MSDS) for all chemicals

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

gloves should be worn when handling the concentrated

acid.

6.0 Equipment and Supplies

6.1 Gas Segmented Continuous Flow Autoanalyzer

Consisting of:

6.1.1 Autosampler.

6.1.2 Analytical cartridge with reaction coils for nitrate

analysis.

6.1.3 Open Tubular Cadmium Reactor (OTCR, 

Alpkem, OR) or laboratory prepared packed coppercoated cadmium reduction column (prepared according

to procedures in Section 7.4 - 7.5 ).

6.1.4 Proportioning pump.

6.1.5 Spectrophotometer equipped with a tungsten

lamp (380-800 nm) or photometer with a 540 nm

interference filter (2 nm bandwidth).

6.1.6 Strip chart recorder or computer based data

acquisition system.

6.1.7 Nitrogen gas ( high-purity grade, 99.99%).

6.2 Glassware and Supplies

6.2.1 All labware used in the analysis must be low in

residual nitrate to avoid sample or reagent contamination.

Soaking with lab grade detergent, rinsing with tap water,

followed by rinsing with 10% HCl (v/v) and thoroughly

rinsing with reagent water is sufficient.

6.2.2 Automatic pipetters capable of delivering volumes

ranging from 100 μL to 1000 μL and 1mL to 10 mL with

an assortment of high quality disposable pipet tips.

6.2.3 Analytical balance, with capability to measure to

0.1 mg, for preparing standards.

glass volumetric flasks and plastic sample tubes.

6.2.5 Drying oven.

6.2.6 Desiccator.

6.2.7 Membrane filters with 0.45 μm nominal pore size.

Plastic syringes with syringe filters.

6.2.8 A pH meter with a glass electrode and a 

reference electrode. A set of standard buffer solutions for

calibration of the pH meter.

7.0 Reagents and Standards

7.1 Stock Reagent Solutions

7.1.1 Stock Sulfanilamide Solution - Dissolved 10 g of

sulfanilamide (C6H8N2O2S, FW 172.21) in 1 L of 10%

HCl. 

7.1.2 Stock Nitrate Solution (100 mg-N/L) -

Quantitatively transfer 0.7217 g of pre-dried (105 C for 1 o

hour) potassium nitrate (KNO , FW 101.099) to a 1000- 3 

mL glass volumetric flask containing approximate 800 mL

of reagent water and dissolve the salt. Dilute the solution

to the mark with reagent water. Store the stock solution in

a polyethylene bottle in refrigerator at 4 C. This solution o

is stable for six months.

7.1.3 Stock Nitrite Solution (100 mg-N/L) -

Quantitatively transfer 0.4928 g of pre-dried (105 C for 1 o

hour) sodium nitrite (NaNO2 , FW 68.99) to a 1000 mL

glass volumetric flask containing approximate 800 mL of

reagent water and dissolve the salt. Dilute the solution to

the mark with reagent water. Store the stock solution in a

polyethylene bottle in a refrigerator at 4

oC. This solution

is stable for three months.

Note: High purity nitrite salts are not available. Assays

given by reagent manufacturers are usually in the range

of 95-97%. The impurity must be taken into account for

calculation of the weight taken.

7.1.4 Low Nutrient Sea Water (LNSW) - Obtain natural

low nutrient seawater from surface water of the Gulf

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

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

this is not available, commercial low nutrient sea water

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

International, Wormley, U.K. ) can be substituted.
 353.4-5 Revision 2.0 September 1997

7.2 Working Reagents 7.2.8 Calibration Standards - Prepare a series of

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

surfactant (ICI Americas, Inc.) to 1000 mL reagent water 100 mL with reagent water or low nutrient seawater.

and mix gently. Prepare these standards daily. The concentration range

Note: Brij-35 is a trade name for polyoxyethylene(23) concentrations of samples and not exceed two orders of

lauryl ether (C H (OCH CH ) OH, FW=1199.57, CASRN magnitude. At least five calibration standards with equal 12 25 2 2 23

9002-92-0). increments in concentration should be used to construct

7.2.2 Working Sulfanilamide Solution - Add 1 mL of 

Brij- 35 solution to 200 mL of stock sulfanilamide solution,

mix gently.

Note: Adding surfactant Brij-35 to sulfanilamide solution

instead of to the buffer solution is to prevent the Brij from

being adsorbed on the cadmium surface, which may

result in decreasing surface reactivity of the cadmium and

reduce the lifetime of the cadmium column.

7.2.3 NED Solution - Dissolve 1 g of NED (N-1-

naphthylethylenediamine Dihydrochloride, C H N .2HCl, 12 14 2

FW 259.18) in 1 L of reagent water. 

7.2.4 Imidazole Buffer Solution - Dissolve 13.6 g of

imidazole (C H N , FW 68.08) in 4 L of reagent water. 3 4 2

Add 2 mL of concentrated HCl. Adjust the pH to 7.8 with

diluted HCl while monitoring the pH with a pH meter.

Store in a refrigerator.

7.2.5 Copper Sulfate Solution (2%) - Dissolve 20 g of

copper sulfate (CuSO .5H O, FW 249.61) in 1 L of 4 2

reagent water. 

7.2.6 Colored SYNC Peak Solution - Add 50 μL of red

food coloring solution to 1000 mL reagent water and mix

thoroughly. Further dilute this solution to obtain a peak

between 25 to 100 percent full scale according to the

AUFS setting used for the refractive index measurement.

7.2.7 Primary Dilution Standard Solution - Prepare a

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

5.0 mL of stock standard solutions to 100 mL with reagent

water. Prepare this solution daily.

Note: This solution should be prepared to give an

appropriate intermediate concentration for further dilution

to prepare the calibration solutions. Therefore the

concentration of a primary dilution standard solution

should be adjusted according to the concentration range

of calibration solutions. 

calibration standards (CAL) by diluting suitable volumes

of a primary dilution standard solution (Section 7.2.7) to

of calibration standards should bracket the expected

the calibration curve.

If nitrate + nitrite and nitrite are analyzed simultaneously

by splitting a sample into two analytical systems, a nitrate

and nitrite mixed standard should be prepared. The total

concentration (nitrate+nitrite) must be assigned to the

concentrations of calibration standards in the

nitrate+nitrite system.

When analyzing samples of varying salinities, it is

recommended that the calibration standard solutions and

sampler wash solution be prepared in reagent water and

corrections for refractive index be made to the sample

concentrations determined (Section 12.2 ).

7.2.9 Saline Nitrate and Nitrite Standards - If CAL

solutions will not be prepared to match sample salinity,

then saline nitrate and nitrite standards must be prepared

in a series of salinities in order to quantify the salt error,

the change in the colorimetric response of nitrate due to

the change in the composition of the solution. The

following dilutions of Primary Dilution Standard Solution

(Section 7.2.7) to 100 mL in volumetric flasks with

reagent water, are suggested:

Salinity Volume of Volume of Conc.

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

_______________________________________

0 0 2 .10

9 25 2 .10

18 50 2 .10

27 75 2 .10

35 98 2 .10

_______________________________________

7.3 Open Tubular Cadmium Reactor

7.3.1 Nitrate in the samples is reduced to nitrite by

either a commercial Open Tubular Cadmium Reactor

(OTCR, Alpkem, OR) or a laboratory-prepared packed

copper-coated cadmium reduction column.
Revision 2.0 September 1997 353.4-6

7.3.2 If an OTCR is employed, the following procedures Connect both ends of the column using a plastic tube

should be used to activate it.10

Prepare reagent water, 0.5N HCl solution and 2% CuSO4

solution in three 50 mL beakers. Fit three 10-mL plastic not been used for several days, it should be reactivated

syringes with unions. First flush the OTCR with 10 mL prior to sample analysis.

reagent water. Then flush it with 10 mL 0.5N HCl

solution in 3 seconds, immediately followed by flushing 7.5 Stabilization of OTCR and Packed Cadmium

with a couple of syringe volumes of reagent water. Slowly Reduction Columns

flush with CuSO4 solution until a large amount of black

precipitated copper come out of OTCR, then stop the 7.5.1 Pump the buffer and other reagent solutions

flushing. Finally flush the OTCR with reagent water. Fill

the OTCR with imidazole buffer for short term storage.

7.4 Packed Cadmium Reduction Column

The following procedures are used for preparation of a

packed cadmium reduction column.13

7.4.1 File a cadmium stick to obtain freshly prepared

cadmium filings.

7.4.2 Sieve the filings and retain the fraction between 25

and 60 mesh size ( 0.25-0.71 mm).

7.4.3 Wash filings two times with 10% HCl followed with

reagent water.

7.4.4 Decant the reagent water and add 50 mL of 2%

CuSO solution. While swirling, brown flakes of colloidal 4

copper will appear and the blue color of the solution will 7.5.5 The reduction efficiency of an OTCR or a packed

fade. Decant the faded solution and add fresh CuSO4

solution and swirl. Repeat this procedure until the blue

color does not fade.

7.4.5 Wash the filings with reagent water until all the

blue color is gone and the supernatant is free of fine Absorbance of Nitrate 

particles. Keep the filings submersed under reagent water Reduction Efficiency = 

and avoid exposure of the cadmium filings to air. Absorbance of Nitrite 

7.4.6 The column can be prepared in a plastic or aglass

tube of 2 mm ID. Plug one end of column with glass wool.

Fill the column with water and transfer Cd filings in

suspension using a 10 mL pipette tip connected to one

end of column. While gently tapping the tube and pipette

tip let Cd filings pack tightly and uniformly in the column

without trapping air bubbles.

7.4.7 Insert another glass wool plug at the top of the

column. If a U- shape tube is used, the pipette tip is

connected to the other end and the procedure repeated.

filled with buffer solution to form a closed loop.

7.4.8 If an OTCR or a packed cadmium column has

through the manifold and obtain a stable baseline.

7.5.2 Pump 0.7 mg-N/L nitrite standard solution

continuously through the sample line and record the

steady state signal.

7.5.3 Stop the pump and install an OTCR or a packed

column on the manifold. Ensure no air bubbles have

been introduced into the manifold during the installation.

Resume the pumping and confirm a stable baseline.

7.5.4 Pump 0.7 mg-N/L nitrate solution continuously

through the sample line and record the signal. The signal

will increase slowly and reach steady state in about 10-15

minutes. This steady state signal should be close to the

signal obtained from the same concentration of a nitrite

solution without the OTCR or packed cadmium column

on line.

cadmium column can be determined by measuring the

absorbance of a nitrate standard solution followed by a

nitrite standard solution of the same concentration.

Reduction efficiency is calculated as follows:

______________________________

8.0 Sample Collection, Preservation and

Storage

8.1 Sample Collection - Samples collected for

nutrient analyses from estuarine and coastal waters are

normally collected using one of two methods: hydrocast

or submersible pump systems.

8.1.1 A hydrocast uses a series of sampling bottles

(Niskin, Go-Flo or equivalent) that are attached at fixed

intervals to a hydro wire. These bottles are sent through

the water column open and are closed either
 353.4-7 Revision 2.0 September 1997

electronically or via a mechanical messenger when the

bottles have reached the desired depth.

8.1.2 In a submersible pump system, a weighted hose is

sent to the desired depth in the water column and water

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

sample processing.

8.1.3 For collecting surface samples, an acid - cleaned

plastic bucket or a large plastic bottle can be used as a

convenient sampler. Wash the sampler three times with

sample water before collecting samples.

8.1.4 Turbid samples should be filtered as soon as

possible after collection.

8.1.5 60-mL high density polyethylene bottles are used

for sample storage. Sample bottles should be rinsed 3

times with about 20 mL of sample, shaking with the cap

in place after each rinse. Pour the rinse water into the cap

to dissolve and rinse away salt crusts trapped in the

threads of the cap. Finally, fill the sample bottle about 3/4

full, and screw the cap on firmly.

8.2 Sample Preservation - After collection and

filtration, samples should be analyzed as soon as

possible. If samples will be analyzed within 3 hours then

keep refrigerated in tightly sealed, high density

polyethylene bottles in the dark at 4 C until analysis can o

be performed.

8.3 Sample Storage - Natural samples usually

contain low concentrations of nitrite (< 14 g N/L) and no

preservation techniques are satisfactory. Samples must 14 

be analyzed within 3 hours of collection to obtain reliable

nitrite concentrations.15

Samples containing high concentrations of ammonia or

nitrite may change in nitrate concentration during storage

due to microbial oxidation of ammonia and nitrite to

nitrate. These samples should be analyzed as soon as

possible.

Natural samples containing low concentrations of nitrite

and ammonia ( < 10% of the nitrate concentration ) can

be preserved for nitrate analysis by freezing. A maximum

holding time for preserved estuarine and coastal water

samples for nitrate analysis is one month.16

The results of preservation of natural samples are shown

in Tables 1 and 2 for nitrate and nitrite, respectively.

9.0 Quality Control

9.1 Each laboratory using this method is required to

implement a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of performance, continued analysis of

Laboratory Reagent Blanks (LRB), laboratory duplicates

and Laboratory Fortified Blanks (LFB) with each set of

samples as a continuing check on performance.

9.2 Initial Demonstration of Performance

(Mandatory)

9.2.1 The initial demonstration of performance is used

to characterize instrument performance by determining

the MDL and LDR and laboratory performance by

analyzing quality control samples prior to analysis of

samples using this method.

9.2.2 A method detection limit (MDL) should be

established for the method analytes using a low level

seawater sample containing, or fortified at, approximately

5 times the estimated detection limit. To determine MDL

values, analyze at least seven replicate aliquots of water

which have been processed through the entire analytical

method. Perform all calculations defined in the method

and report concentration in appropriate units. Calculate

the MDL as follows:

 MDL = (t)(S)

where, S = the standard deviation of the 

 replicate analyses

 t = Student's t value for n-1 degrees of

 freedom at the 99% confidence 

 limit; t = 3.143 for six degrees 

 of freedom. 

MDLs should be determined every six months or

whenever a significant change in background or

instrument response occurs or a new matrix is

encountered.

 

9.2.3 The LDR should be determined by analyzing a

minimum of eight calibration standards ranging from

0.002 to 2.00 mg N/L across all sensitivity settings

(Absorbance Units Full Scale output range setting) of the

detector. Standards and sampler wash solutions should

be prepared in low nutrient seawater with salinities similar

to that of samples, therefore a correction factor for salt

error, or refractive index, will not be necessary. Normalize
Revision 2.0 September 1997 353.4-8

responses by multiplying the response by the Absorbance The optional control limits must be equal to or better than

Units Full Scale output range setting. Perform the linear the required control limits of 90-110%. After each 5 to 10

regression of normalized response vs. concentration and new recovery measurements, new control limits can be

obtain the constants m and b, where m is the slope and calculated using only the most recent 20 to 30 data

b is the y-intercept. Incrementally analyze standards of points. Also the standard deviation (S) data should be

higher concentration until the measured absorbance used to establish an ongoing precision statement for the

response, R, of a standard no longer yields a calculated level of concentrations included in the LFB. These data

concentration C , that is within 100 ± 10% of known must be kept on file and be available for review. c

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

defines the upper limit of the LDR for the instrument. 9.4 Assessing Analyte Recovery -

Should samples be encountered that have a Laboratory Fortified Sample Matrix

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

then these samples must be diluted and reanalyzed. 

9.3 Assessing Laboratory Performance

(Mandatory)

9.3.1 Laboratory Reagent Blank (LRB) - A laboratory

should analyze at least one LRB with each set of

samples. LRB data are used to assess contamination

from the laboratory environment. Should an analyte value

in the LRB exceed the MDL, then laboratory or reagent

contamination should be suspected. When the LRB value

constitutes 10% or more of the analyte concentration

determined for a sample, duplicates of the sample must

be prepared and analyzed again after the source of

contamination has been corrected and acceptable LRB

values have been obtained.

9.3.2 Laboratory Fortified Blank (LFB) - A laboratory

should analyze at least one LFB with each set of

samples. The LFB must be at a concentration that is

within the daily calibration range. The LFB data are used

to calculate accuracy as percent recovery. If the recovery

of the analyte falls outside the required control limits of

90 -110%, the source of the problem should be identified

and resolved before continuing the analyses.

9.3.3 The laboratory must use LFB analyses data to

assess laboratory performance against the required

control limits of 90-110%. When sufficient internal

performance data become available (usually a minimum

of 20 to 30 analyses), optional control limits can be

developed from the percent mean recovery (x) and

standard deviation (S) of the mean recovery. These data

can be used to establish the upper and lower control

limits as follows:

Upper Control Limit = x + 3S

Lower Control Limit = x - 3S

(LFM)

9.4.1 A laboratory should add a known amount of

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

samples or one sample per sample set, whichever is

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

ambient concentration and should be at least four times

greater than the MDL. 

9.4.2 Calculate percent recovery of analyte, corrected

for background concentration measured in a separate

unfortified sample. These values should be compared

with the values obtained from the LFBs. Percent

recoveries may be calculated using the following

equation:

 (C - C) s

R = x 100 ____________

 S

where,

R = percent recovery

C = measured fortified sample concentration s

 (background + addition in mg N/L)

C = sample background concentration (mg N/L)

S = concentration in mg N/L added to the

 environmental sample.

9.4.3 If the recovery of the analyte falls outside the

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

performance for that analyte is within the control limits,

the fortified sample should be prepared again and

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

recovery problem encountered with the fortified sample

is judged to be the matrix related and the sample data

should be flagged.
 353.4-9 Revision 2.0 September 1997

10.0 Calibration and Standardization

10.1 At least five calibration standards should be

prepared fresh daily for system calibration. The

calibration concentrations should bracket the

concentrations of samples and the range should not be

over two orders of magnitude.

10.2 A calibration curve should be constructed for

each sample set by analyzing a series of calibration

standard solutions. A sample set should contain no more

than 60 samples. For a large number of samples make

several sample sets with individual calibration curves. 

10.3 Analyze the calibration standards, in duplicate,

before actual samples.

10.4 The calibration curve containing five or more data

points should have a correlation coefficient, r, of 0.995 or

better. 

10.5 Place a high CAL solution followed by two blank

cups to quantify the carry-over of the system. The

difference in peak heights between two blank cups is due

to the carry over from the high CAL solution. The carryover coefficient, k, is calculated as follows:

 Pb1 - Pb2

k = __________________

 Phigh

where,

P = the peak height of the high high

 nitrate standard

Pb1 = the peak height of the

 first blank sample

Pb2 = the peak height of the

 second blank sample.

The carry over coefficient, k, for a system should be

measured in seven replicates to obtain a statistically

significant number. k should be remeasured with any

change in manifold plumbing or upon replacement of

pump tubung.

The carry over correction (CO) on a given peak i is

proportional to the peak height of the preceding sample,

P . i-1

CO = (k)(P ) i-1

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

carry over correction,17,18

Pi,c = Pi - CO

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

carry over should be applied to all the peak heights

throughout a run. The carry over coefficient should be

less than 5% in this method.

10.6 Place a high standard nitrate solution followed by

a nitrite standard solution of same concentration at the

beginning and end of each sample run to check for

change in reduction efficiency of OTCR or a packed

cadmium column. The decline of reduction efficiency

during a run should be less than 5%.

10.7 Place a high standard solution at the end of each

sample run (60 samples) to check for sensitivity drift.

Apply sensitivity drift correction to all the samples. The

sensitivity drift during a run should be less than 5%.

Note: Sensitivity drift correction is available in most data

acquisition software supplied with autoanalyzers. It is

assumed that the sensitivity drift is linear with time. An

interpolated drift correction factor is calculated for each

sample according to the sample position during a run.

Multiply the sample peak height by the corresponding

sensitivity drift correction factor to obtain the corrected

peak height for each sample.

11.0 Procedure

11.1 If samples are frozen, thaw the samples at room

temperature. If samples are stored in a refrigerator,

remove samples and equilibrate to room temperature.

Mix samples thoroughly prior to analysis.

11.2 Turn on the continuous flow analyzer and data

acquisition components and warm up at least 30 minutes.

11.3 Set up the cartridge according to the type of

cadmium reductor used for nitrate + nitrite analysis

(configuration for OTCR shown in Figure 1 and packed

cadmium column in Figure 2). Configuration for analysis

of nitrite alone is shown in Figure 3.

Note: When a gas segmented flow stream passes

through the OTCR, particles derived from the OTCR

were found to increase baseline noise and to cause
Revision 2.0 September 1997 353.4-10

interference at low level analysis. Packed cadmium stabilization. Ensure reduction efficiencies reach at least

columns are, therefore, preferred for nitrate analysis at 90% before analysis of samples.

low concentrations.

11.4 Set spectrophotometer wavelength at 540 nm. per hour for 60 second sample times and 30 second

11.5 Set the Absorbance Unit Full Scale (AUFS) range

on the spectrophotometer at an appropriate setting 11.10 Use cleaned sample cups or tubes (follow the

according to the highest concentration of nitrate in the procedures outlined in Section 6.2.2). Place CAL

samples. The appropriate setting for this method is 0.2 solutions and saline standards (optional) in sampler.

AUFS for 0.7 mg N/L. Complete filling the sampler tray with samples, laboratory

11.6 Prepare all reagents and standards. fortified sample matrices, and QC samples. Place a blank

11.7 Begin pumping the Brij-35 start-up solution (Section

7.2.1) through the system and obtain a steady baseline. 11.11 Commence analysis.

Place the reagents on-line. The reagent baseline will be

higher than the start-up solution baseline. After the

reagent baseline has been stabilized, reset the baseline.

NOTE: To minimize the noise in the reagent baseline,

clean the flow system by sequentially pumping the

sample line with reagent water, 1 N HCl solution, reagent

water, 1 N NaOH solution for a few minutes each at the

end of the daily analysis. Make sure to rinse the system

well with reagent water after pumping NaOH solution to

prevent precipitation of Mg(OH)2 when seawater is

introduced into the system. Keep the reagents and

samples free of particulate. Filter the reagents and

samples if necessary.

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

seconds to increase back pressure. If absorbance drops

down rapidly when back pressure increases, this indicates

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

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

in the flowcell can often be eliminated by simply attaching

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

the syringe piston. Alternatively, flushing the flowcell with

alcohol was found to be effective in removing air bubbles

from the flow cell.

For samples of varying salinities, it is suggested that the

reagent water used for the sampler wash solution and for

preparing calibration standards and procedures in

Sections 12.2 and 12.3 be employed.

11.8 Check the reduction efficiency of the OTCR or

packed cadmium column following the procedure in

Section 7.5.5. If the reduction efficiency is less than 90%

follow the procedure in Section 7.5 for activation and 

19

11.9 A good sampling rate is approximately 40 samples

wash times.

reagent blanks, laboratory fortified blanks, laboratory

after every ten samples.

12.0 Data Analysis and Calculations

12.1 Concentrations of nitrate in samples are

calculated from the linear regression, obtained from the

standard curve in which the concentrations of the

calibration standards are entered as the independent

variable, and their corresponding peak heights are the

dependent variable.

12.2 Refractive Index Correction for Estuarine and

Coastal Samples

12.2.1 If reagent water is used as the wash solution and

to prepare the calibration standard solutions, the operator

has to quantify the refractive index correction due to the

difference in salinity between sample and standard

solutions. The following procedures are used to measure

the relationship between sample salinity and refractive

index for a particular detector.

12.2.2 First, analyze a set of nitrate or nitrite standards

in reagent water with color reagent using reagent water

as the wash and obtain a linear regression of peak height

versus concentration.

Note: The change in absorbance due to refractive index

is small, therefore low concentration standards should be

used to bracket the expected absorbances due to

refractive index.

12.2.3 Second, replace reagent water wash solution with

Low Nutrient Seawater wash solution.
 353.4-11 Revision 2.0 September 1997

Note: In nitrate and nitrite analysis absorbance of the _______________________________________

reagent water is higher than that of the LNSW. When Salinity Apparent concentration (μg N/L)

using reagent water as a wash solution, the change in (% ) Nitrate Nitrite

refractive index causes the absorbance of seawater to _______________________________________ 

become negative. To measure the absorbance due to

refractive index change in different salinity samples, Low 0.0 0.000 0.000

Nutrient Seawater must be used as a wash solution to 3.8 0.026 0.015

bring the baseline down. 9.2 0.096 0.040

12.2.4 Replace NED solution (Section 7.2.4) with 18.1 0.190 0.086

reagent water. All other reagents remain the same.

Replace the synchronization sample with the colored

SYNC peak solution (Section 7.2.6).

12.2.5 Prepare a set of different salinity samples with

LNSW. Commence analysis and obtain peak heights for

different salinity samples. The peak heights for the

refractive index correction must be obtained at the same

AUFS range setting and on the same spectrophotometer

as the corresponding standards (Section 12.2.2).

12.2.6 Using Low Nutrient Seawater as the wash water,

a maximum absorbance will be observed for reagent

water. No change in refractive index will be observed in

the seawater sample. Assuming the absolute absorbance

for reagent water (relative to the seawater baseline) is

equal to the absorbance for seawater (relative to reagent

water baseline), subtract the absorbances of samples of

various salinities from that of reagent water. The results

are the apparent absorbance due to the change in

refractive index between samples of various salinities

relative to the reagent water baseline.

12.2.7 For each sample of varying salinity, calculate the

apparent nitrate or nitrite concentrations due to refractive

index from its peak height corrected to reagent water

baseline (Section 12.2.5) and the regression equation of

nitrate or nitrite standards obtained with color reagent

being pumped through the system (12.2.2). Salinity is

entered as the independent variable and the apparent

nitrate or nitrite concentration due to refractive index is

entered as the dependent variable. The resulting

regression allows the operator to calculate apparent

nitrate or nitrite concentration due to refractive index when

sample salinity is known. Thus, the operator would not be

required to obtain refractive index peak heights for all

samples.

12.2.8 An example of typical results follows:

o

13.8 0.142 0.055

26.8 0.297 0.153

36.3 0.370 0.187

_______________________________________

Note: You must calculate the refractive index correction

for your particular detector. Moreover, the refractive index

must be redetermined whenever a significant change in

the design of flowcell or a new matrix is encountered.

12.2.9 An example of typical linear equations is:

Apparent nitrate (μg N/L) = 0.01047S

Apparent nitrite (μg N/L) = 0.00513S

where S is sample salinity. The apparent nitrate and nitrite

concentration due to refractive index so obtained should

be added to samples of corresponding salinity when

reagent water is used as wash solution and standard

matrix.

If nitrate and nitrite concentrations are greater than 100

and 50 μg N/L respectively, the correction for refractive

index is negligible and this procedure can be optional.

12.3 Correction for Salt Error in Estuarine and

Coastal Samples 

12.3.1 When calculating concentrations of samples of

varying salinities from standards and the wash solution

prepared in reagent water, it is common to first correct for

refractive index errors, and then correct for any change in

color development due to the differences in composition

between samples and standards (so called salt error). 

12.3.2 Plot the salinity of the saline standards (Section

7.2.9) as the independent variable, and the apparent

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

(corrected for refractive index) calculated from the

regression of standards in reagent water, as the

dependent variable for all saline standards. The resulting

regression equation allows the operator to correct the
Revision 2.0 September 1997 353.4-12

concentrations of samples of known salinity for the color 27.5 0.0335 100.1 0.1052

enhancement due to matrix effect, e.g., salt error.

Following are typical results for the nitrate and nitrite 18.6 0.0167 105.8 0.0523

systems: 18.6 0.0170 101.6 0.0534

_______________________________________ 18.6 0.0229 104.5 0.0719

Salinity Apparent concentration (μg N/L)

(%o) Nitrate Nitrite 9.4 0.0222 105.3 0.0698

_______________________________________ 9.4 0.0229 106.4 0.0720

0.0 569.64 558.15

3.8 570.50 565.50 0.0 0.0260 103.9 0.0817

9.2 572.74 563.00 0.0 0.0306 106.9 0.0961

13.8 568.96 564.94 0.0 0.0160 111.0 0.0501

18.1 566.44 563.00 0.0 0.0248 109.5 0.0780

26.8 558.74 559.06

36.3 559.86 554.67 __________________________________

_______________________________________

12.3.3 As shown in above results, salinity has no

systematic effect on the nitrate and nitrite signal and and Biscayne Bay, Florida. Seven replicates of each

therefore salt error correction is not recommended. sample were processed and analyzed with salinity

12.4 Results of sample analyses should be reported follows:

in mg N/L or in μg N/L.

mg N/L = ppm (parts per million)

 μg N/L = ppb (part per billion) Sample Salinity Concentration RSD

13.0 Method Performance

13.1 Single Laboratory Validation

13.1.1 Method Detection Limit- A method detection limit

(MDL) of 0.075 μg N/L has been determined by one

laboratory from LNSW of five different salinities fortified at

a nitrate concentration of 0.28 μg N/L.

____________________________________

Salinity SD Recovery MDL

(% ) (μg N/L) (%) (μg N/L) o

_____________________________________

36.5 0.0234 103.5 0.0734

36.5 0.0298 98.9 0.0935

36.5 0.0148 110.3 0.0464

36.5 0.0261 103.6 0.0819

27.5 0.0203 105.4 0.0638

27.5 0.0321 102.3 0.1009

27.5 0.0314 103.8 0.0986

18.6 0.0229 106.4 0.0720

9.4 0.0197 91.5 0.0620

13.1.2 Single analyst precision - A single laboratory

analyzed three samples collected from the Miami River

ranging from 0.019 to 32.623‰. The results were as

______________________________________

 (% ) (μg N/L) (%) o

______________________________________

Nitrate

1 32.623 48.22 2.59

2 13.263 206.41 1.07

3 0.019 276.38 1.99

Nitrite

1 32.623 5.21 1.62

2 13.263 31.03 0.58

3 0.019 54.07 0.49

______________________________________

13.1.3 Laboratory fortified sample matrix - Laboratory

fortified sample matrices were processed in three

different salinities ranging from 0.019 to 32.623 and

ambient nitrate concentrations from 48.22 to 276.38 μg

N/L. Seven replicates of each sample were analyzed and

the results were as follows:
 353.4-13 Revision 2.0 September 1997

_______________________________________ waste identification rules and land disposal restrictions.

Salinity Concentration RSD Recovery

 ambient fortified Personnel, available from the American Chemical Society

(% ) (μg N/L) (%) (%) o

______________________________________

32.623 48.22 139.94 1.50 106.4

13.263 206.41 139.94 1.25 102.6

0.019 276.38 139.94 1.19 102.3

______________________________________

13.2 Multi-Laboratory Validation

Multi-laboratory data is unavailable at this time.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

USEPA has established a preferred hierarchy of

environmental management techniques that places

pollution prevention as the management option of first

choice. Whenever feasible, laboratory personnel should

use pollution prevention techniques to address their waste

generation. When wastes cannot be feasibly reduced at

the source, the agency recommends recycling as the next

best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research

institutions, consult Less is Better: Laboratory Chemical

Management for Waste Reduction, available from the naphthyl)ethylenediamine as the coupling agent.

American Chemical Society, Department of Government

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

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

15.0 Waste Management

15.1 The U.S. Environmental Protection Agency

requires that laboratory waste management practices be

conducted consistent with all applicable rules and

regulations. The Agency urges laboratories to protect the

air, water, and land by minimizing and controlling all

releases from hoods and bench operations, complying

with the letter and spirit of any sewer discharge permits

and regulations, and by complying with all solid and

hazardous waste regulations, particularly the hazardous

For further information on waste management consult

The Waste Management Manual for Laboratory

at the address listed in Section 14.2.

16.0 References

1. Morris, A. W. and Riley, J.P., 1963. Determination

of nitrate in sea water. Anal. Chim. Acta. 29:272-

279.

2. Brewer P. G. and J. P. Riley 1965. The automatic

determination of nitrate in seawater. Deep-Sea

Res., 12:765-772.

3. Wood, E.O., Armstrong, F.A.J., and Richards, F.A.,

1967. Determination of nitrate in seawater by

cadmium-copper reduction to nitrite. J. Mar. Biol.

Assn. U.K., 47:23-31.

4. Bendschneider, K. and R. J. Robinson, 1952. A

new spectrophotometric method for the

determination of nitrite in sea water. J. Marine Res.,

11:87-96.

5. Fox, J.B. 1979. Kinetics and mechanisms of the

Griess reaction. Analytical Chem. 51:1493-1502.

6. Norwitz, G., P.N. Keliher,, 1984.

Spectrophotometric determination of nitrite with

composite reagents containing sulphanilamide,

sulphanilic acid or 4- nitroaniline as the diozotisable

aromatic amine and N-(1-

Analyst, 109:1281-1286.

7. Spencer, C.P. 1975, The micronutrient elements.

In Chemical Oceanography (Riley, J. P. and G.

Skirrow, Eds.), Academic Press, London and New

York, 2nd Ed. Vol 2, Chapter 11.

8. 40 CFR, 136 Appendix B. Definition and Procedure

for the Determination of Method Detection Limit.

Revision 1.11.

9. Timmer-ten Hoor, A., 1974. Sulfide interaction on

colorimetric nitrite determination. Marine Chemistry,

2:149-151.
Revision 2.0 September 1997 353.4-14

10. Alpkem Corporation. 1990. RFA Methodology: 18. Zhang, J.-Z. 1997. Distinction and quantification of

Nitrate+Nitrite Nitrogen. Method A303-S170. carry-over and sample interaction in gas

Alpkem Corporation, Clackamas, Oregon. segmented continuous flow analysis . Journal of

11. Olson, R.J. 1980. Phosphate interference in the

cadmium reduction analysis of nitrate. Limnol. 19. Garside, C. 1993. Nitrate reductor efficiency as an

Oceanogr., 25(4)758-760. error source in seawater analysis. Marine

12. Alvarez-Salgado, X.A., F.Fraga and F.F.Perez.

1992, Determination of nutrient salt by automatic

methods both in seawater and brackish water: the

phosphate blank. Marine Chemistry, 39:311-319. 

13. Grasshoff , K. 1983, Determination of Nitrate, In

Methods of Seawater Analysis (Grasshoff, K., M.

Ehrhardt and K. Kremling, Eds) Weinheim, Verlag

Chemie, Germany. pp143-150. 

14. Takenaka, N., A.Ueda and Y. Maeda 1992,

Acceleration of the rate of nitrite oxidation by

freezing in aqueous solution. Nature, Vol. 358,

p736-738.

15. Grasshoff , K. 1983, Determination of Nitrite, In

Methods of Seawater Analysis (Grasshoff, K., M.

Ehrhardt and K. Kremling, Eds) Weinheim, Verlag

Chemie, Germany. pp139-142. 

16. MacDonald, R.W. and F.A. McLaughlin. 1982. The

effect of Storage by freezing on dissolved inorganic

phosphate, nitrate, and reactive silicate for samples

from coastal and estuarine waters. Water

Research, 16:95-104.

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

as a function of system performance for continuous

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

Automataic Chemistry, 19(6):205-212.

Chemistry 44: 25-30.
1

2

3

4

5

6

7

9

10

0.41

0.25

1.57

Pump

mL/min

Debubbler

Detector

Manifold

Sample

Reagent Water 

or Low Nutrient Seawater

Wash To Sampler

To Waste

To Waste

Sample:Wash = 60":30"

540nm

1.12 Buffer

Nitrogen

0.32

0.10 Sulfanilamide

0.10 NED

8

0.41

OTCR

 353.4-15 Revision 2.0 September 1997

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Figure 1. Manifold configuration for nitrate + nitrite analysis using an Open Tubular Cadmium Reactor.
1

2

3

4

5

6

7

9

10

0.41

0.25

1.57

Pump

mL/min

Debubbler

Detector

Manifold

Sample

Reagent Water 

or Low Nutrient Seawater

Wash To Sampler

To Waste

To Waste

Sample:Wash = 60":30"

540nm

1.12 Buffer

Nitrogen

0.32

0.10 Sulfanilamide

0.10 NED

8

0.41

Cd

Column

Revision 2.0 September 1997 353.4-16

Figure 2. Manifold configuration for nitrate + nitrite analysis using a homemade packed copper-coated cadmium

reduction column.
1

2

3

4

5

6

7

9

10

0.41

0.25

1.57

Pump

mL/min

Debubbler

Detector

Manifold Reagent Water 

or Low Nutrient Seawater

Wash To Sampler

To Waste

To Waste

Sample:Wash = 60":30"

540nm

1.01 Sample

Air

0.10 Sulfanilamide

0.10 NED

8

 353.4-17 Revision 2.0 September 1997

Figure 3. Manifold configuration for nitrite analysis.
Revision 2.0 September 1997 353.4-18

Table 1 . Percentage recovery of nitrate from natural water samples preserved by freezing and refrigeration.

Method Sample Salinity Time (Day) A B

0 7 14 21 28 35 46 62 92

25C, P river 0.019 100 192.5 279 287.3 267.5 262.4 300.7 228.1 260.8

estuary 13.263 100 108.5 106.2 124 103.9 139.3 258.9 188.5 229.1

coast 32.623 100 102 128.8 153.8 93.3 89 44.2 72.4 84.9

25C, G river 0.019 100 257 294.9 316.4 298.2 225.4 135.4 77.6 66.9

estuary 13.263 100 108.8 108.5 122.5 90.6 79.2 81.5 56.2 128.2

coast 32.623 100 98 135.2 150.9 98.5 84.3 36.9 56.1 66.6

4C,P river 0.019 100 105 90 111.6 100.7 82.7 112.2 97.3 104.7

estuary 13.263 100 104.5 90.4 107.1 102.6 95.9 109 82.4 101.4

coast 32.623 100 127.6 65.7 149.1 82.3 93.3 43.3 73.5 89.2

4C,G river 0.019 100 158.2 88.1 108.4 99.4

estuary 13.263 100 103.1 84.5 107.4 95.9

coast 32.623 100 100.9 54.4 123 68.9

4C,P, river+ 0.019 100 105.5 99.2 106.1 96.2 91 114.8 98.4 96.9

estuary+ 13.263 100 110.2 116.4 104.8 102.9 93 110.9 85 99.7

coast+ 32.623 100 112.7 112.7 103.8 93.3 90.6 102.4 75.4 98.6

4C,G, river+ 0.019 100 105.7 98.3 101 114.5

estuary+ 13.263 100 100.1 98 93.3 109.1

coast+ 32.623 100 104.4 93.6 90.2 99.5

Fr,P river 0.019 100 100.5 100.4 103.9 95.8 88.6 85.7 95.9

estuary 13.263 100 114.1 115.5 105.6 97.9 104.6 98.8 72.8 87.6

coast 32.623 100 130.5 100.9 128.2 92.7 98.5 42.2 50.9 87.5

Fr,P, river+ 0.019 100 101.9 103.2 103.1 95.4 91.2 82.5 87.4 90.2

estuary+ 13.263 100 102 106.7 102.4 97.4 95 78.5 78 94.7

coast+ 32.623 100 103.2 111.1 101.3 91.5 92.1 104.7 69.6 92.3
 353.4-19 Revision 2.0 September 1997

Table 2 . Percentage recovery of nitrite from natural water samples preserved by freezing and refrigeration

Method Sample Salinity Time(day) A B

0 7 14 21 28 35 46 62 92

25C, P river 0.019 100 220 0.3 0 0 0 0 0 0

estuary 13.263 100 110.6 456.8 920.2 957.8 661.5 58.7 0 0

coast 32.623 100 104.1 92.2 74.1 89.5 74.1 94.6 72.2 0

25C, G river 0.019 100 182.8 0.3 0 0 0 0 0 0

estuary 13.263 100 108.5 519.1 1026.3 1079.1 867.5 843.1 705.7 209.2

coast 32.623 100 100 87.8 73.8 89.5 73.5 95.9 85.7 66.5

4C,P river 0.019 100 104.2 88.2 31.8 93.9 0 65 84.1 0

estuary 13.263 100 102.8 101.8 38.9 0 91 17.8 8.5 0

coast 32.623 100 68.4 65.7 33.2 70.5 50.5 0 0 0

4C,G river 0.019 100 104.9 97.8 99.8 96.7

estuary 13.263 100 104.4 98.8 100.6 91

coast 32.623 100 94.3 87 71.1 97.6

4C,P river+ 0.019 100 47.6 98.9 98.5 97.2 67.8 0 2.2 75.0

estuary+ 13.263 100 95.4 21.1 0 0 0 2.7 0 0

coast+ 32.263 100 0 0 0 0 0 0 0 0

4C,G river+ 0.019 100 97.9 95.8 84.6 85.9

estuary+ 13.263 100 100.6 91.6 94.1 100

coast+ 32.623 100 69.5 97.6 65.9 87.6

Fr,P river 0.019 100 70.6 86.2 98 77.3 68.1 74.9 77.3

estuary 13.263 100 1.3 0.7 0 0 0 96 13.3 57.3

coast 32.623 100 78.6 4.9 0 0 0 8.6 80 27.8

Fr,P river+ 0.019 100 97 87.2 95.4 75.9 75.9 63.1 75.2 69.2

estuary+ 13.263 100 103.5 98.6 95.9 52 90.5 74.2 0 77.6

coast+ 32.623 100 99.7 95.9 56.5 92.2 67 100.5 80 65.9

 Cont’d on

 next page
Revision 2.0 September 1997 353.4-20

Cont’d

Methods of preservation: A

25C,P and G: Store the samples in high density polyethylene carboys (P) or glass bottles (G)

at room temperature (~25 C). o

4C, P and G: Store samples in high density polyethylene bottles (P) or glass bottles (G) in a 

refrigerator (4 C) in the dark. o

Fr,P and Fr,P: Freeze the samples in high density polyethylene bottles (P) and store at -20 C

o

in a freezer in the dark.

Glass and high density polyethylene bottles were used to study the effect of type of sample 

bottles on the recovery of nitrite and nitrate from refrigeration.

For salinity and concentration of nitrate in river, estuary and coast samples see section 13.1.2. B

Sample river+, estuary+ and coast+ are the fortified river, estuary and coast samples,

 respectively, at nitrate concentrations 139.94 μg N/L.
365.5 - 1

Method 365.5

Determination of Orthophosphate in Estuarine and

Coastal Waters by Automated Colorimetric Analysis

Carl F. Zimmermann

Carolyn W. Keefe

University of Maryland System

Center for Environmental and Estuarine Studies

Chesapeake Biological Laboratory

Solomons, MD 20688-0036

Revision 1.4

September 1997

Edited by

Elizabeth J. Arar

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Revision 1.4 September 1997 365.5 - 2

Chemical Abstracts Service

Analyte Registry Numbers (CASRN)

Phosphate 14265-44-2

Method 365.5

Determination of Orthophosphate in Estuarine and Coastal

Waters by Automated Colorimetric Analysis

1.0 Scope and Application

1.1 This method provides a procedure for the determination of low-level orthophosphate concentrations

normally found in estuarine and/or coastal waters. It is

based upon the method of Murphy and Riley adapted for 1

automated segmented flow analysis in which the two

2

reagent solutions are added separately for greater

reagent stability and facility of sample separation.

1.2 A statistically determined method detection limit

(MDL) of 0.0007 mg P/L has been determined by one

laboratory in 3 parts per thousand (ppt) saline water. The 3 

method is linear to 0.39 mg P/L using a Technicon

AutoAnalyzer II system (Bran & Luebbe, Buffalo Grove,

IL).

1.3 Approximately 40 samples per hour can be analyzed.

1.4 This method should be used by analysts experienced in the use of automated colorimetric analyses, and

familiar with matrix interferences and procedures for their

correction. A minimum of 6-months experience under

experienced supervision is recommended.

2.0 Summary of Method

2.1 An automated colorimetric method for the

analysis of low-level orthophosphate concentrations is

described. Ammonium molybdate and antimony potassium tartrate react in an acidic medium with dilute solutions of phosphate to form an antimony-phospho-molybdate complex. This complex is reduced to an intensely

blue-colored complex by ascorbic acid. The color

produced is proportional to the phosphate concentration

present in the sample. Positive bias caused by differences in the refractive index of seawater and reagent

water is corrected for prior to data reporting.

3.0 Definitions American Society of Testing Materials (ASTM). Reverse

3.1 Calibration Standard (CAL) -- A solution

prepared from the stock standard solution that is used to

calibrate the instrument response with respect to analyte

concentration. One of the standards in the standard

curve.

3.2 Dissolved Analyte (DA) -- The concentration of

analyte in an aqueous sample that will pass through a

0.45-Fm membrane filter assembly prior to sample

acidification or other processing.

3.3 Laboratory Fortified Blank (LFB) -- An aliquot

of reagent water to which known quantities of the method

analytes are added in the laboratory. The LFB is analyzed exactly like a sample, and its purpose is to determine whether method performance is within acceptable

control limits. This is basically a standard prepared in

reagent water that is analyzed as a sample.

3.4 Laboratory Fortified Sample Matrix (LFM) -- An

aliquot of an environmental sample to which known

quantities of the method analytes are added in the

laboratory. The LFM is analyzed exactly like a sample,

and its purpose is to determine whether the sample matrix

contributes bias to the analytical results. The background

concentrations of the analytes in the sample matrix must

be determined in a separate aliquot and the measured

values in the LFM corrected for background concentrations.

3.5 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water that is treated exactly as a sample

including exposure to all glassware, equipment, and

reagents that are used with other samples. The LRB is

used to determine if method analytes or other interferences are present in the laboratory environment, the

reagents, or apparatus.

3.6 Linear Dynamic Range (LDR) -- The absolute

quantity or concentration range over which the instrument

response to an analyte is linear.

3.7 Method Detection Limit (MDL) -- The minimum

concentration of an analyte that can be identified, measured, and reported with 99% confidence that the analyte

concentration is greater than zero.

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

water equal to or exceeding standards established by

osmosis systems or distilling units that produce 18

megohm water are two examples of acceptable water

sources.
365.5 - 3 Revision 1.4 September 1997

3.9 Refractive Index (RI) -- The ratio of the velocity 6.1.5 Phototube that can be used for 600-900 nm

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

relative refractive index is the ratio of the velocity of light

in two different media, such as sea or estuarine water

versus reagent water. The correction for this difference

is referred to as the refractive index correction in this

method.

3.10 Stock Standard Solution (SSS) -- A concentrated solution of method analyte prepared in the laboratory using assayed reference compounds or purchased

from a reputable commercial source.

4.0 Interferences

4.1 Interferences caused by copper, arsenate and

silicate are minimal relative to the orthophosphate determination because of the extremely low concentrations

normally found in estuarine or coastal waters. High iron

concentrations can cause precipitation of and subsequent

loss of phosphate from the dissolved phase. Hydrogen

sulfide effects, such as occur in samples collected from

deep anoxic basins, can be treated by simple dilution of

the sample since high sulfide concentrations are most

often associated with high phosphate values.4

4.2 Sample turbidity is removed by filtration prior to

analysis.

4.3 Refractive Index interferences are corrected for

estuarine/coastal samples (Section 12.2).

5.0 Safety

5.1 Water samples collected from the estuarine

and/or ocean environment are generally not hazardous.

However, the individual who collects samples should use

proper technique.

5.2 Good laboratory technique should be used when

preparing reagents. A lab coat, safety goggles, and

gloves should be worn when preparing the sulfuric acid

reagent.

6.0 Equipment and Supplies

6.1 Continuous Flow Automated Analytical System Consisting of:

6.1.1 Sampler.

6.1.2 Manifold or Analytical Cartridge equipped with

37EC heating bath.

6.1.3 Proportioning pump.

6.1.4 Colorimeter equipped with 1.5 X 50 mm tubular

flow cell and a 880 nm filter.

range.

6.1.6 Strip chart recorder or computer based data

system.

6.2 Phosphate-Free Glassware and Polyethylene

Bottles

6.2.1 All labware used in the determination must be

low in residual phosphate to avoid sample or reagent

contamination. Washing with 10% HCI (v/v) and thoroughly rinsing with distilled, deionized water was found to

be effective.

6.2.2 Membrane or glass fiber filters, 0.45 Fm nominal

pore size.

7.0 Reagents and Standards

7.1 Stock Reagent Solutions

7.1.1 Ammonium Molybdate Solution (40 g/L) --

Dissolve 20.0 g of ammonium molybdate tetrahydrate

((NH ) Mo O C4H O, CASRN 12027-67-7) in approxi- 4 6 7 24 2

mately 400 mL of reagent water and dilute to 500 mL.

Store in a plastic bottle out of direct sunlight. This

reagent is stable for approximately three months.

7.1.2 Antimony Potassium Tartrate Solution (3.0 g/L) --

Dissolve 0.3 g of antimony potassium tartrate

[(K(SbO)C H O C1/2H O, CASRN 11071-15-1] in approxi- 4 4 6 2

mately 90 mL of reagent water and dilute to 100 mL. This

reagent is stable for approximately three months.

7.1.3 Ascorbic Acid Solution (18.0 g/L) -- Dissolve 18.0

g of ascorbic acid (C H O , CASRN 50-81-7) in approxi- 6 6 6

mately 800 mL of reagent water and dilute to 1 L. Dispense approximately 75 mL into clean polyethylene

bottles and freeze. The stability of the frozen ascorbic

acid is approximately three months. Thaw overnight in

the refrigerator before use. The stability of the thawed,

refrigerated reagent is less than 10 days.

7.1.4 Sodium Lauryl Sulfate Solution (30.0 g/L) --

Sodium dodecyl sulfate (CH3(CH2)11OSO3Na, CASRN

151-21-3). Dissolve 3.0 g of sodium lauryl sulfate (SLS)

in approximately 80 mL of reagent water and dilute to

100 mL. This solution is the wetting agent and its

stability is approximately three weeks.

7.1.5 Sulfuric Acid Solution (4.9 N) -- Slowly add 136

mL of concentrated sulfuric acid (H SO , CASRN 7664- 2 4

93-9) to approximately 800 mL of reagent water. After

the solution is cooled, dilute to 1 L with reagent water.

7.1.6 Stock Phosphorus Solution -- Dissolve 0.439 g

of pre-dried (105EC for 1 hr) monobasic potassium phosphate (KH2PO4

, CASRN 7778-77-0) in reagent water and
Revision 1.4 September 1997 365.5 - 4

mL of Secondary Conc.

Phosphorus Solution (7.2.4) mg P/L

0.1 0.0010

0.2 0.0020

0.5 0.0050

1.0 0.0100

2.0 0.0200

4.0 0.0400

5.0 0.0500

dilute to 1000 mL. (1.0 mL = 0.100 mg P.) The stability of

this stock standard is approximately three months when

kept refrigerated.

7.1.7 Low Nutrient Seawater -- Obtain natural low

nutrient seawater (36 ppt salinity; <0.0003 mg P/L) or

dissolve 31 g analytical reagent grade sodium chloride,

(NaCl, CASRN 7647-14-5); 10 g analytical grade magnesium sulfate, (MgSO4

) CASRN 10034-99-8); and 0.05 g

analytical reagent grade sodium bicarbonate, (NaHCO ,3

CASRN 144-55-8), in 1 L of reagent water.

7.2 Working Reagents

7.2.1 Reagent A -- Mix the following reagents in the

following proportions for 142 mL of Reagent A: 100 mL of

4.9 N H2SO4 (Section 7.1.5), 30 mL of ammonium

molybdate solution (Section 7.1.1), 10 mL of antimony

potassium tartrate solution (Section 7.1.2), and 2.0 mL of

SLS solution (Section 7.1.4). Prepare fresh daily.

7.2.2 Reagent B -- Add approximately 0.5 mL of the

SLS solution (Section 7.1.4) to the 75 mL of ascorbic acid

solution (Section 7.1.3). Stability is approximately 10 days

when kept refrigerated.

7.2.3 Refractive Reagent A -- Add 50 mL of 4.9 N

H2SO4(Section 7.1.5) to 20 mL of reagent water. Add 1

mL of SLS (Section 7.1.4) to this solution. Prepare fresh

every few days.

7.2.4 Secondary Phosphorus Solution -- Take 1.0 mL

of Stock Phosphorus Solution (Section 7.1.6) and dilute

to 100 mL with reagent water. (1.0 mL = 0.0010 mg P.)

Refrigerate and prepare fresh every 10 days.

7.2.5 Prepare a series of standards by diluting suitable

volumes of standard solutions (Section 7.2.4) to 100 mL

with reagent water. Prepare these standards daily. When

working with samples of known salinity, it is recommended that the standard curve concentrations be prepared in low-level natural seawater (Section 7.1.7) diluted

to match the salinity of the samples. Doing so obviates

the need to perform the refractive index correction

outlined in Section 12.2. When analyzing samples of

varying salinities, it is recommended that the standard

curve be prepared in reagent water and refractive index

corrections be made to the sample concentrations (Section 12.2). The following dilutions are suggested.

8.0 Sample Collection, Preservation and

Storage

8.1 Sample Collection -- Samples collected for

nutrient analyses from estuarine and coastal waters are

normally collected using one of two methods: hydrocast

or submersible pump systems. Filtration of the sample

through a 0.45-Fm membrane or glass fiber filter immediately after collection is required.

8.1.1 A hydrocast uses a series of sampling bottles

(Niskin, Nansen, Go-Flo or equivalent) that are attached

at fixed intervals to a hydro wire. These bottles are sent

through the water column open and are closed either

electronically or via a mechanical “messenger” when the

bottles have reached the desired depth.

8.1.2 When a submersible pump system is used, a

weighted hose is sent to the desired depth in the water

column and water is pumped from that depth to the deck

of the ship for processing.

8.1.3 Another method used to collect surface samples

involves the use of a plastic bucket or large plastic bottle.

While not the most ideal method, it is commonly used in

citizen monitoring programs.

8.2 Sample Preservation -- After collection and

filtration, samples should be analyzed as quickly as

possible. If the samples are to be analyzed within 24 hr

of collection, then refrigeration at 4EC is acceptable.

8.3 Sample Storage -- Long-term storage of frozen

samples should be in clearly labeled polyethylene bottles

or polystyrene cups compatible with the analytical system’s automatic sampler (Section 6.1.1). If samples

cannot be analyzed within 24 hr, then freezing at -20EC

for a maximum period of two months is acceptable.5-8

9.0 Quality Control

9.1 Each laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of laboratory capability, the continued

analysis of LRBs, laboratory duplicates, and LFBs as a

continuing check on performance.

9.2 Initial Demonstration of Performance 

(Mandatory)

9.2.1 The initial demonstration of performance is used

to characterize instrument performance (MDLs and linear

dynamic range) and laboratory performance (analysis of

QC samples) prior to analyses of samples using this

method.

9.2.2 MDLs should be established using a low-level

estuarine water sample fortified to approximately five
R'

(Cs&C)

S

x 100

365.5 - 5 Revision 1.4 September 1997

where, S = the standard deviation of the 

replicate analyses.

t = the Student’s t value for n-1 

degrees of freedom at the 99% 

confidence limit. t = 3.143 for six 

degrees of freedom.

where, R = percent recovery

 C = measured fortified sample S

concentration (background + 

concentrated addition in mg P/L)

C = sample background concentration 

(mg P/L)

S = concentration in mg P/L added to 

the environmental sample.

times the estimated detection limit. To determine MDL of control and the source of the problem should be

3

values, analyze seven replicate aliquots of water and identified and resolved before continuing the analyses.

process through the entire analytical method. Perform all

calculations defined in the method and report the con- 9.3.3 The laboratory must use LFB data to assess

centration values in the appropriate units. Calculate the

MDL as follows:

MDL = (t)(S)

MDLs should be determined every six months or whenever a significant change in background or instrument

response occurs or when a new matrix is encountered.

9.2.3 Linear Dynamic Range (LDR) -- The LDR should

be determined by analyzing a minimum of five calibration used to establish an ongoing precision statement for the

standards ranging in concentration from 0.001 mg P/L to level of concentrations included in the LFB. These data

0.20, mg P/L across all sensitivity settings of the auto- must be kept on file and be available for review.

analyzer. Normalize responses by dividing the response

by the sensitivity setting multiplier. Perform the linear 9.4 Assessing Analyte Recovery - Laboratory

regression of normalized response vs. concentration and Fortified Sample Matrix

obtain the constants m and b, where m is the slope and b

is the y-intercept. Incrementally analyze standards of

higher concentration until the measured absorbance analyte to a minimum of 5% of the routine samples or one

response, R, of a standard no longer yields a calculated sample per sample set, whichever is greater. The analyte

concentration CC, that is ± 10% of the known concentra- concentration should be two to four times the ambient

tion, C, where CC = (R - b)/m. That concentration defines

the upper limit of the LDR for your instrument. Should

samples be encountered that have a concentration that

is $90% of the upper limit of the LDR, then these samples

must be diluted and reanalyzed.

9.3 Assessing Laboratory Performance 

(Mandatory)

9.3.1 Laboratory Reagent Blank (LRB) -- A laboratory

should analyze at least one LRB (Section 3.5) with each

set of samples. LRB data are used to assess contamination from the laboratory environment. Should an analyte

value in the LRB exceed the MDL, then laboratory or

reagent contamination should be suspected. When LRB

values constitute 10% or more of the analyte level determined for a sample, fresh samples or field duplicates of

the samples must be prepared and analyzed again after

the source of contamination has been corrected and

acceptable LRB values have been obtained.

9.3.2 Laboratory Fortified Blank (LFB) -- A laboratory

should analyze at least one LFB (Section 3.3) with each

batch of samples. Calculate accuracy as percent recovery. If the recovery of the analyte falls outside the required control limits of 90-110%, the analyte is judged out

laboratory performance against the required control limits

of 90-110% (Section 9.3.2). When sufficient internal performance data become available (usually a minimum of

20 to 30 analyses), optional control limits can be developed from the percent mean recovery (x) and the standard deviation (S) of the mean recovery. These data can

be used to establish the upper and lower control limits as

follows:

Upper Control Limit = x + 3S

Lower Control Limit = x - 3S

The optional control limits must be equal to or better than

the required control limits of 90-110%. After each 5 to 10

new recovery measurements, new control limits can be

calculated using only the most recent 20 to 30 data

points. Also, the standard deviation (S) data should be

9.4.1 A laboratory should add a known amount of

concentration and should be at least four times the MDL.

9.4.2 Calculate the percent recovery of the analyte,

corrected for background concentrations measured in the

unfortified sample, and compare these values with the

values obtained from the LFBs.

Percent recoveries may be calculated using the following

equation:

9.4.3 If the recovery of the analyte falls outside the

designated range of 90-110% recovery, but the laboratory

performance for that analyte is in control, the fortified
Revision 1.4 September 1997 365.5 - 6

Apparent orthophosphate

conc. due to refractive

Salinity (ppt) index (mg P/L)

1 0.0002

5 0.0006

10 0.0009

20 0.0017

 sample should be prepared again and analyzed. If the standards are entered as the independent variable and

result is the same after reanalysis, the recovery problem the corresponding peak height is the dependent variable.

encountered with the fortified sample is judged to be

matrix related, not system related. 12.2 Refractive Index Correction for Estuarine/

10.0 Calibration and Standardization

10.1 Calibration (Refer to Sections 11.5 and 12.0).

10.2 Standardization (Refer to Section 12.2).

11.0 Procedure

11.1 If samples are frozen, thaw the samples to room

temperature.

11.2 Set up manifold as shown in Figure 1. The tubing,

flow rates, sample:wash ratio, sample rate, etc., are

based on a Technicon AutoAnalyzer II system. Specifications for similar segmented flow analyzers vary, so

slight adjustments may be necessary.

11.3 Allow both colorimeter and recorder to warm up

for 30 min. Obtain a steady baseline with reagent water

pumping through the system, add reagents to the sample

stream and after the reagent water baseline has equilibrated, note that rise (reagent water baseline), and adjust

baseline.

For analysis of samples with a narrow salinity range, it is

advisable to use low nutrient seawater matched to

sample salinity as wash water in the sampler in place of

reagent water. For samples with a large salinity range, it

is suggested that reagent wash water and procedure

(Section 12.2) be employed.

11.4 A good sampling rate is approximately 40 samples/hr with a 9:1, sample:wash ratio.

11.5 Place standards (Section 7.2.5) in sampler in

order of decreasing concentration. Complete filling the

sampler tray with samples, LRBs, LFBs, and LFMs.

11.6 Commence analysis.

11.7 Obtain a second set of peak heights for all

samples and standards with Refractive Reagent A

(Section 7.2.3) being pumped through the system in place

of Reagent A (Section 7.2.1). This “apparent” concentration due to coloration of the water should be subtracted

from concentrations obtained with Reagent A pumping

through the system.

12.0 Data Analysis and Calculations

12.1 Concentrations of orthophosphate are calculated

from the linear regression obtained from the standard

curve in which the concentrations of the calibration 

Coastal Systems

12.2.1 Obtain a second set of peak heights for all

samples and standards with Refractive Reagent A (Section 7.2.3) being pumped through the system in place of

Reagent A (Section 7.2.1). Reagent B (Section 7.2.2)

remains the same and is also pumped through the

system. Peak heights for the refractive index correction

must be obtained at the same Standard Calibration

Setting and on the same colorimeter as the corresponding samples and standards.9

12.2.2 Subtract the refractive index peak heights from

the heights obtained for the orthophosphate determination. Calculate the regression equation using the corrected standard peak heights. Calculate the concentration of samples from the regression equation using the

corrected sample peak heights.

12.2.3 When a large data set has been amassed in

which each sample’s salinity is known, a regression for

the refractive index correction on a particular colorimeter

can be calculated. For each sample, the apparent orthophosphate concentration due to refractive index is

calculated from its peak height obtained with Refractive

Reagent A (Section 7.2.3) and Reagent B (Section 7.2.2)

and the regression of orthophosphate standards obtained

with orthophosphate Reagent A (Section 7.2.1) and

Reagent B (Section 7.2.2) for each sample. Its salinity is

entered as the independent variable and its apparent

orthophosphate concentration due to its refractive index

in that colorimeter is entered as the dependent variable.

The resulting regression equation allows the operator to

subtract an apparent orthophosphate concentration when

the salinity is known, as long as other matrix effects are

not present. Thus, the operator would not be required to

obtain the refractive index peak heights for all samples

after a large data set has been found to yield consistent

apparent orthophosphate concentrations due to salinity.

An example follows:

12.2.4 An example of a typical equation is:

mg P/L apparent PO4 = 0.000087 X Salinity

3-

(ppt) where, 0.000087 is the slope of the line.

where, 0.000087 is the slope of the line.
365.5 - 7 Revision 1.4 September 1997

Salinity Concentration Percent Relative

Sample (ppt) (mg P/L) Standard Deviation

1 36 0.0040 6.5

2 18 0.0024 10

3 3 0.0007 24

12.3 Results should be reported in mg PO - P/L or μg prevention as the management option of first choice. 4

3-

PO4- P/L. Whenever feasible, laboratory personnel should use

3-

mg PO - P/L = ppm (parts per million) generation. When wastes cannot be feasibly reduced at 4

3-

μg PO - P/L = ppb (parts per billion) 4

3-

13.0 Method Performance

13.1 Single Analyst Precision -- A single laboratory

analyzed three samples collected from Chesapeake Bay,

Maryland, and East Bay, Florida. Seven replicates of

each sample were processed and analyzed randomly

throughout a group of 75 samples with salinities ranging

from 3 to 36 ppt. The results were as follows:

13.2 Multilaboratory Testing

13.2.1 This method was tested by nine laboratories

using reagent water, high salinity seawater from the

Sargasso Sea (36 ppt) and three different salinity waters

from Chesapeake Bay, Maryland (8.3 ppt, 12.6 ppt, and

19.5 ppt). The reagent water and the Sargasso Seawater

were fortified at four Youden pair concentrations ranging

from 0.0012 mg P/L to 0.1000 mg P/L. The Chesa10 

peake Bay waters were fortified at three Youden pair

concentrations ranging from 0.0050 mg P/L to 0.0959 mg

P/L with the highest salinity waters containing the lowest

Youden pair and the lowest salinity waters containing the

highest Youden pair. Analysis of variance (ANOVA) at the

95% confidence level found no statistical differences

between water types indicating that the refractive index

correction for different salinity waters is an acceptable

procedure. Table 1 contains the linear equations that

describe the single-analyst standard deviation, overall

standard deviation, and mean recovery of orthophosphate

from each water type.

13.2.2 Pooled Method Detection Limit (p-MDL) -- The pMDL is derived from the pooled precision obtained by

single laboratories for the lowest analyte concentration

level used in the multilaboratory study. The p-MDLs using

reagent water and Sargasso Sea water were 0.00128 and

0.00093 mg P/L, respectively.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

EPA has established a preferred hierarchy of environmental management techniques that places pollution

pollution prevention techniques to address their waste

the source, the Agency recommends recycling as the next

best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research institutions, consult Less is Better. Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society’s Department of Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036, (202)872-4477.

15.0 Waste Management

15.1 The Environmental Protection Agency requires

that laboratory waste management practices be conducted consistent with all applicable rules and regulations. The Agency urges laboratories to protect the air,

water, and land by minimizing and controlling all releases

from hoods and bench operations, complying with the

letter and spirit of any sewer discharge permits and

regulations, and by complying with all solid and hazardous waste regulations, particularly the hazardous waste

identification rules and land disposal restrictions. For

further information on waste management, consult The

Waste Management Manual for Laboratory Personnel,

available from the American Chemical Society at the

address listed in Section 14.2.

16.0 References

1. Murphy, J. and J.P. Riley. 1962. A Modified Single

Solution Method for the Determination of Phosphate in Natural Waters. Analytica Chim. Acta 27,

31-36.0.

2. Technicon Industrial Systems. 1973. Orthophosphate in Water and Seawater. Industrial Method

155-71 W. Technicon Industrial Systems, Tarrytown, NY 10591.

3. 40 CFR, 136 Appendix B. Definition and Procedure

for the Determination of the Method Detection

Limit. Revision 1.11.

4. Grasshoff, K., M. Ehrhardt, and K. Kremling. 1983.

Methods of Seawater Analysis. Verlag Chemie,

Federal Republic of Germany, 419 pages.

5. Klingamann, E.D. and D.W. Nelson. 1976. Evaluation of Methods for Preserving the Levels of Soluble Inorganic Phosphorus and Nitrogen in Unfiltered Water Samples. J. Environ. Qual., 5:1, 42-

46.
Revision 1.4 September 1997 365.5 - 8

6. MacDonald, R.W. and F.A. McLaughlin. 1982. The

Effect of Storage by Freezing on Dissolved Inorganic

Phosphate, Nitrate, and Reactive Silicate for Samples

from Coastal and Estuarine Waters. Water Research, 16:95-104.

7. Thayer, G.W. 1979. Comparison of Two Storage

Methods for the Analysis of Nitrogen and Phosphorus

Fractions in Estuarine Water. Ches. Sci., 11:3, 155-

158.

8. Salley, B.A., J.G. Bradshaw, and B.J. Neilson. 1986.

Results of Comparative Studies of Preservation

Techniques for Nutrient Analysis on Water Samples.

VIMS, Gloucester Point, VA 23062. 32 pp.

9. Froelich, P.N. and M.E.Q. Pilson. 1978. Systematic

Absorbance Errors with Technicon AutoAnalyzer II

Colorimeters. Water Research 12: 599-603.

10. Edgell, K.W., E.J. Erb, and J.E. Longbottom, “Determination of Orthophosphate in Estuarine and

Coastal Waters by Automated Colorimetric Analysis:

Collaborative Study,” submitted in November 1992

for publication in Marine Chemistry.
365.5 - 9 Revision 1.4 September 1997

Table 1. Single-Analyst Precision, Overall Precision and Recovery from Multilaboratory Study

Reagent Water

(0.0012 - 0.100 mg P/L)

Mean Recovery X = 0.972C - 0.000018

Overall Standard Deviation S = 0.033X + 0.000505 R

Single-Analyst Standard Deviation S = 0.002X + 0.000448 r

Sargasso Sea Water

(0.0012 - 0.100 mg P/L)

Mean Recovery X = 0.971C - 0.000002

Overall Standard Deviation S = 0.021X + 0.000550 R

Single-Analyst Standard Deviation S = 0.010X + 0.000249 r

Chesapeake Bay Water

( 0.005 - 0.100 mg P/L)

Mean Recovery

X = 1.019C - 0.000871

Overall Standard Deviation S = 0.066X + 0.000068 R

Single-Analyst Standard Deviation S = 0.030X + 0.000165 r

C True value of spike concentration, mg P/L

X Mean concentration found, mg P/L, exclusive of outliers.

S Overall standard deviation, mg P/L, exclusive of outliers. R

S Single-analyst standard deviation, mg P/L, exclusive of outliers. r

17.0 Tables, Diagrams, Flowcharts, and Validation Data
366.0-1

Method 366.0

 Determination of Dissolved Silicate in Estuarine and Coastal Waters

 by Gas Segmented Continuous Flow Colorimetric Analysis

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

Rosenstiel School of Marine and Atmospheric Science, Atlantic Oceanographic and

Meteorological Laboratory, National Oceanic and Atmospheric Administration,

University of Miami, Miami, FL 33149

George A. Berberian, National Oceanic and Atmospheric Administration, Atlantic

Oceanographic and Meteorological Laboratory, Ocean Chemistry Division, Miami, FL

33149

Project Officer

Elizabeth J. Arar

Version 1.0

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Version 1.0 September 1997 366.0-2

Method 366.0

 Determination of Dissolved Silicate in Estuarine and Coastal Waters

 by Gas Segmented Continuous Flow Colorimetric Analysis

1.0 Scope and Application

1.1 This method provides a procedure for the

determination of dissolved silicate concentration in

estuarine and coastal waters. The dissolved silicate is

mainly in the form of silicic acid, H4SiO4, in estuarine and

coastal waters. All soluble silicate, including colloidal

silicic acid, can be determined by this method. Long chain

polymers containing three or more silicic acid units do not

react at any appreciable rate , but no significant amount 1

of these large polymers exists in estuarine and coastal

waters. This method is based upon the method of

2,3

Koroleff, adapted to automated gas segmented

4

continuous flow analysis.5-7

Chemical Abstracts Service

Analyte Registry Numbers (CASRN)

_______________________________________

Silicate 12627-13-3

_______________________________________

1.2 A statistically determined method detection limit

(MDL) of 0.0012 mg Si/L has been determined by one

laboratory in seawaters of three different salinities. The 8

method is linear to 6.0 mg Si/L using a Flow Solution

System (Perstorp Analytical Inc., Silver Spring, MD).

1.3 Approximately 60 samples per hour can be

analyzed.

1.4 This method should be used by analysts

experienced in the use of automated gas segmented

continuous flow colorimetric analyses, and familiar with

matrix interferences and procedures for their correction.

A minimum of 6-months experience under supervision is

recommended.

2.0 Summary of Method 

2.1 An automated gas segmented continuous flow

colorimetric method for the analysis of dissolved silicate

concentration is described. In the method, $-

molybdosilicic acid is formed by reaction of the silicate

contained in the sample with molybdate in acidic solution.

The $-molybdosilicic acid is then reduced by ascorbic

acid to form molybdenum blue. The absorbance of the

molybdenum blue, measured at 660 nm, is linearly

proportional to the concentration of silicate in the sample.

A small positive error caused by differences in the

refractive index of seawater and reagent water, and

negative error caused by the effect of salt on the color

formation, are corrected prior to data reporting.

3.0 Definitions

3.1 Calibration Standard (CAL) -- A solution

prepared from the primary dilution standard solution or

stock standard solution containing analytes. The CAL

solutions are used to calibrate the instrument response

with respect to analyte concentration.

3.2 Dissolved Analyte (DA) -- The concentration of

analyte in an aqueous sample that will pass through a

0.45 Fm membrane filter assembly prior to sample

acidification or other processing.

3.3 Laboratory Fortified Blank (LFB) -- An aliquot

of reagent water to which known quantities of the method

analytes are added in the laboratory. The LFB is analyzed

exactly like a sample, and its purpose is to determine

whether method performance is within acceptable control

limits, and whether the laboratory is capable of making

accurate and precise measurements. 

This is basically a standard prepared in reagent water that

is analyzed as a sample.

3.4 Laboratory Fortified Sample Matrix (LFM) -- An

aliquot of an environmental sample to which known

quantities of the method analytes are added in the

laboratory. The LFM is analyzed exactly like a sample,

and its purpose is to determine whether the sample matrix

contributes bias to the analytical results. The background

concentrations of the analytes in the sample matrix must

be determined in a separate aliquot and the measured

values in the LFM corrected for background

concentrations. 
 366.0-3 Version 1.0 September 1997

3.5 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water that is treated exactly as a sample

including exposure to all labware, equipment, and

reagents that are used with other samples. The LRB is

used to determine if method analytes or other

interferences are present in the laboratory environment,

the reagents, or apparatus.

3.6 Linear Dynamic Range (LDR) -- The absolute

quantity or concentration range over which the instrument

response to an analyte is linear.

3.7 Method Detection Limit (MDL) -- The minimum

concentration of an analyte that can be identified,

measured and reported with 99% confidence that the

analyte concentration is greater than zero.8

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

water equal to or exceeding standards established by

American Society for Testing and Materials (ASTM).

Reverse osmosis systems or distilling units followed by

Super-Q Plus Water System that produce water with 18

megohm resistance are examples of acceptable water

sources.

3.9 Refractive Index (RI) -- The ratio of velocity of

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

refractive index is the ratio of the velocity of light in two

different media, such as estuarine or sea water versus

reagent water. The correction for this difference is

referred to as refractive index correction in this method. 

3.10 Stock Standard Solution (SSS) -- A

concentrated solution of method analyte prepared in the

laboratory using assayed reference compounds or

purchased from a reputable commercial source. 

3.11 Quality Control Sample (QCS) - A solution of

method analyte of known concentrations which is used to

fortify an aliquot of LRB or sample matrix. The QCS is

obtained from a source external to the laboratory and

different from the source of calibration standards. It is

used to check laboratory performance with externally

prepared test materials.

3.12 SYNC Peak Solution - A colored solution used

to produce a synchronization peak in the refractive index

measurement. A synchronization peak is required by

most data acquisition programs to initialize the peak

finding parameters. The first cup in every run must always

be identified as a SYNC sample. The SYNC sample is

usually a high standard, but can be any sample that

generates a peak at least 25% of full scale. 

4.0 Interferences

4.1 Interferences caused by hydrogen sulfide, such

as occur in samples taken from deep anoxic basins can

be eliminated by oxidation with bromine or stripping with

nitrogen gas after acidification. Interferences of

phosphate at concentrations larger than 0.15 mg P/L is

eliminated by the use of oxalic acid in the color

development step of this method. Interferences of fluoride

at concentrations greater than 50 mg F/L can be reduced

by complexing the fluoride with boric acid.4

4.2 Glassware made of borosilicate glass should be

avoided for use in silicate analysis. Plastic labware such

as polyethylene volumetric flasks and plastic sample

vials, should be used. 

4.3 Sample turbidity and particles are removed by

filtration through a 0.45 μm non-glass membrane filters

after sample collection. 

4.4 This method corrects for refractive index and salt

error interferences which occur if sampler wash solution

and calibration standards are not matched with samples

in salinity.

4.5 Frozen samples should be filled about 3/4 full in

the sample bottles. The expansion of water on freezing

will squeeze some of the brine out of the bottle if the

bottle was overfilled. The overfill of the sample bottle

during freezing will drastically alter the nutrient

concentrations in the sample that remains.

5.0 Safety

5.1 Water samples collected from the estuarine and

coastal environment are generally not hazardous.

However, the individual who collects samples should use

proper technique.

5.2 Good laboratory technique should be used when

preparing reagents. A lab coat, safety goggles, and

gloves should be worn when preparing the sulfuric acid

reagent.

6.0 Equipment and Supplies

6.1 Gas Segmented Continuous Flow

Autoanalyzer Consisting of:

6.1.1 Autosampler.
Version 1.0 September 1997 366.0-4

6.1.2 Analytical cartridge with reaction coils for silicate plate using a Teflon-coated stirring bar. Complete

analysis.

6.1.3 Proportioning pump.

6.1.4 Monochromator or spectrophotometer equipped

with a tungsten lamp ( 380-800 nm) and a low refractive

index flowcell.

6.1.5 Strip chart recorder or computer based data

acquisition system.

6.2 Glassware and Supplies

6.2.1 All labware used in the analysis must be low in

residual silicate to avoid sample or reagent

contamination. Soaking with lab grade detergent, rinsing

with tap water, followed by rinsing with 10% HCl (v/v) and

thoroughly rinsing with reagent water was found to be

effective.

6.2.2 Glassware made of borosilicate glass should be

avoided for storage of solutions for silicate analysis.

Plastic containers are preferable for silicate analysis.

6.2.3 Non-glass membrane filters with 0.45 μm

nominal pore size. Plastic syringes with syringe filters,

pipets, 60 mL polyethylene bottles, and polyethylene

volumetric flasks, plastic sample vials.

6.2.4 Drying oven, desiccator and analytical balance.

7.0 Reagents and Standards

7.1 Stock Reagent Solutions

7.1.1 Sulfuric Acid Solution (0.05 M) - Cautiously add

2.8 mL of concentrated Analytical Reagent Grade sulfuric

acid (H SO ) to approximately 800 mL of reagent water, 2 4

mix then bring up to 1 L with reagent water.

7.1.2 Ammonium Molybdate Solution (10 g/L) -

Dissolve 10 g of ammonium molybdate (VI) tetrahydrate

((NH4)6Mo7O24.4H2O) in approximately 800 mL of 0.05 M

sulfuric acid solution and dilute to 1000 mL with 0.05 M

sulfuric acid solution. Store in an amber plastic bottle.

This solution is stable for one month. Inspect the solution

before use. If a white precipitation forms on the wall of

container, discard the solution and make a fresh one. 

7.1.3 Stock Silicate Solution (100 mg Si/L) -

Quantitatively transfer 0.6696 g of pre-dried (105 C for 2 o

hours) sodium hexafluorosilicate (Na2SiF6) to a 1000 mL

polypropylene flask containing approximate 800 mL of

reagent water, cover with plastic film and dissolve on a stir

dissolution usually takes 2-24 hours. Dilute the solution to

1000 mL in polyethylene volumetric flask with reagent

water. Store the stock solution in a plastic bottle. This

solution is stable for one year if care is taken to prevent

contamination and evaporation.

7.1.4 Low Nutrient Sea Water (LNSW) - Obtain natural

low nutrient seawater from surface seawater in the Gulf

Stream or Sargasso Sea (salinity 36 %o, < 0.03 mg Si/L)

and filter through 0.45 μm pore size non-glass membrane

filters. In addition, commercially available low nutrient sea

water ( < 0.03 mg Si/L) with salinity of 35 %o (Ocean

Scientific International, Wormley, U.K. ) can be used. 

7.2 Working Reagents

7.2.1 Dowfax Start-up Solution - Add 2 mL of Dowfax

2A1 surfactant (Dow Chemical Company) to 1000 mL

reagent water and mix gently.

Note: Dowfax 2A1 contains (w/w) 47% benzene, 1,1-

oxybis, tetrapropylene derivatives, sulfonate, sodium salt,

1% sodium sulfate, 3% sodium chloride and 49% water.

7.2.2 Working Molybdate Reagent - Add 0.5 mL

Dowfax 2A1 to 250 mL of ammonium molybdate solution,

mix gently. Prepare this solution daily. This volume of

solution is sufficient for an 8-hour run.

7.2.3 Ascorbic Acid Solution - Dissolve 4.4 g of

ascorbic acid (C6H8O6) in 200 mL of reagent water and

12.5 mL of acetone(C H O), dilute to 250 mL with reagent 3 6

water. Store in a plastic container. This solution is stable

for one week if stored at 4 C. Discard the solution if it

o

turns brown.

7.2.4 Oxalic Acid Solution - Dissolve 50 g of oxalic acid

(C2H2O4) in approximately 800 mL of reagent water and

dilute to 1000 mL with reagent water. Store in a plastic

container. This solution is stable for approximately 3-

months.

7.2.5 Refractive Index Matrix Solution - Add 0.5 mL

Dowfax 2A1 to 250 mL of 0.05 M sulfuric acid solution

and mix gently.

7.2.6 Colored SYNC Peak Solution - Add 50 μL of blue

food coloring solution to 1000 mL reagent water and mix

thoroughly. The solution should give a peak of between

25 to 100 percent full scale, otherwise the volume of food

coloring added needs to be adjusted.

7.2.7 Calibration Standards - Prepare a series of

calibration standards (CAL) by diluting suitable volumes

of Stock Silicate Solution (Section 7.1.3) to 100 mL with
 366.0-5 Version 1.0 September 1997

reagent water or low nutrient seawater. Prepare these intervals to a hydro wire. These bottles are sent through

standards daily. The concentration range of calibration the water column open and are closed either

standards should bracket the expected concentrations of electronically or via a mechanical messenger when the

samples and not exceed two orders of magnitude. At bottles have reached the desired depth.

least five calibration standards with equal increments in

concentration should be used to construct the calibration 8.1.2 In a submersible pump system, a weighted hose

curve.

When working with samples of a narrow range of

salinities (± 2 %o), it is recommended that the CAL

solutions be prepared in Low Nutrient Seawater (Section

7.1.4) diluted to the salinity of samples, and the Sampler

Wash Solution also be Low Nutrient Seawater (Section

7.1.4) diluted to that salinity. If this procedure is

performed, it is not necessary to perform the salt error

and refractive index corrections outlined in Sections 12.2

and 12.3. 

When analyzing samples of varying salinities, it is

recommended that the calibration standard solutions and

sampler wash solution be prepared in reagent water and

corrections for salt error and refractive index be made to

the sample concentrations (Section 12.2 and 12.3).

7.2.8 Saline Silicate Standards - If CAL solutions will

not be prepared to match sample salinity, then saline

silicate standards must be prepared in a series of

salinities in order to quantify the salt error, the change in

the colorimetric response of silicate due to the change in

the ionic strength of the solution. The following dilutions

prepared in 100 mL volumetric flasks, diluted to volume

with reagent water, are suggested.

Salinity Volume of Volume(mL) Conc.

(%o) LNSW(mL) Si stock std mg Si/L

_______________________________________

0 0 1.5 1.5

9 25 1.5 1.5

18 50 1.5 1.5

27 75 1.5 1.5

35 98 1.5 1.5

_______________________________________

8.0 Sample Collection, Preservation 

and Storage

8.1 Sample Collection - Samples collected for

nutrient analyses from estuarine and coastal waters are

normally collected using one of two methods: hydrocast

or submersible pump systems.

8.1.1 A hydrocast uses a series of sampling bottles

(Niskin, Go-Flo or equivalent) that are attached at fixed

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

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

sample processing.

8.1.3 For collecting surface samples, an acid - cleaned

plastic bucket or a large plastic bottle can be used as

convenient samplers. Wash the sampler three times with

sample water before collecting samples.

8.1.4 Samples must be filtered through a 0.45 μm nonglass membrane filters as soon as possible after

collection.

8.1.5 60-mL high density polyethylene bottles are used

for sample storage. Sample bottles should be rinsed 3

times with about 20 mL of sample, shaking with the cap

in place after each rinse. Pour the rinse water into the cap

to dissolve and rinse away salt crusts trapped in the

threads of the cap. Finally, fill the sample bottle about 3/4

full, and screw the cap on firmly. The expansion of water

on freezing will squeeze some of the brine out of the

bottle if the bottle was overfilled.

8.2 Sample Preservation - After collection and

filtration, samples should be analyzed as soon as

possible. If samples will be analyzed within 24 hours then

keep refrigerated in tightly sealed, high density

polyethylene bottles in the dark at 4 C until analysis can o

be performed.

8.3 Sample Storage - If samples are to be frozen

for long-term storage ensure that none of the sample

bottles are filled more than 3/4 full and the cap is firmly

screwed on. Place the bottles upright on a rack and store

in the freezer (-20 C). o

Before analysis, frozen samples must be taken out of the

freezer and allowed to thaw in a refrigerator at 4 C in the o

dark. Thawing times depend upon sample salinities. It

was found that the frozen low salinity estuarine water took

4 days to thaw. After completely thawing, take samples

out of the refrigerator and mix thoroughly. Keep samples

in the dark at room temperature overnight before

analysis.

Effects of thawing conditions on the recoveries of frozen

samples are more pronounced in low salinity estuarine 
Version 1.0 September 1997 366.0-6

waters than high salinity coastal waters as shown in each set of samples as a continuing check on

following results: performance.

_______________________________________

Day Recovery (%) Remark 9.2 Initial Demonstration of Performance

S=35.85 S=18.07 S=2.86 (Mandatory)

_______________________________________

0 100.00 100.00 100.00 9.2.1 The Initial demonstration of performance is used

7 102.44 102.65 89.37 a

14 98.59 101.06 86.49 a

21 99.51 99.30 83.49 a

27 98.86 a

98.86 91.43 b

35 98.70 b

98.66 92.98 b

42 100.87 b

49 102.44 79.12 c

103.92 79.10 d

99.92 89.68 e

56 103.47 c

104.12 d

99.35 e

84 100.80 91.71 f

99.90 93.81 g

91 100.65 f

100.22 g

______________________________________

S = Salinity

a, overnight thawing at room temperature

b, 20 hours thawing at room temperature

c, 24 hours thawing at room temperature

d, 8 hours thawing at room temperature then

 heating at 80 C for 16 hours o

e, 24 hours thawing at room temperature in the dark

f, 4 days thawing at room temperature in the dark

g, 4 days thawing at 4 C in a refrigerator in the dark o

To ensure a slow process of depolymerization of

polysilicate to occur, thawing the frozen samples in the

dark at 4 C for 4 days is critical condition for obtaining

o

high recoveries of silicate in frozen samples. A maximum

holding time for frozen estuarine and coastal waters is

two months.9-11

9.0 Quality Control

9.1 Each laboratory using this method is required to

implement a formal quality control(QC) program. The

minimum requirements of this program consists of an

initial demonstration of performance, continued analysis

of Laboratory Reagent Blanks (LRB), laboratory

duplicates and Laboratory Fortified Blanks (LFB) with

to characterize instrument performance by determining

the MDL and LDR and laboratory performance by

analyzing quality control samples prior to analysis of

samples using this method.

9.2.2 Method Detection Limits (MDLs) should be

established using a low level seawater sample containing,

or fortified at, approximately 5 times the estimated

detection limit. To determine MDL values, analyze at least

seven replicate aliquots of water which have been

processed through the entire analytical method. Perform

all calculations defined in the method and report

concentration in appropriate units. Calculate the MDL as

follows:

MDL = (t)(S)

where, S = the standard deviation of the 

replicate analyses

 t = Student's t value for n-1 degrees of 

freedom at the 99% confidence 

limit; t = 3.143 for six degrees of 

 freedom. 

MDLs should be determined every 6-months or whenever

a significant change in background or instrument

response occurs or a new matrix is encountered.

 

9.2.3 The LDR should be determined by analyzing a

minimum of eight calibration standards ranging from 0.03

to 5.00 mg Si/L across all sensitivity settings (Absorbance

Units Full Scale) of the detector. Standards and sampler

wash solutions should be prepared in low nutrient

seawater with salinities similar to that of samples,

therefore a correction factor for salt error, or refractive

index, will not be necessary. Normalize responses by

multiplying the response by the Absorbance Units Full

Scale output range setting. Perform the linear regression

of normalized response vs. concentration and obtain the

constants m and b, where m is the slope and b is the yintercept. Incrementally analyze standards of higher

concentration until the measured absorbance response,

R, of a standard no longer yields a calculated

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

concentration, C, where C = (R-b)/m. That concentration c 
 366.0-7 Version 1.0 September 1997

defines the upper limit of the LDR for the instrument. 9.4 Assessing Analyte Recovery -

Should samples be encountered that have a Laboratory Fortified Sample Matrix 

concentration that is 90% of the upper limit of LDR, then (LFM)

these samples must be diluted and reanalyzed. 

9.3 Assessing Laboratory Performance

(Mandatory)

9.3.1 Laboratory Reagent Blank (LRB) - A laboratory

should analyze at least one LRB with each set of greater than the MDL. 

samples. LRB data are used to assess contamination

from the laboratory environment. Should an analyte value 9.4.2 Calculate percent recovery of analyte, corrected

in the LRB exceed the MDL, then laboratory or reagent

contamination should be suspected. When the LRB value

constitutes 10% or more of the analyte concentration

determined for a sample, duplicates of the sample must

be prepared and analyzed again after the source of

contamination has been corrected and acceptable LRB

values have been obtained.

9.3.2 Laboratory Fortified Blank (LFB) - A laboratory

should analyze at least one LFB with each set of S

samples. The LFB must be at a concentration that is

within the daily calibration range. The LFB data are used where, R = percent recovery

to calculate accuracy as percent recovery. If the recovery

of the analyte falls outside the required control limits of C = measured fortified sample

90 -110%, the source of the problem should be identified concentration (background + 

and resolved before continuing the analyses. addition in mg Si/L)

9.3.3 The laboratory must use LFB analyses data to C = sample background concentration

assess laboratory performance against the required

control limits of 90 -110%. When sufficient internal

performance data become available (usually a minimum

of 20 to 30 analyses), optional control limits can be

developed from the percent mean recovery (x) and

standard deviation (S) of the mean recovery. These data

can be used to establish the upper and lower control required control limits of 90-110%, but the laboratory

limits as follows: performance for that analyte is within the control limits,

Upper Control Limit = x + 3S analyzed. If the result is the same after reanalysis, the

Lower Control Limit = x - 3S recovery problem encountered with the fortified sample is

The optional control limits must be equal to or better than be flagged.

the required control limits of 90-110%. After each 5 to 10

new recovery measurements, new control limits can be 10.0 Calibration and Standardization

calculated using only the most recent 20 to 30 data

points. Also the standard deviation (S) data should be 10.1 At least five calibration standards should be

used to establish an ongoing precision statement for the prepared daily for system calibration. 

level of concentrations included in the LFB. These data

must be kept on file and be available for review. 10.2 A calibration curve should be constructed for

9.4.1 A laboratory should add a known amount of

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

samples or one sample per sample set, whichever is

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

ambient concentration and should be at least four times

for background concentration measured in a separate

unfortified sample. These values should be compared

with the values obtained from the LFBs. Percent

recoveries may be calculated using the following

equation:

(C - C) s

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

s 

 (mg Si/L)

S = concentration in mg Si/L added to the

 environmental sample.

9.4.3 If the recovery of the analyte falls outside the

the fortified sample should be prepared again and

judged to be matrix related and the sample data should

each run by analyzing a set of calibration standard

solutions. A run should contain no more than 60 samples.
Version 1.0 September 1997 366.0-8

It is suggested that a large set of samples be analyzed in

several sets with individual calibration curves. 

10.3 Place the calibration standards before samples

for each run. All the calibration solutions should be

analyzed in duplicate.

10.4 The calibration curve containing five data points

or more should have a correlation coefficient 0.995. 

10.5 Place a high standard solution cup and follow by

two blank cups to quantify the carry-over of the system.

The difference in peak heights between two blank cups is

due to the carry over from the high standard cup. The

carry-over coefficient, k, is calculated as follows:

Pb1 - Pb2

k = -----------------X 100

 Phigh 

where, P = the peak height of the high high

silicate standard

P = the peak height of the first b1 

blank sample

P = the peak height of the second b2 

blank sample.

The carry over coefficient, k, for a system should be

measured in seven replicates in order to obtain a

statistically significant number. The k should be

remeasured when a change in the plumbing of the

manifold or replacement of pump tube occur.

The carry over correction (CO) on a given peak, i, is

proportional to the peak height of the preceding sample,

P . i-1

CO = kxPi-1

To correct a given peak height reading, Pi, one subtracts

the carry over correction.12,13

Pi,c = Pi - CO

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

carry over should be applied to all the peak heights

throughout a run. The carry over should be less than 2%.

10.6 Place a high standard solution at the end of a run

to check sensitivity drift. The sensitivity drift should be ±

5% during the run.

11.0 Procedure

11.1 If samples are frozen, thaw the sample at 4 C in o

the dark as outlined in Section 8.3. Mix samples

thoroughly prior to analyses.

11.2 Turn on the continuous flow analyzer and PC

components and warm up at least 30 minutes.

11.3 Set up the cartridge and pump tubes as shown in

Figure 1. 

Note: Fluctuation of ambient temperature can cause

erratic results due to the effect of temperature on kinetics

of color development. The laboratory temperature should

be maintained as close to a constant temperature as

possible. The cartridge should be away from the direct

path of air flow from a heater or air conditioner. In cases

such as on a ship where the fluctuation of temperature

can be extreme, the temperature effect can be minimized

by increasing the length of mixing coil 1 (Figure 1) to bring

the formation of silicomolybdic acid reaction to

completion.

11.4 Set the wavelength at 660 nm on the

spectrometer/monochrometer.

Note: The absorption spectra of silicomolybdeum blue

complex has two maxima at 820 nm and 660 nm with 820

nm higher than 660 nm. This method measures

absorbance at 660 nm because the detector works in the

range of 380 to 800 nm. The sensitivity of the method is

satisfactory at 660 nm. The sensitivity, however, can be

improved by using 820 nm if this wavelength is available

on the detector.

11.5 On the monochromator, set the Absorbance Unit

Full Scale at an appropriate setting according to the

highest concentration of silicate in the samples. The

highest setting used in this method was 0.2 for 6 mg Si/L.

11.6 Prepare all reagents and standards.

11.7 Begin pumping the Dowfax start-up solution

(Section 7.2.1) through the system and obtain a steady

baseline. Place the reagents on-line. The reagent

baseline will be higher than the start-up solution baseline.

After the reagent baseline has stabilized, reset the

baseline.

NOTE: To minimize the noise in the reagent baseline,

clean the flow system by sequentially pumping the
 366.0-9 Version 1.0 September 1997

sample line with reagent water, 1 N HCl solution, reagent standards and samples. The peak heights obtained from

water, 1N NaOH solution for a few minutes each at the these measurements must be subtracted from the peak

end of the daily analysis. Make sure to rinse the system heights of samples analyzed with color developing

well with reagent water after pumping NaOH solution to reagent pumping through the system. If a low refractive

prevent precipitation of Mg(OH)2 when seawater is index flowcell is used, the correction for refractive index is

introduced into the system. Keep the reagents and negligible. This procedure is optional.

samples free of particulate. Filter the reagents and

samples if necessary.

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

seconds to increase back pressure. If absorbance drops

down rapidly when back pressure increases, this indicates

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

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

in the flowcell can often be eliminated by simply attaching

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

pumping the syringe pistion. Alternatively, flushing the

flowcell with alcohol was found to be effective in removing

air bubbles from the flowcell.

For analysis of samples with a narrow range of salinities

(± 2 %o), it is recommended that the wash water in the

sampler be prepared in Low Nutrient Seawater diluted to

the salinity of samples in place of reagent water. For

samples with varying salinities, it is suggested that reagent

waters and procedures in Sections 12.2 and 12.3 be

employed.

11.8 A good sampling rate is approximately 60

samples per hour with 40 seconds of sample time and 20

seconds of wash time.

11.9 Use 10% HCl followed by reagent water to rinse

sample cups. Place CAL solutions and saline standards

(optional) in sampler. Complete filling the sampler tray

with samples, laboratory reagent blanks, laboratory

fortified blanks, laboratory fortified sample matrices, and

QC samples. Place a blank every ten samples and

between samples of high and low concentrations. 

11.10 Commence analysis.

11.11 If the reagent water is used as wash solution

instead of Low Nutrient Seawater and an operator wants

to quantify the refractive index correction due to the

difference in salinities between sample and wash solution,

the following procedures are used. Replace ammonium

molybdate solution (Section 7.1.2) with refractive index

matrix solution (Section 7.2.5). All other reagents remain

the same. Replace the synchronization cup with the

colored SYNC peak solution (Section 7.2.6). Commence

analysis and obtain a second set of peak heights for all

12.0 Data Analysis and Calculations

12.1 Concentrations of silicate are calculated from the

linear regression, obtained from the standard curve in

which the concentrations of the calibration standards are

entered as the independent variable, and their

corresponding peak heights are the dependent variable.

12.2 Refractive Index Correction for Estuarine and

Coastal Samples (optional)

12.2.1 Obtain a second set of peak heights for all

standards and samples with refractive index matrix

solution being pumped through the system in place of

color reagent (ammonium molybdate solution). All other

reagents remain the same. The peak heights for the

refractive index correction must be obtained at the same

Absorbance Unit Full Scale range setting and on the

same monochromator as the corresponding samples and

standards.

12.2.2 Subtract the refractive index peak heights from

the peak heights obtained from the silicate determination.

12.2.3 An alternative approach is to measure the

relationship between the sample salinity and refractive

index on a particular detector.

First analyze a set of silicate standards in reagent water

with color reagent and obtain a linear regression from the

standard curve. 

Prepare a set of different salinity samples with LNSW.

Analyze these samples with refractive index matrix

solution being pumped through the system in place of

color reagent (ammonium molybdate solution). All other

reagents remain the same. The peak heights for the

refractive index correction must be obtained at the same

Absorbance Unit Full Scale setting and on the same

monochromator as the corresponding standards.

For each sample, the apparent silicate concentration due

to refractive index is then calculated from its peak height

obtained with refractive index reagent and the regression

of silicate standards obtained with color reagent pumping
Version 1.0 September 1997 366.0-10

through the system. Salinity is entered as the independent _______________________________________

variable and the apparent silicate concentration due to Salinty Peak height of Uncorrected Si 

refractive index in that detector is entered as the (% ) 1.50 mg Si/L conc.calculated 

dependent variable. The resulting regression allows the from standards

operator to calculate apparent silicate concentration due in reagent water

to refractive index when the salinity is known. Thus, the _______________________________________

operator would not be required to obtain refractive index 0 2503 1.50

peak heights for all samples. 9 2376 1.32

12.2.4 Refractive index correction can be minimized by 27 2250 1.25

using a low refractive index flowcell. An example of typical

results using a low refractive index flowcell follows:

______________________________________

Salinity Apparent silicate conc. due 

(% ) to refractive index (mg Si/L) salt error is: o

_______________________________________ 

4.5 0.0003 Uncorrected mg Si/L

9.0 0.0005 Corrected mg Si/L = ------------------------------------

18.0 0.0016 1 - 0.02186x/S

27.0 0.0027

36.0 0.0042 where S is salinity.

_______________________________________

12.2.5 An example of a typical equation is:

Apparent silicate (mg Si/L) = 0.00001953*S1.5

where S is sample salinity. The form of fitted equation

might vary as the design of flowcell, so the operators are

advised to obtain the appropriate equation which has the

best fit of their own data with the least fitting coefficients.

12.3 Correction for Salt Error in Estuarine and

Coastal Samples 

12.3.1 When calculating concentrations of samples of

varying salinities from standards and wash solution

prepared in reagent water, it is usual to first correct for

refractive index errors, then correct for the change in color

development due to the differences in ionic strength

between samples and standards (salt error). The

refractive index correction is negligible, so is optional, but

correction for salt error is necessary. 

12.3.2 Plot the salinity of the saline standards (Section

7.2.8) as the independent variable, and the apparent

concentration of silicate (mg Si/L) from the peak height

(corrected for refractive index) calculated from the

regression of standards in reagent water, as the

dependent variable for all 1.50 mg Si/L standards. The

resulting regression equation allows the operator to

correct the concentrations of samples of known salinity

for the color suppression due to salinity effect, e.g., salt

error. An example of typical results follows:

o

18 2282 1.27

36 2202 1.23

______________________________________

12.3.3 An example of a typical equation to correct for

12.3.4 Results of sample analyses should be reported

in mg Si/L or in μg Si/L.

mg Si/L = ppm (parts per million)

μg Si/L = ppb (part per billion)

13.0 Method Performance

13.1 Single Laboratory Validation

13.1.1 Method Detection Limit - A method detection limit

(MDL) of 0.0012 mg Si/L has been determined by one

laboratory in seawaters of three different salinities.

_____________________________________

 Salinity SD Recovery MDL

(% ) (μg/L) (%) (μg/L) o

_____________________________________

36 0.3924 105 1.233

36 0.4980 107 1.565

27 0.2649 104 0.832

27 0.3362 104 1.056

27 0.4671 100 1.468

18 0.3441 101 1.081

18 0.2809 105 0.883

18 0.2432 104 0.764

3 0.3441 101 1.081

3 0.2331 102 0.733

3 0.1963 98 0.617

3 0.2809 99 0.883

_____________________________________

13.1.2 Single Analyst Precision - A single laboratory

analyzed three samples collected from the Miami River

and Biscayne Bay areas of Florida. Seven replicates of
 366.0-11 Version 1.0 September 1997

each sample were processed and analyzed with salinities choice. Whenever feasible, laboratory personnel should

ranging from 2.86 to 35.85. The results were as follows: use pollution prevention techniques to address their waste

______________________________________ generation. When wastes cannot be feasibly reduced at

the source, the Agency recommends recycling as the next

Sample Salinity Concentration RSD best option.

 (% ) (mg Si/L) (%) o

______________________________________ 14.2 For information about pollution prevention that

1 35.85 0.097 1.2 institutions, consult Less is Better: Laboratory Chemical

2 18.07 1.725 1.4

3 2.86 3.322 0.9

______________________________________

13.1.3 Laboratory Fortified Sample Matrix - Laboratory

fortified sample matrixes were processed in three different

salinities ranging from 2.86 to 35.85 and ambient

concentrations from 0.095 to 3.322 mg Si/L with three

fortified levels at each salinity. Seven replicates of each

sample were analyzed and the results were as follows:

_______________________________________

Concentration

Salinity (mg Si/L) RSD Recovery

(% ) Ambient Fortified (%) (%) o

______________________________________

35.85 0.095 0.1647 0.82 99.37

35.85 0.095 0.2196 1.34 100.61

35.85 0.095 0.2747 1.74 99.62

18.07 1.725 0.5517 1.11 107.18

18.07 1.725 1.1008 0.77 104.69

18.07 1.725 1.6508 0.98 103.62

2.86 3.322 0.5421 0.99 101.03

2.86 3.322 1.0801 1.26 103.22

2.86 3.322 1.6188 0.98 100.59

______________________________________

13.2 Multi-Laboratory Validation

Multi-laboratory validation has not been conducted for this

method and, therefore, multi-laboratory data is currently

unavailable.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

USEPA has established a preferred hierarchy of

environmental management techniques that places

pollution prevention as the management option of first

may be applicable to laboratories and research

Management for Waste Reduction, available from the

American Chemical Society, Department of Government

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

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

15.0 Waste Management

15.1 The U.S. Environmental Protection Agency

requires that laboratory waste management practices be

conducted consistent with all applicable rules and

regulations. The Agency urges laboratories to protect the

air, water, and land by minimizing and controlling all

releases from hoods and bench operations, complying

with the letter and spirit of any sewer discharge permits

and regulations, and by complying with all solid and

hazardous waste regulations, particularly the hazardous

waste identification rules and land disposal restrictions.

For further information on waste management consult

The Waste Management Manual for Laboratory

Personnel, available from the American Chemical Society

at the address listed in Section 14.2.

16.0 References

1 Chow, D. T-W., and Robinson, R.J. 1953, Forms of

silicate available for colorimetric determination.

Analytical Chemistry. 25, 646-648.

2. Burton, J. D., T.M. Leatherland and P.S. Liss, 1970.

The reactivity of dissolved silicon in some natural

waters. Limnology and Oceanography, 15, 473-

476.

3 Isshiki, K., Sohrin, Y, and Nakayama, E., 1991.

Form of dissolved silicon in seawater. Marine

Chemistry, 32, 1-8.

4. Koroleff, F. 1983, Determination of silicon, In

Methods of Seawater Analysis (Grasshoff, K., M.

Ehrhardt and K. Kremling, Eds) Weinheim, Verlag

Chemie, Germany. pp174 -187. 
Version 1.0 September 1997 366.0-12

5. Grasshoff, K. 1965. On the automatic

determination of silicate, phosphate and fluoride in

seawater. I.C.E.S. Hydrographic Committee

Report, No. 129, Rome. (Mimeographed).

 

6. Brewer P. G. and J. P. Riley. 1966. The automatic

determination of silicate-silicon in natural water with

special reference to sea water. Anal. Chim. Acta,

35, 514-519.

7. Hansen, H.P., K.Grasshoff, Statham and P.J.LeB.

Williams. 1983, Automated chemical analysis, In

Methods of Seawater Analysis (Grasshoff, K., M.

Ehrhardt and K. Kremling, Eds) Weinheim, Verlag

Chemie, Germany. pp374 -395. 

8. 40 CFR, 136 Appendix B. Definition and Procedure

for the Determination of Method Detection Limit.

Revision 1.11.

9. MacDonald, R.W. and F.A. McLaughlin. 1982. The

effect of storage by freezing on dissolved inorganic

phosphate, nitrate, and reactive silicate for samples

from coastal and estuarine waters. Water

Research, 16:95-104.

10. MacDonald, R.W. , F.A. McLaughlin and C. S.

Wong. 1986. The storage of reactive silicate

samples by freezing. Limnol. Oceanogr.,

31(5):1139-1142.

11. Salley, B.A., J.G. Bradshaw, and B.J. Neilson.

1987. Results of comparative studies of

preservation techniques for nutrient analysis on

water samples. Virginia Institute of Marine Science,

Gloucester Point, VA 23062. USEPA, CBP/TRS

6/87, 32pp.

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

as a function of system performance for continuous

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

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

carry-over and sample interaction in gas

segmented continuous flow analysis. J. Automatic

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

2

3

4

5

6

7

8

9

10

0.41

0.41

0.41

0.32

0.25

0.41

1.57

Pump

ml/min

Debubbler

Detector

660nm

Manifold

Ascorbic Acid 

Oxalic Acid 

Sample

Air

Molybdate 

Reagent Water 

or Low Nutrient Seawater

Wash To Sampler

To Waste

To Waste

Coil

Sample:Wash = 20":40"

Coil3

Coil1

Coil2

 366.0-13 Version 1.0 September 1997

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Figure 1. Manifold Configuration for Silicate Analysis.
440.0-1

Method 440.0

Determination of Carbon and Nitrogen in Sediments and Particulates

 of Estuarine/Coastal Waters Using Elemental Analysis

Carl F. Zimmermann

Carolyn W. Keefe

University of Maryland System

Center for Environmental Estuarine Studies

Chesapeake Biological Laboratory

Solomns, MD 20688-0038

and

Jerry Bashe

Technology Applications, Inc.

26 W. Martin Luther King Drive

Cincinnati, OH 45219

Revision 1.4

September 1997

Work Assignment Manager

Elizabeth J. Arar

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Revision 1.4 September 1997 440.0-2

Chemical Abstracts Service

Analyte Registry Numbers (CASRN)

Carbon 7440-44-0

Nitrogen 1333-74-0

Method 440.0

Determination of Carbon and Nitrogen in Sediments and Particulates

of Estuarine/Coastal Waters Using Elemental Analysis

1.0 Scope and Application

1.1 Elemental analysis is used to determine particulate carbon (PC) and particulate nitrogen (PN) in estuarine and coastal waters and sediment. The method

measures the total carbon and nitrogen irrespective of

source (inorganic or organic).

1.2 The need to qualitatively or quantitatively deter- be passed through a number 10 sieve or a 2-mm mesh

mine the particulate organic fraction from the total sieve.

particulate carbon and nitrogen depends on the dataquality objectives of the study. Section 11.4 outlines 3.2 Material Safety Data Sheet (MSDS) -- Written

procedures to ascertain the organic/inorganic particulate information provided by vendors concerning a chemical’s

ratio. The method performance presented in the method toxicity, health hazards, physical properties, fire, and

was obtained on particulate samples with greater than reactivity data including storage, spill, and handling pre80% organic content. Performance on samples with a cautions.

greater proportion of particulate inorganic versus organic

carbon and nitrogen has not been investigated. 3.3 Instrument Detection Limit (IDL) -- The mini1.3 Method detection limits (MDLs) of 10.5 μg/L and which gives an analyte signal equal to three times the

1

62.3 μg/L for PN and PC, respectively, were obtained for standard deviation of the background signal at the sea 200-mL sample volume. Sediment MDLs of PN and lected wavelength, mass, retention time, absorbance line,

PC are 84 mg/kg and 1300 mg/kg, respectively, for a etc.

sediment sample weight of 10.00 mg. The method has

been determined to be linear to 4800 μg of C and 700 μg 3.4 Method Detection Limit (MDL) -- The minimum

of N in a sample. Multilaboratory study validation data are concentration of an analyte that can be identified, meain Section 13. sured, and reported with 99% confidence that the analyte

1.4 This method should be used by analysts experienced in the theory and application of elemental analysis. 3.5 Linear Dynamic Range (LDR) -- The absolute

A minimum of 6 months experience with an elemental quantity over which the instrument response to an analyte

analyzer is recommended. is linear.

1.5 Users of the method data should set the data- 3.6 Calibration Standard (CAL) -- An accurately

quality objectives prior to analysis. Users of the method weighed amount of a certified chemical used to calibrate

must document and have on file the required initial the instrument response with respect to analyte mass.

demonstration of performance data described in Section

9.2 prior to using the method for analysis. 3.7 Conditioner -- A standard chemical which is not

2.0 Summary of Method

2.1 An accurately measured amount of particulate

matter from an estuarine water sample or an accurately

weighed dried sediment sample is combusted at 975EC

using an elemental analyzer. The combustion products

are passed over a copper reduction tube to convert the 

oxides of N into molecular N. Carbon dioxide, water vapor

and N are homogeneously mixed at a known volume,

temperature and pressure. The mixture is released to a

series of thermal conductivity detectors/traps, measuring

in turn by difference, hydrogen (as water vapor), C (as

carbon dioxide) and N (as N2). Inorganic and organic C

may be determined by two methods which are also

presented.

3.0 Definitions

3.1 Sediment Sample -- A fluvial, sand, or humic

sample matrix exposed to a marine, brackish or fresh

water environment. It is limited to that portion which may

mum quantity of analyte or the concentration equivalent

concentration is greater than zero.

necessarily accurately weighed that is used to coat the

surfaces of the instrument with the analytes (water vapor,

carbon dioxide, and nitrogen).

3.8 External Standards (ES) -- A pure analyte(s)

that is measured in an experiment separate from the

experiment used to measure the analyte(s) in the sample.

The signal observed for a known quantity of the pure 

external standard(s) is used to calibrate the instrument
440.0-3 Revision 1.4 September 1997

response for the corresponding analyte(s). The instru- which has been certified for specific analytes by a variety

ment response is used to calculate the concentrations of of analytical techniques and/or by numerous laboratories

the analyte(s) in the sample. using similar analytical techniques. These may consist of

3.9 Response Factor (RF) -- The ratio of the re- These materials are used as an indication of the accuracy

sponse of the instrument to a known amount of analyte. of a specific analytical technique.

3.10 Laboratory Reagent Blank (LRB) -- A blank 3.17 Quality Control Sample (QCS) -- A solution of

matrix (i.e., a precombusted filter or sediment capsule) method analytes of known concentrations which is used

that is treated exactly as a sample including exposure to to fortify an aliquot of LRB or sample matrix. The QCS is

all glassware, equipment, solvents, and reagents that are obtained from a source external to the laboratory and

used with other samples. The LRB is used to determine different from the source of calibration standards. It is

if method analytes or other interferences are present in used to check laboratory performance with externally

the laboratory environment, the reagents, or the appa- prepared test materials.

ratus.

3.11 Field Reagent Blank (FRB) -- An aliquot of

reagent water or other blank matrix that is placed in a

sample container in the laboratory and treated as a

sample in all respects, including shipment to the sampling

site, exposure to sampling site conditions, storage,

preservation, and all analytical procedures. The purpose

of the FRB is to determine if method analytes or other

interferences are present in the field environment.

3.12 Laboratory Duplicates (LD1 and LD2) -- Two

aliquots of the same sample taken in the laboratory and

analyzed separately with identical procedures. Analyses

of LD1 and LD2 indicate precision associated with laboratory procedures, but not with sample collection, preservation, or storage procedures.

3.13 Field Duplicates (FD1 and FD2) -- Two separate samples collected at the same time and place under

identical circumstances and treated exactly the same

throughout field and laboratory procedures. Analyses of

FD1 and FD2 give a measure of the precision associated

with sample collection, preservation and storage, as well

as with laboratory procedures.

3.14 Laboratory Fortified Blank (LFB) -- An aliquot

of reagent water or other blank matrices to which known

quantities of the method analytes are added in the

laboratory. The LFB is analyzed exactly like a sample,

and its purpose is to determine whether the method is in

control, and whether the laboratory is capable of making

accurate and precise measurements.

3.15 Laboratory Fortified Sample Matrix (LFM) -- An

aliquot of an environmental sample to which known

quantities of the method analytes are added in the

laboratory. The LFM is analyzed exactly like a sample,

and its purpose is to determine whether the sample matrix

contributes bias to the analytical results. The background

concentrations of the analytes in the sample matrix must

be determined in a separate aliquot and the measured

values in the LFM corrected for background concentrations.

3.16 Standard Reference Material (SRM) -- Material

pure chemicals, buffers or compositional standards.

4.0 Interferences

4.1 There are no known interferences for estuarine/coastal water or sediment samples. The presence of

C and N compounds on laboratory surfaces, on fingers,

in detergents and in dust necessitates the utilization of

careful techniques (i.e., the use of forceps and gloves) to

avoid contamination in every portion of this procedure.

5.0 Safety

5.1 The toxicity or carcinogenicity of each reagent

used in this method has not been fully established. Each

chemical should be regarded as a potential health hazard

and exposure to these compounds should be as low as

reasonably achievable. Each laboratory is responsible for

maintaining a current awareness file of OSHA regulations

regarding the safe handling of the chemicals specified in

this method. A reference file of material safety data

2-5

sheets (MSDS) should also be made available to all

personnel involved in the chemical analysis.

5.2 The acidification of samples containing reactive

materials may result in the release of toxic gases, such as

cyanides or sulfides. Acidification of samples should be

done in a fume hood.

5.3 All personnel handling environmental samples

known to contain or to have been in contact with human

waste should be immunized against known disease

causative agents.

5.4 Although most instruments are adequately

shielded, it should be remembered that the oven temperatures are extremely high and that care should be

taken when working near the instrument to prevent

possible burns.

5.5 It is the responsibility of the user of this method to

comply with relevant disposal and waste regulations. For

guidance see Sections 14.0 and 15.0.
Revision 1.4 September 1997 440.0-4

6.0 Apparatus and Equipment

6.1 Elemental Analyzer

6.1.1 An elemental analyzer capable of maintaining a

combustion temperature of 975EC and analyzing particulate samples and sediment samples for elemental C and

N. The Leeman Labs Model 240 XA Elemental Analyzer

was used to produce the data presented in this method.

6.2 A gravity convection drying oven. Capable of

maintaining 103-105EC for extended periods of time.

6.3 Muffle furnace. Capable of maintaining 875EC ±

15EC.

6.4 Ultra-micro balance. Capable of accurately

weighing to 0.1 μg. Desiccant should be kept in the

weighing chamber to prevent hygroscopic effects.

6.5 Vacuum pump or source capable of maintaining

up to 10 in. Hg of vacuum.

6.6 Mortar and pestle.

6.7 Desiccator, glass.

6.8 Freezer, capable of maintaining -20EC ± 5EC.

6.9 47-mm or 25-mm vacuum filter apparatus made

up of a glass filter tower, fritted glass disk base and 2-L

vacuum flask.

6.10 13-mm Swinlok filter holder.

6.11 Teflon-tipped, flat blade forceps.

6.12 Labware -- All reusable labware (glass, quartz,

polyethylene, PTFE, FEP, etc.) should be sufficiently

clean for the task objectives. Several procedures found

to provide clean labware include washing with a detergent

solution, rinsing with tap water, soaking for 4 hr or more

in 20% (v/v) HCI, rinsing with reagent water and storing

clean. All traces of organic material must be removed to

prevent C-N contamination.

6.12.1 Glassware -- Volumetric flasks, graduated

cylinders, vials and beakers.

6.12.2 Vacuum filter flasks -- 250 mL and 2 L, glass.

6.12.3 Funnel, 6.4 mm i.d., polyethylene.

6.12.4 Syringes, 60-mL, glass.

7.0 Reagents and Standards

7.1 Reagents may contain elemental impurities which

affect analytical data. High-purity reagents that conform

to the American Chemical Society specifications should 6

be used whenever possible. If the purity of a reagent is in

question, analyze for contamination. The acid used for

this method must be of reagent grade purity or equivalent.

A suitable acid is available from a number of manufacturers.

7.2 Hydrochloric acid, concentrated (sp. gr. 1.19)-

HCI.

7.3 Acetanilide, 99.9% + purity, C8H19NO (CASRN

103-84-4).

7.4 Blanks -- Three blanks are used for the analysis.

Two blanks are instrument related. The instrument zero

response (ZN) is the background response of the instrument without sample holding devices such as capsules

and sleeves. The instrument blank response (BN) is the

response of the instrument when the sample capsule,

sleeve and ladle are inserted for analysis without standard

or sample. The BN is also the laboratory reagent blank

(LRB) for sediment samples. The LRB for water samples

includes the capsule, sleeve, ladle and a precombusted

filter without standard or sample. These blanks are

subtracted from the uncorrected instrument response

used to calculate concentration in Sections 12.3 and 12.4.

7.4.1 Laboratory fortified blank (LFB) -- The third blank

is the laboratory fortified blank. For sediment analysis,

add a weighed amount of acetanilide in an aluminum

capsule and analyze for PC and PN (Section 9.3.2). For

aqueous samples, place a weighed amount of acetanilide

on a glass fiber filter the same size as used for the

sample filtration. Analyze the fortified filter for PC and PN

(Section 9.3.2)

7.5 Quality Control Sample (QCS) -- For this method, the QCS can be any assayed and certified sediment

or particulate sample which is obtained from an external

source. The Canadian Reference Material, BCSS-1, is

just such a material and was used in this capacity for the

data presented in this method. The percent PC has been

certified in this material but percent PN has not.

8.0 Sample Collection, Preservation and

Storage

8.1 Water Sample Collection -- Samples collected

for PC and PN analyses from estuarine/coastal waters

are normally collected from a ship using one of two

methods; hydrocast or submersible pump systems. Follow the recommended sampling protocols associated

with the method used. Whenever possible, immediately

filter the samples as described in Section 11.1.1. Store

the filtered sample pads by freezing at -20EC or storing in

a desiccator after drying at 103-105E C for 24 hr. No

significant difference has been noted in comparing the

two storage procedures for a time period of up to 100

days. If storage of the water sample is necessary, place
440.0-5 Revision 1.4 September 1997

MDL = (t) X (S)

where, S = Standard deviation of the replicate analyses.

t = Student’s t value for n-1 

degrees of freedom at the 

99% confidence limit; t = 3.143 

for six degrees of freedom.

the sample into a clean amber bottle and store at 4EC

until filtration is done. When beginning the use of this method, on a quarterly

8.1.1 The volume of water sample collected will vary calibration standards and acceptable instrument perforwith the type of sample being analyzed. Table 1 provides

a guide for a number of matrices of interest. If the matrix

cannot be classified by this guide, collect 2 x 1L of water

from each site. A minimum filtration volume of 200 mL is

recommended.

8.2 Sediment Sample Collection -- Estuarine/coastal sediment samples are collected with benthic

samplers. The type of sampler used will depend on the be established for PC and PN using a low level estuarine

type of sample needed by the data-quality objectives. water sample, typically three to five times higher than the 7

Store the wet sediment in a clean jar and freeze at -20EC estimated MDL. The same procedure should be followed

until ready for analysis. for sediments. To determine MDL values, analyze seven

8.2.1 The amount of sediment collected will depend on through the entire analytical procedure (Section 11).

the sample matrix and the elemental analyzer used. A

minimum of 10 g is recommended.

9.0 Quality Control

9.1 Each laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of laboratory capability and the continued

analysis of laboratory reagent blanks, laboratory duplicates, field duplicates and calibration standards analyzed

as samples as a continuing check on performance. The

laboratory is required to maintain performance records

that define the quality of data thus generated.

9.2 Initial Demonstration of Performance 

(Mandatory)

9.2.1 The initial demonstration of performance is used

to characterize instrument performance (MDLs, linear dynamic range) and laboratory performance (analysis of QC

samples) prior to the analyses conducted by this method.

9.2.2 Linear dynamic range (LDR) -- The upper limit of

the LDR must be established by determining the signal

responses from a minimum of three different concentration standards across the range, one of which is close to

the upper limit of the LDR. Determined LDRs must be

documented and kept on file. The LDR which may be

used for the analysis of samples should be judged by the

analyst from the resulting data. The upper LDR limit

should be an observed signal no more than 10% below

the level extrapolated from the lower standards. Determined sample analyte concentrations that are 90% and

above the upper LDR must be reduced in mass and

reanalyzed. New LDRs should be determined whenever

there is a significant change in instrument response and

for those analytes that periodically approach the upper

LDR limit, every 6 months or whenever there is a change

in instrument analytical hardware or operating conditions.

9.2.3 Quality control sample (QCS) (Section 7.5) --

basis or as required to meet data quality needs, verify the

mance with the analyses of a QCS. If the determined

concentrations are not within ± 5% of the stated values,

performance of the determinative step of the method is

unacceptable. The source of the problem must be identified and corrected before either proceeding with the

initial determination of MDLs or continuing with analyses.

9.2.4 Method detection limits (MDLs) -- MDLs should

replicate aliquots of water or sediment and process

These replicates should be randomly distributed throughout a group of typical analyses. Perform all calculations

defined in the method (Section 12) and report the concentration values in the appropriate units. Calculate the

MDL as follows:1

MDLs should be determined whenever a significant

change in instrumental response, change of operator, or

a new matrix is encountered.

9.3 Assessing Laboratory Performance 

(Mandatory)

9.3.1 Laboratory reagent blank (LRB) -- The laboratory

must analyze at least one LRB (Section 3.10) with each

batch of 20 or fewer samples of the same matrix. LRB

data are used to assess contamination from the laboratory environment. LRB values that exceed the MDL

indicate laboratory or reagent contamination. When LRB

values constitute 10% or more of the analyte level determined for a sample, fresh samples or field duplicates of

the samples must be prepared and analyzed again after

the source of contamination has been corrected and

acceptable LRB values have been obtained. For aqueous samples the LRB is a precombusted filter of the

same type and size used for samples.
Revision 1.4 September 1997 440.0-6

Response factor (μv/μg) = RN-ZN-BN

 WTN

where, RN = Average instrument response to 

standard (μv)

ZN = Instrument zero response (μv)

BN = Instrument blank response (μv)

and, WTN = (M)(N )(AW/MW) a

where, M = The mass of standard material in 

μg

N = Number of atoms of C, N or H, in a

a molecule of standard material

AW = Atomic weight of C (12.01), N 

(14.01) or H (1.01)

MW = Molecular weight of standard 

material (135.2 for acetanilide)

9.3.2 Laboratory fortified blank (LFB) -- The laboratory

must analyze at least one LFB (Section 7.4.1) with each

batch of samples. Calculate accuracy as percent recovery. If the recovery of any analyte falls outside the

required control limits of 85-115%, that analyte is judged

out of control, and the source of the problem should be

identified and resolved before continuing analyses.

9.3.3 The laboratory must use LFB analyses data to

assess laboratory performance against the required control limits of 85-115% (Section 9.3.2). When sufficient

internal performance data become available (usually a

minimum of 20-30 analyses), optional control limits can

be developed from the percent mean recovery (x) and the

standard deviation (S) of the mean recovery. These data

can be used to establish the upper and lower control

limits as follows:

Upper Control Limit = x + 3S

Lower Control Limit = x - 3S

The optional control limits must be equal to or better than

the required control limits of 85-115%. After each five to

ten new recovery measurements, new control limits can

be calculated using only the most recent 20-30 data

points. Also the standard deviation (S) data should be

used to establish an ongoing precision statement for the

level of concentrations included in the LFB. These data

must be kept on file and be available for review.

9.4 Assessing Analyte Recovery and Data

Quality

9.4.1 Percent recoveries cannot be readily obtained

from particulate samples. Consequently, accuracy can

only be assessed by analyzing check standards as

samples and quality control samples (QCS). The use of

laboratory fortified matrix samples has not been assessed.

10.0 Calibration and Standardization

10.1 Calibration -- After following manufacturer’s

installation and temperature stabilization procedures,

daily calibration procedures must be performed and

evaluated before sample analysis may begin. Single

point or standard curve calibrations are possible, depending on instrumentation.

10.1.1 Establish single response factors (RF) for each

element (C,H, and N) by analyzing three weighed portions

of calibration standard (acetanilide). The mass of

calibration standard should provide a response within

20% of the response expected for the samples being

analyzed. Calculate the (RF) for each element using the

following formula:

If instrument response is in units other than μv, then

change the formula accordingly.

10.1.2 For standard curve preparation, the range of

calibration standard masses used should be such that the

low concentration approaches but is above the MDL and

the high concentration is above the level of the highest

sample, but no more than 90% of the linear dynamic

range. A minimum of three concentrations should be

used in constructing the curve. Measure response versus

mass of element in the standard and perform a

regression on the data to obtain the calibration curve.

11.0 Procedure

11.1 Aqueous Sample Preparation

11.1.1 Water Sample Filtration -- Precombust GF/F

glass fiber filters at 500EC for 1.5 hr. The diameter of filter

used will depend on the sample composition and instrument capabilities (Section 8.1.1). Store filters covered if

not immediately used. Place a precombusted filter on

fritted filter base of the filtration apparatus and attach the

filtration tower. Thoroughly shake the sample container

to suspend the particulate matter. Measure and record

the required sample volume using a graduated cylinder.

Pour the measured sample into the filtration tower, no

more than 50 mL at a time. Filter the sample using a

vacuum no greater than 10 in. of Hg. Vacuum levels

greater than 10 in. of Hg can cause filter rupture. If less

than the measured volume of sample can be practically

filtered due to clogging, measure and record the actual

volume filtered. Do not rinse the filter following filtration.

It has been demonstrated that sample loss occurs when

the filter is rinsed with an isotonic solution or the filtrate.8

Air dry the filter after the sample has passed through by

continuing the vacuum for 30 sec. Using Teflon-coated

flat-tipped forceps, fold the filters in half while still on the

fritted glass base of the filter apparatus. Store filters as

described in Section 8.
440.0-7 Revision 1.4 September 1997

Corrected

Concentration (μg/L) = sample response (μv)

Sample volume (L) x RF (μv/μg)

where, RF = Response Factor (Section 10.1.1)

Corrected Sample Response (Section 

7.4)

Corrected

Concentration (mg/kg) = sample response (μv)

Sample weight (g) x RF (μv/μg)

where, RF = Response Factor (Section 10.1.1) 

Corrected Sample Response (Section 

7.4)

Note: Units of μg/g = mg/kg

11.1.2 If the sample has been stored frozen, place the Analyze the samples for particulate C. The resultant data

sample in a drying oven at 103-105E C for 24 hr before are particulate organic carbon.

analysis and dry to a constant weight. Precombust one

nickel sleeve at 875E C for 1 hr for each sample.

11.1.3 Remove the filter pads containing the particulate

material from the drying oven and insert into a precombusted nickel sleeve using Teflon-coated flat-tipped

forceps. Tap the filter pad using a stainless steel rod.

The sample is ready for analysis.

11.2 Sediment Samples Preparation

11.2.1 Thaw the frozen sediment sample in a 102-

105EC drying oven for at least 24 hr before analysis and

dry to a constant weight. After drying, homogenize the

dry sediment with a mortar and pestle. Store in a desiccator until analysis. Precombust aluminum capsules at

550EC in a muffle furnace for 1.5 hr for each sediment

sample being analyzed. Precombust one nickel sleeve

at 875EC for 1 hr for each sediment sample.

11.2.2 Weigh 10 mg of the homogenized sediment to

the nearest 0.001 mg with an ultra-micro balance into a

precombusted aluminum capsule. Crimp the top of the

aluminum capsule with the Teflon-coated flat-tipped forceps and place into a precombusted nickel sleeve. The

sample is ready for analysis.

11.3 Sample Analysis

11.3.1 Measure instrument zero response (Section 7.4)

and instrument blank response (Section 7.4) and record

values. Condition the instrument by analyzing a conditioner. Calibrate the instrument according to Section 10

and analyze all preliminary QC samples as required by

Section 9. When satisfactory control has been established, analyze samples according to the instrument

manufacturer’s recommendations. Record all response

data.

11.3.2 Report data as directed in Section 12.

11.4 Determination of Particulate Organic and

Inorganic Carbon

11.4.1 Method 1: Thermal Partitioning -- The difference

found between replicate samples, one of which has been

analyzed for total PC and PN and the other which was

muffled at 550EC and analyzed is the particulate organic

component of that sample. This method of thermally

partitioning organic and inorganic PC may underestimate

slightly the carbonate minerals’ contribution in the

inorganic fraction since some carbonate minerals

decompose below 500EC, although CaCO does not. 3

9

11.4.2 Method 2: Fuming HCI -- Allow samples to dry

overnight at 103-105EC and then place in a desiccator

containing concentrated HCI, cover and fume for 24 hr in

a hood. The fuming HCI converts inorganic carbonate in

the samples to water vapor, CO and calcium chloride. 2

10

12.0 Data Analysis and Calculations

12.1 Sample data should be reported in units of μg/L

for aqueous samples and mg/kg dry weight for sediment

samples. 

12.2 Report analyte concentrations up to three significant figures for both aqueous and sediment samples.

12.3 For aqueous samples, calculate the sample concentration using the following formula:

12.4 For sediment samples, calculate the sample concentration using the following formula:

12.5 The QC data obtained during the analyses

provide an indication of the quality of the sample data and

should be provided with the sample results.

13.0 Method Performance

13.1 Single Laboratory Performance

13.1.1 Single laboratory performance data for aqueous

samples from the Chesapeake Bay are provided in Table

2.

13.1.2 Single-laboratory precision and accuracy data for

the marine sediment reference material, BCSS-1, are

listed in Table 3.

13.2 Multilaboratory Performance

13.2.1 In a multilab study, 13 participants analyzed

sediment and filtered estuarine water samples for

particulate carbon and nitrogen. The data were analyzed
Revision 1.4 September 1997 440.0-8

using the statistical procedures recommended in ASTM letter and spirit of any sewer discharge permits and

D2777-86 for replicate designs. See Table 4 for regulations, and by complying with all solid and hazardsummary statistics. ous waste regulations, particularly the hazardous waste

13.2.2 Accuracy as mean recovery was estimated from further information on waste management consult The

the analyses of the NRC of Canada Marine Sediment

Reference Material, BCSS-1. Mean recovery was 98.2% available from the American Chemical Society at the

of the certified reference carbon value and 100% of the address listed in Section 14.2.

noncertified nitrogen value.

13.2.3 Overall precision for analyses of carbon and

nitrogen in sediments was 1-11% RSD, while the

analyses of both particulate carbon and nitrogen in

estuarine water samples was 9-14% RSD.

13.2.4 Single analyst precision for carbon and nitrogen

in sediment samples was 1-8% RSD and 4-9% for water

samples.

13.2.5 Pooled method detection limits (p-MDLs) were

calculated using the pooled single analyst standard

deviations. The p-MDLs for particulate nitrogen and

carbon in estuarine waters were 0.014 mg N/L and 0.064

mg C/L , respectively. The p-MDLs for percent carbon

and nitrogen in estuarine sediments were not estimated

because the lowest concentration sediment used in the

study was still 20 times higher than the estimated MDLs.

Estimates of p-MDLs from these data would be

unrealistically high.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

EPA has established a preferred hierarchy of environmental management techniques that places pollution

prevention as the management option of first choice.

Whenever feasible, laboratory personnel should use

pollution prevention techniques to address their waste

generation. When wastes cannot be feasibly reduced at

the source, the Agency recommends recycling as the next

best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society’s Department of Government Relations and Science Policy, 1155 16th Street N.W.,

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

15.0 Waste Management

15.1 The Environmental Protection Agency requires

that laboratory waste management practices be conducted consistent with all applicable rules and regulations. The Agency urges laboratories to protect the air,

water and land by minimizing and controlling all releases

from hoods and bench operations, complying with the

identification rules and land disposal restrictions. For

Waste Management Manual for Laboratory Personnel,

16.0 References

1. 40 CFR, Part 136, Appendix B. Definition and

Procedure for the Determination of the Method

Detection Limit. Revision 1.11.

2. Carcinogens - Working With Carcinogens,

Department of Health, Education, and Welfare,

Public Health Service, Center for Disease Control,

National Institute for Occupational Safety and

Health, Publication No. 77-206, Aug. 1977.

3. OSHA Safety and Health Standards, General

Industry, (29 CFR 1910), Occupational Safety and

Health Administration, OSHA 2206, (Revised,

January 1976).

4. Safety in Academic Chemistry Laboratories,

American Chemical Society Publication, Committee

on Chemical Safety, 3rd Edition, 1979.

5. Proposed OSHA Safety and Health Standards,

Laboratories, Occupational Safety and Health

Administration, Federal Register, July 24, 1986.

6. Rohrdough, W.G. et al. Reagent Chemicals,

American Chemical Society Specifications, 7th

Edition. American Chemical Society, Washington,

DC, 1986.

7. Holme, N.A. and A.D. McIntyre (eds). 1971.

Methods for the Study of Marine Benthos.

International Biome Program. IBP Handbook #16.

F.A. Davis Co., Philadelphia, PA.

8. Hurd, D.C. and D.W. Spencer (eds). 1991. Marine

Particles: Analysis and Characterization.

Geophysical Monograph: 63, American Geophysical

Union, Washington, DC 472p.

9. Hirota, J. and J.P. Szyper. 1975. Separation of total

particulate carbon into inorganic and organic

components. Limnol. and Oceanogr. 20: 896-900.

10. Grasshoff, K., M. Ehrhardt and K. Kremling (eds).

1983. Methods of Seawater Analysis. Verlag

Chemie.
440.0-9 Revision 1.4 September 1997

 Filter diameter

Sample matrix 47mm 25mm 13mm

Sample matrix volume

Open ocean 2000 mL 500 mL 100 mL

Coastal 1000 mL 400-500 mL 100 mL

Estuarine 500-700 mL 250-400 mL 50 mL

 (low particulate)

Estuarine 100-400 mL 75-200 mL 25 mL

 (high particulate)

Measured Measured

nitrogen carbon

concentration S.D. concentration S.D. A A

Sample (μg/L) (μg/L) (μg/L) (μg/L)

1 147 ± 4 1210 ± 49

2 148 ± 11 1240 ± 179

3 379 ± 51 3950 ± 269

4 122 ± 9 1010 ± 63

 Standard deviation based on 7 replicates. A

Mean

measured

Element T.V. value (%) %RSD %Recovery A B C

Carbon 2.19% 2.18 ± 3.3 99.5

Nitrogen 0.195% 0.194 ± 3.9 99.5

 True value. Carbon value is certified; nitrogen value is listed but A

 not certified

 Percent relative standard deviation based on 10 replicates. B

 As calculated from T.V. C

17.0 Tables, Diagrams, Flowcharts, and

Validation Data

Table 1. Filter Diameter Selection Guide

Table 2. Performance Data--Chesapeake Bay Aqueous

 Samples

Table 3. Precision and Accuracy Data - Canadian

 Sediment Reference Material BCSS-1
Revision 1.4 September 1997 440.0-10

Table 4. Overall and Single Analyst Precision Estimates from Collaborative Study

Analyte Sample N Mean Overall Overall Analyst Analyst (1) (2)

Conc. Std. Dev. %RSD Std. Dev. %RSD

Particulate A 11 0.0655 0.0081 12.4% 0.0050 7.6%

Nitrogen

(as N) in 

Estuarine 

Waters

B 12 0.0730 0.0076 10.3% 0.0057 7.7%

C 12 0.0849 0.0110 12.9% 0.0060 7.1%

D 12 0.126 0.0138 11.0% 0.0071 5.6%

E 11 0.182 0.0245 13.5% 0.0157 8.6%

Nitrogen 1 10 0.178 0.0190 10.7% 0.0131 7.3%

(as %N) in

Estuarine 

Water

2 10 0.295 0.0114 3.9% 0.0046 1.6%

3 10 0.436 0.0178 4.1% 0.0104 2.4%

4 10 0.497 0.0183 3.7% 0.0082 1.6%

5 10 0.580 0.0207 3.6% 0.0150 2.6%

Particulate B 12 0.369 0.0505 13.7% 0.0222 6.0%

Carbon 

(as C) in 

Estuarine

Waters

A 12 0.417 0.0490 11.8% 0.0230 5.5%

D 12 0.619 0..0707 11.4% 0.0226 3.6%

C 12 0.710 0.0633 8.9% 0.0367 5.2%

E 12 0.896 0.1192 13.3% 0.0569 6.4%

Carbon 1 13 1.78 0.1517 8.5% 0.1346 7.6%

(as %C) in

Estuarine

Sediments

2 13 2.55 0.0372 1.5% 0.0204 0.8%

3 13 3.18 0.0435 1.4% 0.0348 1.1%

4 13 4.92 0.1201 2.4% 0.0779 1.6%

5 13 5.92 0.0621 1.1% 0.0547 0.9%

(1) N = Number of participants whose data was used.

(2) Concentration in mg/L or percent, as indicated.
445.0-1

Method 445.0

In Vitro Determination of Chlorophyll a and Pheophytin a

in Marine and Freshwater Algae by Fluorescence

Elizabeth J. Arar

and

Gary B. Collins

Revision 1.2

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Revision 1.2 September 1997 445.0-2

Method 445.0

In Vitro Determination of Chlorophyll a and Pheophytin a

in Marine and Freshwater Algae by Fluorescence

1.0 Scope and Application

1.1 This method provides a procedure for low level

determination of chlorophyll a (chl a) and its magnesiumfree derivative, pheophytin a (pheo a), in marine and

freshwater phytoplankton using fluorescence detection.(1,2)

Phaeophorbides present in the sample are determined

collectively as pheophytin a. For users primarily

interested in chl a there is currently available a set of very

narrow bandpass excitation and emission filters (Turner

Designs, Sunnyvale, CA) that nearly eliminates the

spectral interference caused by the presence of pheo a

and chlorophyll b. The difference between the modified

method and the conventional fluorometric method is that

the equations used for the determination of chlor a

without pheo a correction (uncorrected chlor a), are used

instead of the equations for "corrected chlor a". This EPA

laboratory has evaluated the modified filters and found

the technique to be an acceptable alternative to the

conventional fluorometric method using pheo a

correction.(3)

 

Chemical Abstracts Service

Analyte Registry Number (CASRN)

 

Chlorophyll a 479-61-8

 

1.2 Instrumental detection limits (IDL) of 0.05 μg chl

a/L and 0.06 μg pheo a/L in a solution of 90% acetone

were determined by this laboratory. Method detection

limits (MDL) using mixed assemblages of algae provide

little information because the fluorescence of other

pigments interferes in the fluorescence of chlorophyll a

and pheophytin a. A single lab estimated detection limit (4)

for chlorophyll a was determined to be 0.11 μg/L in 10 mL

of final extraction solution. The upper limit of the linear

dynamic range for the instrumentation used in this

method evaluation was 250 μg chl a/L.

1.3 This method was multilaboratory validated in

1996. Results from that study may be found in Section (5)

13. Additional QC procedures also have been added as

a result of that study.

1.4 This method uses 90% acetone as the extraction

solvent because of its efficiency for most types of algae.

There is evidence that certain chlorophylls and

carotenoids are more thoroughly extracted with

methanol or dimethyl sulfoxide. Bowles, et al. (5-8) (9) (8)

found that for chlorophyll a, however, 90% acetone was

an effective extractant when the extraction period was

optimized for the dominant species present in the sample.

1.5 Depending on the type of algae under

investigation, this method can have uncorrectable

interferences (Sect. 4.0). In cases where taxomonic

classification is unavailable, a spectrophotometric or high

performance liquid chromatographic (HPLC) method may

provide more accurate data for chlorophyll a and

pheophytin a.

1.6 This method is for use by analysts experienced in

the handling of photosynthetic pigments and in the

operation of fluorescence detectors or by analysts under

the close supervision of such qualified persons.

2.0 Summary of Method

2.1 Chlorophyll-containing phytoplankton in a

measured volume of sample water are concentrated by

filtering at low vacuum through a glass fiber filter. The

pigments are extracted from the phytoplankton in 90%

acetone with the aid of a mechanical tissue grinder and

allowed to steep for a minimum of 2 h, but not to exceed

24 h, to ensure thorough extraction of the chlorophyll a.

The filter slurry is centrifuged at 675 g for 15 min (or at

1000 g for 5 min) to clarify the solution. An aliquot of the

supernatant is transferred to a glass cuvette and

fluorescence is measured before and after acidification to

0.003 N HCl with 0.1 N HCl. Sensitivity calibration factors,

which have been previously determined on solutions of
 445.0-3 Revision 1.2 September 1997 

pure chlorophyll a of known concentration, are used to interferences are present in the laboratory environment,

calculate the concentration of chlorophyll a and

pheophytin a in the sample extract. The concentration in

the natural water sample is reported in μg/L.

3.0 Definitions

3.1 Estimated Detection Limit (EDL) -- The

minimum concentration of an analyte that yields a

fluorescence 3X the fluorescence of blank filters which

have been extracted according to this method.

3.2 Linear Dynamic Range (LDR) -- The absolute

quantity or concentration range over which the instrument

response to an analyte is linear.

3.3 Instrument Detection Limit (IDL) -- The

minimum quantity of analyte or the concentration

equivalent which gives an analyte signal equal to three

times the standard deviation of the background signal at

the selected wavelength, mass, retention time,

absorbance line, etc. For this method the background is

a solution of 90% acetone.

3.4 Stock Standard Solution (SSS) -- A

concentrated solution containing one or more method

analytes prepared in the laboratory using assayed

reference materials or purchased from a reputable

commercial source.

3.5 Primary Dilution Standard Solution (PDS) -- A

solution of the analytes prepared in the laboratory from

stock standard solutions and diluted as needed to

prepare calibration solutions and other needed analyte

solutions. vary with the taxonomic composition of the phytoplankton.

3.6 Calibration Standard (CAL) -- A solution

prepared from the primary dilution standard solution or

stock standard solutions containing the internal standards

and surrogate analytes. The CAL solutions are used to

calibrate the instrument response with respect to analyte

concentration.

3.7 Response Factor (RF) -- The ratio of the

response of the instrument to a known amount of analyte.

3.8 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water or other blank matrices that are treated

exactly as a sample including exposure to all glassware,

equipment, solvents, reagents, internal standards, and

surrogates that are used with other samples. The LRB is

used to determine if method analytes or other

reagents, or apparatus.

3.9 Field Duplicates (FD1 and FD2) -- Two separate

samples collected at the same time and place under

identical circumstances and treated exactly the same

throughout field and laboratory procedures. Analyses of

FD1 and FD2 give a measure of the precision associated

with sample collection, preservation and storage, as well

as with laboratory procedures.

3.10 Quality Control Sample (QCS) -- A solution of

method analytes of known concentrations which is used

to fortify an aliquot of LRB or sample matrix. Ideally, the

QCS is obtained from a source external to the laboratory

and different from the source of calibration standards. It

is used to check laboratory performance with externally

prepared test materials.

3.11 Material Safety Data Sheet (MSDS) -- Written

information provided by vendors concerning a chemical's

toxicity, health hazards, physical properties, fire, and

reactivity data including storage, spill, and handling

precautions.

4.0 Interferences

4.1 Any substance extracted from the filter or

acquired from laboratory contamination that fluoresces in

the red region of the spectrum may interfere in the

accurate measurement of both chlorophyll a and

pheophytin a.

4.2 The relative amounts of chlorophyll a, b and c

Chlorophylls b and c may significantly interfere with

chlorophyll a measurements depending on the amount

present. Due to the spectral overlap of chlorophyll b with

pheophytin a and chlorophyll a, underestimation of

chlorophyll a occurs accompanied by overestimation of

pheophytin a when chlorophyll b is present in the sample.

The degree of interference depends upon the ratio of a:b.

This laboratory found that at a ratio of 5:1, using the

acidification procedure to correct for pheophytin a,

chlorophyll a was underestimated by approximately 5%.

Loftis and Carpenter reported an underestimation of

(10)

16% when the a:b ratio was 2.5:1. A ratio of 1:1 is the

highest ratio likely to occur in nature. They also reported

overestimation of chlorophyll a in the presence of

chlorophyll c of as much as 10% when the a:c ratio was

1:1 (the theoretical maximum likely to occur in nature).

The presence of chlorophyll c also causes the under-
Revision 1.2 September 1997 445.0-4

estimation of pheophytin a. The effect of chlorophyll c is

not as severe as the effect of chlorophyll b on the

measurement of chlorophyll a and pheophytin a.

Knowledge of the taxonomy of the algae under

consideration will aid in determining if the

spectrophotometric method using trichromatic equations

to determine chlorophyll a, b, and c or an HPLC method

would be more appropriate. In the presence of

(11-16) 

chlorophyll b or pheopigments, the modified fluorometric

method described here is also appropriate.(5)

4.3 Quenching effects are observed in highly

concentrated solutions or in the presence of high

concentrations of other chlorophylls or carotenoids.

Minimum sensitivity settings on the fluorometer should be

avoided; samples should be diluted instead.

4.4 Fluorescence is temperature dependent with

higher sensitivity occurring at lower temperatures.

Samples, standards, LRBs and QCSs must be at the

same temperature to prevent errors and/or low precision.

Analyses of samples at ambient temperature is

recommended in this method. Ambient temperature

should not fluctuate more than ± 3EC between

calibrations or recalibration of the fluorometer will be

necessary.

4.5 Samples must be clarified by centrifugation prior

to analysis.

4.6 All photosynthetic pigments are light and

temperature sensitive. Work must be performed in

subdued light and all standards, QC materials and filter

samples must be stored in the dark at -20 C or -70 C to o o

prevent degradation.

5.0 Safety

5.1 The toxicity or carcinogenicity of the chemicals

used in this method have not been fully established.

Each chemical should be regarded as a potential health

hazard and handled with caution and respect. Each

laboratory is responsible for maintaining a current

awareness file of Occupational Safety and Health

Administration (OSHA) regulations regarding the safe

handling of the chemicals specified in this method. A (17-20)

file of MSDS should also be made available to all

personnel involved in the chemical analysis.

5.2 The grinding of filters during the extraction step of

this method should be conducted in a fume hood due to

the volatilization of acetone by the tissue grinder.

6.0 Apparatus and Equipment

6.1 Fluorometer -- Equipped with a high intensity

F4T.5 blue lamp, red-sensitive photomultiplier, and filters

for excitation (CS-5-60) and emission (CS-2-64). A

Turner Designs Model 10 Series fluorometer was used in

the evaluation of this method. The modified method

requires excitation filter (436FS10) and emission filter

(680FS10).

6.2 Centrifuge, capable of 675 g.

6.3 Tissue grinder, Teflon pestle (50 mm X 20 mm)

with grooves in the tip with 1/4" stainless steel rod long

enough to chuck onto a suitable drive motor and 30-mL

capacity glass grinding tube.

6.4 Filters, glass fiber, 47-mm or 25-mm, nominal

pore size of 0.7 μm unless otherwise justified by data

quality objectives. Whatman GF/F filters were used in this

work.

6.5 Petri dishes, plastic, 50 X 9-mm, or some other

solid container for transporting and storing sampled

filters.

6.6 Aluminum foil.

6.7 Laboratory tissues.

6.8 Tweezers or flat-tipped forceps.

6.9 Vacuum pump or source capable of maintaining

a vacuum up to 6 in. Hg.

6.10 Room thermometer.

6.11 Labware -- All reusable labware (glass,

polyethylene, Teflon, etc.) that comes in contact with

chlorophyll solutions should be clean and acid free. An

acceptable cleaning procedure is soaking for 4 h in

laboratory grade detergent and water, rinsing with tap

water, distilled deionized water and acetone.

6.11.1 Assorted Class A calibrated pipets.

6.11.2 Graduated cylinders, 500-mL and 1-L.

6.11.3 Volumetric flasks, Class A calibrated, 25-mL, 50-

mL, 100-mL and 1-L capacity.

6.11.4 Glass rods.
 445.0-5 Revision 1.2 September 1997 

6.11.5 Pasteur type pipets or medicine droppers. the solution must be determined spectrophotometrically

6.11.6 Disposable glass cuvettes for the fluorometer.

6.11.7 Filtration apparatus consisting of 1 or 2-L filtration

flask, 47-mm fritted glass disk base and a glass filter

tower.

6.11.8 Centrifuge tubes, polypropylene or glass, 15-mL

capacity with nonpigmented screw-caps.

6.11.9 Polyethylene squirt bottles.

7.0 Reagents and Standards

7.1 Acetone, HPLC grade, (CASRN 67-64-1).

7.2 Hydrochloric acid (HCl), concentrated (sp. gr.

1.19), (CASRN 7647-01-0).

7.3 Chlorophyll a free of chlorophyll b. May be

obtained from a commercial supplier such as Sigma

Chemical (St. Louis, MO). Turner Designs (Sunnyvale,

CA) supplies ready-made standards.

7.4 Water -- ASTM Type I water (ASTM D1193) is

required. Suitable water may be obtained by passing

distilled water through a mixed bed of anion and cation

exchange resins.

7.5 0.1 N HCl Solution -- Add 8.5 mL of

concentrated HCl to approximately 500 mL water and

dilute to 1 L.

7.6 Aqueous Acetone Solution -- 90% acetone

/10% water. Carefully measure 100 mL of water into the

1-L graduated cylinder. Transfer to a 1-L flask or storage

bottle. Measure 900 mL of acetone into the graduated

cylinder and transfer to the flask or bottle containing the

water. Mix, label and store.

7.7 Chlorophyll Stock Standard Solution (SSS) --

Chlorophyll a from a commercial supplier will be shipped

in an amber glass ampoule which has been flame sealed.

This dry standard should be stored at -20 or -70EC in the

dark and the SSS prepared just prior to use. Tap the

ampoule until all the dried chlorophyll is in the bottom of

the ampoule. In subdued light, carefully break the tip off

the ampoule. Transfer the entire contents of the

ampoule into a 50-mL volumetric flask. Dilute to volume

with 90% acetone, label the flask and wrap with

aluminum foil to protect from light. The concentration of

using a multiwavelength spectrophotometer. When

(10)

stored in a light and airtight container at freezer

temperatures, the SSS is stable for at least six months.

The concentration of all dilutions of the SSS must be

determined spectrophotometrically each time they are

made. 

7.8 Laboratory Reagent Blank (LRB) -- A blank

filter which is extracted and analyzed just as a sample

filter. The LRB should be the last filter extracted of a

sample set. It is used to assess possible contamination

of the reagents or apparatus.

7.9 Chlorophyll a Primary Dilution Standard

Solution (PDS) -- Add 1 mL of the SSS (Sect. 7.8) to a

clean 100-mL flask and dilute to volume with the aqueous

acetone solution (Sect. 7.7). If exactly 1 mg of pure

chlorophyll a was used to prepare the SSS, the

concentration of the PDS is 200 μg/L. Prepare fresh just

prior to use.

7.10 Quality Control Sample (QCS) -- Since there

are no commercially available QCSs, dilutions of a stock

standard of a different lot number from that used to

prepare calibration solutions may be used.

8.0 Sample Collection, Preservation and

Storage

8.1 Water Sample Collection -- Water may be

obtained by a pump or grab sampler. Data quality

objectives will determine the depth at which samples are

taken. Healthy phytoplankton, however, are generally

obtained from the photic zone (depth at which the

illumination level is 1% of surface illumination). Enough

water should be collected to concentrate phytoplankton

on at least three filters so that precision can be assessed.

Filtration volume size will depend on the particulate load

of the water. Four liters may be required for open ocean

water where phytoplankton density is usually low,

whereas 1 L or less is generally sufficient for lake, bay or

estuary water. All apparatus should be clean and acidfree. Filtering should be performed in subdued light as

soon as possible after sampling since algal poulations,

thus chlorophyll a concentration, can change in relatively

short periods of time. Aboard ship filtration is highly

recommended.

Assemble the filtration apparatus and attach the vacuum

source with vacuum gauge and regulator. Vacuum

filtration should not exceed 6 in. Hg (20 kPa). Higher
Revision 1.2 September 1997 445.0-6

filtration pressures and excessively long filtration times (> laboratory performance (analyses of QCSs) prior to

10 min) may damage cells and result in loss of sample analyses.

chorophyll. 

Prior to drawing a subsample from the water sample

container, thoroughly but gently agitate the container to

suspend the particulates (stir or invert several times).

Pour the subsample into a graduated cylinder and

accurately measure the volume. Pour the subsample into

the filter tower of the filtration apparatus and apply a

vacuum (not to exceed 20 kPa). A sufficient volume has

been filtered when a visible green or brown color is

apparent on the filter. Do not suck the filter dry with the

vacuum; instead slowly release the vacuum as the final

volume approaches the level of the filter and completely

release the vacuum as the last bit of water is pulled

through the filter. Remove the filter from the fritted base

with tweezers, fold once with the particulate matter inside,

lightly blot the filter with a tissue to remove excess

moisture and place it in the petri dish or other suitable

container. If the filter will not be immediately extracted,

then wrap the container with aluminum foil to protect the

phytoplankton from light and store the filter at -20 or

 -70 C. Short term storage (2 to 4 h) on ice is acceptable, o

but samples should be stored at -20 or -70EC as soon as

possible.

8.2 Preservation -- Sampled filters should be stored

frozen (-20 C or -70 C) in the dark until extraction. o o

8.3 Holding Time -- Filters can be stored frozen at

-20 or -70 C for as long as 31⁄2 weeks without significant o

loss of chlorophyll a.(21)

9.0 Quality Control

9.1 Each Laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of laboratory capability and the continued

analysis of laboratory reagent blanks, field duplicates and

quality control samples as a continuing check on

performance. The laboratory is required to maintain

performance records that define the quality of the data

thus generated.

9.2 Initial Demonstration of Performance

(Mandatory)

9.2.1 The initial demonstration of performance is used

to characterize instrument performance (instrumental

detection limits, linear dynamic range and MDLs) and

9.2.2 Linear Dynamic Range (LDR) -- The LDR should

be determined by analyzing a minimum of 5 calibration

standards ranging in concentration from 0.2 μg/L to 200

μg chl a/L across all sensitivity settings of the fluorometer.

If using an analog fluorometer or a digital fluorometer

requiring manual changes in sensitivity settings, normalize

responses by dividing the response by the sensitivity

setting multiplier. Perform the linear regression of

normalized response vs. concentration and obtain the

constants m and b, where m is the slope and b is the yintercept. Incrementally analyze standards of higher

concentration until the measured fluorescence response,

R, of a standard no longer yields a calculated

concentration, Cc, that is ± 10% of the known

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

concentration defines the upper limit of the LDR for your

instrument. Should samples be encountered that have a

concentration which is 90% of the upper limit of the LDR,

these samples must be diluted and reanalyzed. 

9.2.3 Instrumental Detection Limit (IDL) -- Zero the

fluorometer with a solution of 90% acetone on the

maximum sensitivity setting. Pure chlorophyll a in 90%

acetone should be serially diluted until it is no longer

detected by the fluorometer on a maximum sensitivity

setting.

9.2.4 Estimated Detection Limit (EDL) -- Several blank

filters should be extracted according to the procedure in

Sect. 11, using clean glassware and apparatus, and the

fluorescence measured. A solution of pure chlorophyll a

in 90% acetone should be serially diluted until it yields a

response which is 3X the average response of the blank

filters. 

9.2.5 Quality Control Sample (QCS) -- When beginning

to use this method, on a quarterly basis or as required to

meet data quality needs, verify the calibration standards

and acceptable instrument performance with the analysis

of a QCS (Sect. 7.10). If the determined value is not

within the confidence limits established by project data

quality objectives, then the determinative step of this

method is unacceptable. The source of the problem

must be identified and corrected before continuing

analyses.

9.2.6 Extraction Proficiency -- Personnel performing

this method for the first time should demonstrate

proficiency in the extraction of sampled filters (Sect. 11.1).
 445.0-7 Revision 1.2 September 1997 

Twenty to thirty natural samples should be obtained using the instrument to warm up for at least 15 min. Measure

the procedure outlined in Sect. 8.1 of this method. Sets the fluorescence of each standard at sensitivity settings

of 10 or more samples should be extracted and analyzed that provide midscale readings. Obtain response factors

according to Sect. 11.2. The percent relative standard for chlorophyll a for each sensitivity setting as follows:

deviation (%RSD) of uncorrected values of chlorophyll a

should not exceed 15% for samples that are F = C /R

approximately 10X the IDL. RSD for pheophytin a might

typically range from 10 to 50%.

 

9.2.7 Corrected Chl a -- Multilaboratory testing of this

method revealed that many analysts do not adequately

mix the acidified sample when determining corrected chl

a. The problem manifests itself by highly erratic pheo-a

results, high %RSDs for corrected chl a and poor

agreement between corrected and uncorrected chl a. To

determine if a new analyst is performing the acidification

step properly, perform the following QC procedure:

Prepare 100 mL of a 50 ppb chl a solution in 90%

acetone. The new analyst should analyze 5-10 separate

aliquots, using separate cuvettes, according to

instructions in Section 11.2. Process the results

according to Section 12 and calculate separate means

and %RSDs for corrected and uncorrected chl a. If the

means differ by more than 10%, then the stock chl a has

probably degraded and fresh stock should be prepared.

The %RSD for corrected chl a should not exceed 5%. If

the %RSD exceeds 5%, repeat the procedure until the

%RSD 5%.

9.3 Assessing Laboratory Performance

(Mandatory)

9.3.1 Laboratory Reagent Blank (LRB) -- The

laboratory must analyze at least one blank filter with each

sample batch. The LRB should be the last filter

extracted. LRB data are used to assess contamination

from the laboratory environment. LRB values that exceed

the IDL indicate contamination from the laboratory

environment. When LRB values constitute 10% or more

of the analyte level determined for a sample, fresh

samples or field duplicates must be analyzed after the

contamination has been corrected and acceptable LRB

values have been obtained.

10.0 Calibration and Standardization

10.1 Calibration -- Calibration should be performed

bimonthly or when there has been an adjustment made

to the instrument, such as replacement of lamp, filters or

photomultiplier. Prepare 0.2, 2, 5, 20 and 200 μg chl a/L

calibration standards from the PDS (Sect. 7.11). Allow

s a s

where:

F = response factor for sensitivity setting, S. s

Rs = fluorometer reading for sensitivity

 setting, S.

C = concentration of chlorophyll a. a

NOTE: If you are using special narrow bandpass filters

for chl a determination, DO NOT acidify. Use the

“uncorrected” chl a calculation described in Section 12.1.

If pheophytin a determinations will be made, it will be

necessary to obtain before-to-after acidification response

ratios of the chlorophyll a calibration standards as follows:

(1) measure the fluorescence of the standard, (2) remove

the cuvette from the fluorometer, (3) acidify the solution

to .003 N HCl with the 0.1 N HCl solution, (4) use a

(6)

pasteur type pipet to thoroughly mix the sample by

aspirating and dispensing the sample into the cuvette,

keeping the pipet tip below the surface of the liquid to

avoid aerating the sample, (5) wait 90 sec and measure

the fluorescence of the standard solution again. Addition

of the acid may be made using a medicine dropper. It will

be necessary to know how many drops are equal to 1 mL

of acid. For a cuvette that holds 5 mL of extraction

solution, it will be necessary to add 0.15 mL of 0.1 N HCl

to reach a final acid concentration of 0.003N in the 5 mL.

Calculate the ratio, r, as follows:

 r = Rb/Ra

where:

Rb = fluorescence of pure chlorophyll a

 standard solution before acidification. 

R = fluorescence of pure chlorophyll a a

 standard solution after acidification.
Revision 1.2 September 1997 445.0-8

11.0 Procedure

11.1 Extraction of Filter Samples

11.1.1 If sampled filters have been frozen, remove them

from the freezer but keep them in the dark. Set up the

tissue grinder and have on hand tissues and squirt bottles

containing water and acetone. Workspace lighting should

be the minimum that is necessary to read instructions and

operate instrumentation. Remove a filter from its

container and place it in the glass grinding tube. The filter

may be torn into smaller pieces to facilitate extraction.

Push it to the bottom of the tube with a glass rod. With a

volumetric pipet, add 4 mL of the aqueous acetone

solution (Sect. 7.6) to the grinding tube. Grind the filter

until it has been converted to a slurry. (NOTE: Although

grinding is required, care must be taken not to overheat

the sample. Good judgement and common sense will

help you in deciding when the sample has been

sufficiently macerated.) Pour the slurry into a 15-mL

screw-cap centrifuge tube and, using a 6-mL volumetric

pipet, rinse the pestle and the grinding tube with 90%

acetone. Add the rinse to the centrifuge tube containing

the filter slurry. Cap the tube and shake it vigorously.

Place it in the dark before proceeding to the next filter

extraction. Before placing another filter in the grinding

tube, use the acetone and water squirt bottles to

thoroughly rinse the pestle, grinding tube and glass rod.

The last rinse should be with acetone. Use a clean tissue

to remove any filter residue that adheres to the pestle or

to the steel rod of the pestle. Proceed to the next filter

and repeat the steps above. The entire extraction with

transferring and rinsing steps takes 5 min. Approximately

500 mL of acetone and water waste are generated per 20

samples from the rinsing of glassware and apparatus.

11.1.2 Shake each tube vigorously before placing them

to steep in the dark at 4 C. Samples should be allowed o

to steep for a minimum of 2 h but not to exceed 24 h.

The tubes should be shaken at least once during the

steeping period.

11.1.3 After steeping is complete, shake the tubes

vigorously and centrifuge samples for 15 min at 675 g or

for 5 min at 1000 g. Samples should be allowed to come

to ambient temperature before analysis. This can be

done by placing the tubes in a constant temperature

water bath or by letting them stand at room temperature

for 30 min. Recalibrate the fluorometer if the room

temperature fluctuated ± 3EC from the last calibration

date. 

11.2 SAMPLE ANALYSIS

11.2.1 After the fluorometer has warmed up for at least

15 min, use the 90% acetone solution to zero the

instrument on the sensitivity setting that will be used for

sample analysis. 

11.2.2 Pour or pipet the supernatant of the extracted

sample into a sample cuvette. The volume of sample

required in your instrument's cuvette should be known so

that the correct amount of acid can be added in the

pheophytin a determinative step. For a cuvette that holds

5 mL of extraction solution, 0.15 mL of the 0.1 N HCl

solution should be used. Choose a sensitivity setting that

yields a midscale reading when possible and avoid the

minimum sensitivity setting. If the concentration of

chlorophyll a in the sample is 90% of the upper limit of

the LDR, then dilute the sample with the 90% acetone

solution and reanalyze. Record the fluorescence

measurement and sensitivity setting used for the sample.

Remove the cuvette from the fluorometer and acidify the

extract to a final concentration of 0.003 N HCl using the

0.1 N HCl solution. Use a pasteur type pipet to

thoroughly mix the sample by aspirating and dispensing

the sample into the cuvette, keeping the pipet tip below

the surface of the liquid to avoid aerating the sample.

Wait 90 sec before measuring fluorescence again.

NOTE: Proper mixing is critical for precise and accurate

results. Twenty-five to thirty-five samples can be

extracted and analyzed in one 8 hr day.

NOTE: If you are using special narrow bandpass filters

for chl a determination, DO NOT acidify samples. Use

the “uncorrected” chl a calculations described in Section

12.1.

12.0 Data Analysis and Calculations

12.1 For “uncorrected chlorophyll a,” calculate the

chlorophyll a concentration in the extract as:

CE,u = Rb x Fs

where CE,u = uncorrected chlorophyll a concentration

 (μg/L) in the extract solution analyzed,

 R = fluoresence response of sample extract b

 before acidification, and 

 Fs = fluoresence respnse factor for sensitivity

 setting S.
CS,u

'

CE,ux extract volume (L) X DF

sample volume (L)

Cs,c '

CE,ux extract volume (L) X DF

sample volume (L)

Ps'

PE X extract volume (L) X DF

sample volume (L)

 445.0-9 Revision 1.2 September 1997 

Calculate the “uncorrected” concentration of chlorophyll extract volume = volume (L) of extract prepared

a in the whole water sample as follows: before dilution,

where CS,u = uncorrected chlorophyll a concentration

 (μg/L) in the whole water sample,

 extract volume = volume (L) of extraction

 prepared before any dilutions,

 DF = dilution factor, sample extract; and 

 sample volume = volume (L) of whole water P = pheophytin a concentration (μg/L) in the

 sample.

12.2 For “corrected chlorophyll a”, calculate the

chlorophyll a concentration in the extract as :

CE,c = F (r/r-1) (R - R ) s b a

where:

CE,c = corrected chlorophyll a concentration (μg/L) in the

extract solution analyzed,

F = response factor for the sensitivity setting S, s

r = the before-to-after acidification ratio of a

 pure chlorophyll a solution (Sect. 10.1),

Rb = fluorescence of sample extract before

 acidification, and

R = fluorescence of sample extract after a

 acidification.

Calculate the “corrected” concentration of chlorophyll a

in the whole water sample as follows:

where CS,c = corrected chlorophyll a concetration (μg/L)

 in the whole water sample,

12.3 Calculate the pheophytin a concentration as

follows:

 PE = F (r/r-1) (rR - R ) s a b

where PE = pheophytin a concentration (μg/L) in the

s 

 whole water sample.

12.4 LRB and QCS data should be reported with each

sample data set.

13.0 Method Performance

13.1 The single lab EDL for the instrument used in the

evaluation of this method was 0.05 μg/L for chlorophyll a

and 0.06 μg/L pheophytin a.

13.2 The precision (%RSD) for chlorophyll a in mostly

blue-green and green phytoplankton natural samples

which were steeped for 2 h vs 24 h is reported in Table 1.

Although the means were the same, precision was better

for samples which were allowed to steep for 24 h prior to

analysis. Since pheophytin a was found in the samples,

the chlorophyll a values are "corrected" (Sect. 12.2).

Table 2 contains precision data for pheophytin a. A

statistical analysis of the pheophytin a data indicated a

significant difference in the mean values at the 0.05

significance level. The cause of the lower pheophytin a

values in samples extracted for 24 h is not known.

13.3 Three QCS ampoules obtained from the USEPA

were analyzed and compared to the reported confidence

limits in Table 3. NOTE: The USEPA no longer provides

these QCSs.

13.4 Multilaboratory Testing - A multilaboratory

validation and comparison study of EPA Methods 445.0,

446.0 and 447.0 for chlorophyll a was conducted in 1996

by Research Triangle Institute, Research Triangle Park,

N.C. (EPA Contract No. 68-C5-0011). There were 21

volunteer participants in the fluorometric methods
Revision 1.2 September 1997 445.0-10

component that returned data; 10 that used the modified HPLC results for the highest concentration algae sample

fluorometric method and 11 that used the conventional since chlorophyll a is separated from other interfering

method. The primary goals of the study were to

determine estimated detection limits and to assess

precision (%RSD) and bias (as percent recovery) for

select unialgal species, and natural seawater.

13.4.1 The term, pooled estimated detection limit (pEDL), is used in this method to distinguish it from the EPA

defined method detection limit (MDL). An EPA MDL

determination is not possible nor practical for a natural

water or pure species sample due to known spectral

interferences and to the fact that it is impossible to

prepare solutions of known concentrations that

incorporate all sources of error (sample collection,

filtration, processing). The statistical approach used to

determine the p-EDL was an adaptation of the Clayton,

et.al. method that does not assume constant error

(22)

variances across concentration and controls for Type II

error. The statistical approach used involved calculating

an estimated DL for each lab that had the desired Type I

and Type II error rates (0.01 and 0.05, respectively). The

median DLs over labs was then determined and is

reported in Table 4. It is referred to as pooled-EDL (pEDL).

Solutions of pure chlorophyll a in 90% acetone were

prepared at three concentrations (0.11, 0.2 and 1.6 ppm) definitive explanation can be offered at this time for this

and shipped with blank glass fiber filters to participating phenomenon. A possible explanation for the

laboratories. Analysts were instructed to spike the filters Phaeodactylum is that it contained significant amounts of

in duplicate with a given volume of solution and to chlorophyllide a which is determined as chlorophyll a in

process the spiked filters according to the method. The

results from these data were used to determine a p-EDL

for each method. Results (in ppm) are given in Table 4.

The standard fluorometric and HPLC methods gave the

lowest p-EDLs while the spectrophotometric

(monochromatic equations) gave the highest p-EDLs.

Due to the large dilutions required to analyze these

solutions, the fluorometric p-EDLs are unrealistically high

compared to what is achievable by a single lab. Typical

single lab EDLs can easily be 1000 fold lower than the pEDL reported in Table 4.

13.4.2 To address precision and bias in chlorophyll a

determination for different algal species, three pure

unialgal cultures (Amphidinium, Dunaliella and

Phaeodactylum) were cultured and grown in the

laboratory. Four different “concentrations” of each

species were prepared by filtering varying volumes of the

algae. The filters were frozen and shipped to participant

labs. Analysts were instructed to extract and analyze the

filters according to the respective methods. The “true”

concentration was assigned by taking the average of the

pigments prior to determination. Pooled precision (as

determined by %RSD) data are presented in Tables 5-7

and accuracy data (as percent recovery) are presented in

Table 8. No significant differences in precision were

observed across concentrations for any of the species. It

should be noted that there was considerable lab-to-lab

variation (as exhibited by the min and max recoveries in

Table 8) and in this case the median is a better measure

of central tendency than the mean. 

In summary, the mean and median concentrations

determined for Amphidinium carterae (class

dinophyceae) are similar for all methods. No method

consistently exhibited high or low values relative to the

other methods. The only concentration trend observed

was that the spectrophotometric method-trichromatic

equations (SP-T) showed a slight percent increase in

recovery with increasing algae filtration volume.

For Dunaliella tertiolecti (class chlorophyceae) and

Phaeodactylum tricornutum (class bacillariophyceae)

there was generally good agreement between the

fluorometric and the spectrophotometric methods,

however, the HPLC method yielded lower recoveries with

increasing algae filtration volume for both species. No

the fluorometric and spectrophotometric methods. The

conventional fluorometric method (FL-STD) showed a

slight decrease in chlorophyll a recovery with increasing

Dunaliella filtration volume. The spectrophotometrictrichromatic equations (SP-T) showed a slight increase in

chlorophyll a recovery with increasing Dunaliella filtration

volume. The fluorometric and the spectrophotometric

methods both showed a slight decrease in chlorophyll a

recovery with increasing Phaeodactylum filtration volume.

Results for the natural seawater sample are presented in

Table 9. Only one filtration volume (100 mL) was

provided in duplicate to participant labs.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

EPA has established a preferred hierarchy of
 445.0-11 Revision 1.2 September 1997 

environmental management techniques that places 4. Trees, C.C., M.C. Kennicutt, and J.M. Brooks,

pollution prevention as the management option of first "Errors associated with the standard fluorometric

choice. Whenever feasible, laboratory personnel should determination of chlorophylls and

use pollution prevention techniques to address their waste phaeopigments", Mar. Chem., 17 (1985) pp. 1-

generation (e.g., Sect. 11.1.1). When wastes cannot be 12.

feasibly reduced at the source, the Agency recommends

recycling as the next best option. 5. Method 445, “Multi-Laboratory Comparison and

14.2 For information about pollution prevention that USEPA Contract 68-C5-0011, WA1-03, August

may be applicable to laboratories and research 1997.

institutions, consult Less is Better: Laboratory Chemical

Management for Waste Reduction, available from the

American Chemical Society's Department of Government

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

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

15.0 Waste Management

15.1 The Environmental Protection Agency requires

that laboratory waste management practices be

conducted consistent with all applicable rules and

regulations. The Agency urges laboratories to protect the

air, water, and land by minimizing and controlling all

releases from hoods and bench operations, complying

with the letter and spirit of any sewer discharge permits

and regulations, and by complying with all solid and

hazardous waste regulations, particularly the hazardous

waste identification rules and land disposal restrictions.

For further information on waste management consult

The Waste Management Manual for Laboratory

Personnel, available from the American Chemical Society

at the address listed in the Sect. 14.2.

16.0 References

1. Yentsch, C.S. and D.W. Menzel, "A method for

the determination of phytoplankton chlorophyll

and pheophytin by fluorescence", Deep Sea

Res., 10 (1963), pp. 221-231.

2. Strickland, J.D.H. and T.R. Parsons, A Practical

Handbook of Seawater Analysis, Bull. Fish. Res.

Board Can., 1972, No.167, p. 201.

3. Arar, E., "Evaluation of a new technique that uses

highly selective interference filters for measuring

chlorophyll a in the presence of chlorophyll b and

pheopigments," USEPA Summary Report, 1994,

NTIS No. PB94-210622.

Validation of Chlorophyll Methods,” Final Report,

6. Holm-Hansen, O., “Chlorophyll a determination:

improvements in methodology”, OKIOS, 30

(1978), pp. 438-447.

7. Wright, S.W. and J.D. Shearer, "Rapid extraction

and HPLC of chlorophylls and carotenoids from

marine phytoplankton", J. Chrom., 294 (1984),

pp. 281-295.

8. Bowles, N.D., H.W. Paerl, and J. Tucker,

"Effective solvents and extraction periods

employed in phytoplankton carotenoid and

chlorophyll determination", Can. J. Fish. Aquat.

Sci., 42 (1985) pp. 1127-1131.

9. Shoaf, W.T. and B.W. Lium, "Improved extraction

of chlorophyll a and b from algae using dimethyl

sulfoxide", Limnol. and Oceanogr., 21(6) (1976)

pp. 926-928.

10. Loftis, M.E. and J.H. Carpenter, "A fluorometric

method for determining chlorophylls a, b, and c, "

1

J. Mar. Res., 29 (1971) pp.319-338.

11. Standard Methods for the Analysis of Water and

Wastes, 17th Ed., 1989, 10200H, Chlorophyll.

12. Wright, S.W., S.W. Jeffrey, R.F.C. Manntoura,

C.A. Llewellyn, T. Bjornland, D. Repeta, and N.

Welschmeyer, "Improved HPLC method for the

analysis of chlorophylls and carotenoids from

marine phytoplankton", paper submitted for

publication in 1991.

13. Mantoura, R.F.C. and C.A. Llewellyn, "The rapid

determination of algal chlorophyll and carotenoid

pigments and their breakdown products in

natural waters by reverse-phase high

performance liquid chromatography", Anal.

Chim. Acta., 151 (1983) pp. 297-314.
Revision 1.2 September 1997 445.0-12

14. Brown, L.M., B.T. Hargrave, and M.D. 20. "Proposed OSHA Safety and Health Standards,

MacKinnon, "Analysis of chlorophyll a in Laboratories", Occupational Safety and Health

sediments by high-pressure liquid

chromatography", Can. J. Fish. Aquat. Sci., 38

(1981) pp. 205-214.

15. Bidigare, R.R., M.C. Kennicutt, II, and J.M.

Brooks, "Rapid determination of chlorophylls and

their degradation products by HPLC", Limnol.

Oceanogr., 30(2) (1985) pp. 432-435.

16. Minguez-Mosquera, M.I., B. Gandul-Rojas, A.

Montano-Asquerino, and J. Garrido-Fernandez,

"Determination of chlorophylls and carotenoids

by HPLC during olive lactic fermentation", J.

Chrom., 585 (1991) pp. 259-266.

17. Carcinogens - Working With Carcinogens,

Department of Health, Education and Welfare,

Public Health Service, Center for Disease

Control, National Institute for Occupational Safety

and Health, Publication No. 77-206, 1977.

18. "OSHA Safety and Health Standards, General

Industry", (29 CFR 1910), Occupational Safety

and Health Administration, OSHA 2206, revised

January 1976.

19. Safety in Academic Chemistry Laboratories,

American Chemical Society publication,

Committee on Chemical Safety, 3rd Edition,

1979.

Administration, Federal Register, July 24, 1986.

21. Weber, C.I., L.A. Fay, G.B. Collins, D.E. Rathke,

and J. Tobin, "A Review of Methods for the

Analysis of Chlorophyll in Periphyton and

Plankton of Marine and Freshwater Systems",

work funded by the Ohio Sea Grant Program,

Ohio State University. Grant No.NA84AA-D00079, 1986, 54 pp.

22. Clayton, C.A., J.W. Hines and P.D. Elkins,

“Detection limits within specified assurance

probabilities,” Analytical Chemistry, 59 (1987),

pp. 2506-2514. 
 445.0-13 Revision 1.2 September 1997 

17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA

TABLE 1. COMPARISON OF PRECISION OF TWO EXTRACTION PERIODS

CORRECTED CHLOROPHYLL a

 Sample A Sample B (1) (2)

2 h 24 h 2 h 24 h (3) (3) (3) (3)

Mean Concentration (μg/L) 49.6 52.9 78.6 78.8

Standard Deviation (μg/L) 4.89 2.64 6.21 2.77 

Relative Standard Deviation (%) 9.9 5.0 7.9 3.5

Values reported are the mean measured concentrations (n=6) of chlorophyll a in the natural water based 1

on a 100-mL filtration volume. 

Values reported are the mean measured concentrations (n=9) of the extraction solution. Sample filtration 2

volume was 300 mL. 

The length of time that the filters steeped after they were macerated. 3
Revision 1.2 September 1997 445.0-14

TABLE 2. COMPARISON OF PRECISION OF TWO EXTRACTIONS PERIODS FOR Pheophytin a

Pheophytin a

 Sample A Sample B (1) (2)

2 h 24 h 2 h 24 h (3) (3) (3) (3)

Mean Concentration (μg/L) 9.22 8.19 13.1 10.61

Standard Deviation (μg/L) 2.36 3.55 3.86 2.29

Relative Standard Deviation (%) 25.6 43.2 29.5 21.6

Values reported are the mean measured concentrations (n=6) of pheophytin a in the natural water based 1

on a 100-mL filtration volume. 

Values reported are the mean measured concentrations (n=9) of pheophytin a the extraction solution.

2

Sample filtration volume was 300 mL. 

The length of time that the filters steeped after they were macerated. 3
 445.0-15 Revision 1.2 September 1997 

TABLE 3. ANALYSES OF USEPA QC SAMPLES

ANALYTE REFERENCE VALUE CONFIDENCE LIMITS

Chlorophyll a 2.1 μg/L 0.5 to 3.7 μg/L

Pheophytin a 0.3 μg/L -0.2 to 0.8 μg/L

 

MEAN % Relative Standard 

1

ANALYTE MEASURED VALUE Deviation

Chlorophyll a 2.8 μg/L 1.5

Pheophytin a 0.3 μg/L 33

 

 N = 3 1
Revision 1.2 September 1997 445.0-16

TABLE 4. POOLED ESTIMATED DETECTION LIMITS FOR CHLOROPHYLL A METHODS(1)

_____________________________________________________________

Method N p-EDL (mg/L) (2) (3) (4)

FL -Mod 8 0.096 (5)

FL - Std 9 0.082 (5)

HPLC 4 0.081

SP-M 15 0.229

SP-T 15 0.104

_____________________________________________________________

(1) See Section 13.4.1 for a description of the statistical approach used to determine p-EDLs. 

(2) FL-Mod = fluorometric method using special interference filters.

 FL-Std = conventional fluorometric method with pheophytin a correction.

 HPLC = EPA method 447.0

 SP-M = EPA method 446.0, monochromatic equation.

 SP-T = EPA method 446.0, trichromatic equations.

(3) N = number of labs whose data was used.

(4) The p-EDL was determined with p = 0.01 and q (type II error rate) = 0.05.

(5) Due to the large dilutions required to analyze the solutions by fluorometry, the fluormetric p-EDLs are

 unrealistically high.
 445.0-17 Revision 1.2 September 1997 

TABLE 5. POOLED PRECISION FOR DUNALIELLA TERTIOLECTI SAMPLES

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

Fl-Mod 5 7 0.163 0.037 22.8

 10 7 0.298 0.080 26.7

 50 7 1.684 0.385 22.9

100 7 3.311 0.656 19.8

Fl-Std 5 8 0.185 0.056 30.4

 10 8 0.341 0.083 24.4

 50 8 1.560 0.311 19.9

100 8 3.171 0.662 20.9

___________________________________________________________________________________

(1) Fl-Mod = fluorometric method using special interference filters.

 Fl-Std = conventional fluorometric method with pheophytin a correction.

(2) N = number of volunteer labs whose data was used.
Revision 1.2 September 1997 445.0-18

TABLE 6. POOLED PRECISION FOR AMPHIDINIUM CARTERAE SAMPLES

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

Fl-Mod 5 7 0.066 0.010 14.6

 10 7 0.142 0.045 31.5

 50 7 0.757 0.208 27.5

100 7 1.381 0.347 25.1

Fl-Std 5 8 0.076 0.018 23.2

 10 8 0.165 0.040 24.3

 50 8 0.796 0.140 17.5

100 8 1.508 0.324 21.5

___________________________________________________________________________________

(1) Fl-Mod = fluorometric method using special interference filters.

 Fl-Std = conventional fluorometric method with pheophytin a correction.

(2) N = number of volunteer labs whose data was used.
 445.0-19 Revision 1.2 September 1997 

TABLE 7. POOLED PRECISION FOR PHAEODACTYLUM TRICORNUTUM SAMPLES

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

Fl-Mod 5 7 0.221 0.040 18.0

 10 7 0.462 0.094 20.3

 50 7 2.108 0.491 23.3

100 7 3.568 1.186 33.2

Fl-Std 5 8 0.214 0.053 24.8

 10 8 0.493 0.091 18.4

 50 8 2.251 0.635 28.2

100 8 4.173 0.929 22.3

___________________________________________________________________________________

(1) Fl-Mod = fluorometric method using special interference filters.

 Fl-Std = conventional fluorometric method with pheophytin a correction.

(2) N = number of volunteer labs whose data was used.

NOTE: The phaeodactylum extract contained significant amounts of chlorophyll c and chlorophyllide a which

interferes in chlorophyll a measurement in the fluorometric method, therefore, the concentration of chlorophyll a is

overestimated compared to the HPLC method which separates the three pigments. The FL-Mod interference filters

minimize this interference more so than the conventional filters.
Revision 1.2 September 1997 445.0-20

TABLE 8. MINIMUM, MEDIAN, AND MAXIMUM PERCENT RECOVERIES BY GENERA, METHOD, AND 

 CONCENTRATION LEVEL

Species Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 Level 4

Amphidinium Minimum FL-MOD 70 73 75 76

FL-STD 66 91 91 90

HPLC 82 85 87 88

SP-M 36 48 68 64

SP-T 21 63 71 70

Median FL-MOD 105 112 105 104

FL-STD 109 107 111 109

HPLC 102 106 112 105

SP-M 99 101 101 101

SP-T 95 96 106 107

Maximum FL-MOD 121 126 143 146

FL-STD 156 154 148 148

HPLC 284 210 131 116

SP-M 141 133 126 125

SP-T 115 116 119 117

Dunaliella Minimum FL-MOD 162 159 157 156

FL-STD 179 171 165 164

HPLC 165 109 64 41

SP-M 120 188 167 164

SP-T 167 169 166 165

Median FL-MOD 206 246 227 223

FL-STD 250 228 224 210

HPLC 252 177 89 80
Table 8 cont’d

 445.0-21 Revision 1.2 September 1997 

Species Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 Level 4

SP-M 240 247 247 243

SP-T 225 244 256 256

Dunaliella Maximum FL-MOD 295 277 287 288

FL-STD 439 385 276 261

HPLC 392 273 172 154

SP-M 342 316 296 293

SP-T 291 283 283 283

Phaeodactylum Minimum FL-MOD 216 183 157 154

FL-STD 189 220 223 219

HPLC 150 119 84 75

SP-M 161 138 156 160

SP-T 203 195 216 244

Median FL-MOD 292 285 250 245

FL-STD 296 263 254 254

HPLC 225 203 114 90

SP-M 287 274 254 253

SP-T 286 281 277 274

Maximum FL-MOD 357 337 320 318

FL-STD 371 415 415 334

HPLC 394 289 182 139

SP-M 446 344 330 328

SP-T 357 316 318 299
Revision 1.2 September 1997 445.0-22

TABLE 9. CHLOROPHYLL A CONCENTRATIONS IN MG/L DETERMINED IN FILTERED SEAWATER 

 SAMPLES

Method Con. No. Obs. No. Labs Mean Std. Dev. RSD(%) Minimum Median Maxium (1)

FL-MOD 100 14 7 1.418 0.425 30.0 0.675 1.455 2.060

FL-STD 100 15 8 1.576 0.237 15.0 1.151 1.541 1.977

HPLC 100 10 5 1.384 0.213 15.4 1.080 1.410 1.680

SP-M 100 38 19 1.499 0.219 14.6 0.945 1.533 1.922

SP-T 100 36 18 1.636 0.160 9.8 1.250 1.650 1.948

All Methods 100 113 57 1.533 0.251 16.4 0.657 1.579 2.060

(1) Con = mLs of seawater filtered.
446.0-1

Method 446.0

In Vitro Determination of Chlorophylls a, b, c1 + c2 and Pheopigments in

Marine And Freshwater Algae by Visible Spectrophotometry

Adapted by

Elizabeth J. Arar

Revision 1.2

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Revision 1.2 September 1997 446.0-2

Method 446.0

In Vitro Determination of Chlorophylls a, b, c1 + c2 and Pheopigments in

Marine and Freshwater Algae by Visible Spectrophotometry

1.0 Scope and Application

1.1 This method provides a procedure for

determination of chlorophylls a (chl a), b (chl b), c1 + c2

(chl c + c ) and pheopigments of chlorophyll a (pheo a) 1 2

found in marine and freshwater phytoplankton.

Chlorophyllide a is determined as chl a. Visible

wavelength spectrophotometry is used to measure the

pigments in sub-parts per million (ppm) concentrations.

The trichromatic equations of Jeffrey and Humphrey are (1)

used to calculate the concentrations of chl a, chl b, and

chl c +c . Modified monochromatic equations of 1 2

Lorenzen are used to calculate pheopigment-corrected (2)

chl a and pheo a. 

1.2 This method differs from previous descriptions of

the spectrophotometric technique in several important

aspects. Quality assurance/quality control measures are

described in Sect. 9.0. Detailed sample collection and

extraction procedures are described in Sect. 8.0, and

most importantly, interference data, heretofore only

presented in research journals, is included so the analyst

may know the potential limitations of the method.

Multilaboratory data is included in Section 13.

 

 Chemical Abstracts Service

Analyte Registry Number (CASRN)

 

Chlorophyll a 479-61-8

Chlorophyll b 519-62-0

Chlorophyll c1 18901-56-9

Chlorophyll c2 27736-03-4

 

1.3 Instrumental detection limits (IDLs) of 0.08 mg

chl a/L, 0.093 mg chl b/L and 0.085 mg pheo a/L in pure

solutions of 90% acetone were determined by this

laboratory using a 1-cm glass cell. Lower detection limits

can be obtained using 2, 5 or 10-cm cells. An IDL for

chlorophylls c1+c2 was not determined due to

commercial unavailability of the pure pigments.

Estimated detection limit (EDL) determinations were

made by analyzing seven replicate filtered phytoplankton

samples containing the pigments of interest. Singlelaboratory EDLs (S-EDL) were as follows: chl a - 0.037

mg/L, chl b - 0.07 mg/L, chl c + c - 0.087 mg/L, 1 2

pheopigment-corrected chl a - 0.053 mg/L, and pheo a -

0.076 mg/L. The trichromatic equations lead to

inaccuracy in the measurement of chlorophylls b and

c1+c2 at chl a concentrations greater than ~5X the

concentration of the accessory pigment or in the

presence of pheo a. The upper limit of the linear dynamic

range (LDR) for the instrumentation used in this method

evaluation was approximately 2.0 absorbance units (AU)

which corresponded to pigment concentrations of 27 mg

chl a/L, 30 mg chl b/L and approximately 45 mg pheo a/L.

No LDR for chl c1 + c2 was determined. It is highly

unlikely that samples containing chl c +c at 1 2

concentrations approaching the upper limit of the LDR will

be encountered in nature.

1.4 Chl c +c is not commercially available, 1 2 

therefore, the minimum indicator of laboratory

performance for this pigment is precision of chl c1 + c2

determinations in natural samples known to contain the

pigments.

1.5 This method uses 90% acetone as the extraction

solvent because of its efficiency for extracting chl a from

most types of algae. (NOTE: There is evidence that

certain chlorophylls and carotenoids are more thoroughly

extracted with methanol or dimethyl sulfoxide. Using (3-5) (6)

high performance liquid chromatography (HPLC),

Mantoura and Llewellyn found that methanol led to the (7)

formation of chl a derivative products, whereas 90%

acetone did not. Bowles, et al.(5) found that for chl a 90%

acetone was an effective solvent when the steeping

period was optimized for the predominant species

present.) 

1.6 One of the limitations of absorbance

spectrophotometry is low sensitivity. It may be preferable
 446.0-3 Revision 1.2 September 1997

to use a fluorometric or HPLC method if high selected wavelength, mass, retention time, absorbance

(8-10) (11-15)

volumes of water (>4 L) must be filtered to obtain line, etc. In this method the instrument is zeroed on a

detectable quantities of chl a. The user should be aware background of 90% acetone resulting in no signal at the

of the inaccuracies of fluorometric methods when chl b is

also present in the sample.

1.7 This method is for use by analysts experienced in

handling photosynthetic pigments and in the operation of

visible wavelength spectrophotometers or by analysts

under the close supervision of such qualified persons.

2.0 Summary of Method

2.1 Chlorophyll-containing phytoplankton in a

measured volume of sample water are concentrated by

filtration at low vacuum through a glass fiber filter. The

pigments are extracted from the phytoplankton in 90%

acetone with the aid of a mechanical tissue grinder and

allowed to steep for a minimum of 2 h, but not exceeding

24 h, to ensure thorough extraction of the pigments. The

filter slurry is centrifuged at 675 g for 15 min (or at 1000

g for 5 min) to clarify the solution. An aliquot of the

supernatant is transferred to a glass cell and absorbance

is measured at four wavelengths (750, 664, 647 and 630

nm) to determine turbidity, chlorophylls a, b, and c + c , 1 2

respectively. If pheopigment-corrected chl a is desired,

the sample's absorbance is measured at 750 and 664 nm

before acidification and at 750 and 665 nm after

acidification with 0.1 N HCl. Absorbance values are

entered into a set of equations that utilize the extinction

coefficients of the pure pigments in 90% acetone to

simultaneously calculate the concentrations of the

pigments in a mixed pigment solution. No calibration of

the instrument with standard solutions is required.

Concentrations are reported in mg/L (ppm).

3.0 Definitions

3.1 Field Replicates -- Separate samples collected

at the same time and place under identical circumstances

and treated exactly the same throughout field and

laboratory procedures. Analyses of field replicates give

a measure of the precision associated with sample

collection, preservation and storage, as well as with

laboratory procedures.

3.2 Instrument Detection Limit (IDL) -- The

minimum quantity of analyte or the concentration

equivalent that gives an analyte signal equal to three

times the standard deviation of a background signal at the

measured wavelengths. The IDL is determined instead

by serially diluting a solution of known pigment

concentration until the signal at the selected wavelength

is between .005 and .008 AU.

3.3 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water or other blank matrices that are treated

exactly as a sample including exposure to all glassware,

equipment, solvents, reagents, internal standards, and

surrogates that are used with other samples. The LRB is

used to determine if method analytes or other

interferences are present in the laboratory environment,

reagents, or apparatus. For this method the LRB is a

blank filter that has been extracted as a sample.

3.4 Linear Dynamic Range (LDR) -- The absolute

quantity or concentration range over which the instrument

response to an analyte is linear.

3.5 Material Safety Data Sheet (MSDS) -- Written

information provided by vendors concerning a chemical's

toxicity, health hazards, physical properties, fire, and

reactivity data including storage, spill, and handling

precautions.

3.6 Estimated Detection Limit (EDL) -- The EDL

is determined in a manner similar to an EPA MDL. It is

not called an MDL in this method because there are

known spectral interferences inherent to this method that

make 99% confidence that the chlorophyll concentration

is greater than zero impossible.

3.7 Quality Control Sample (QCS) -- A solution of

method analytes of known concentrations that is used to

fortify an aliquot of LRB or sample matrix. Ideally, the

QCS is obtained from a source external to the laboratory

and different from the source of calibration standards. It

is used to check laboratory performance with externally

prepared test materials. The USEPA no longer provides

QCSs for this method.

4.0 Interferences

4.1 Any compound extracted from the filter or

acquired from laboratory contamination that absorbs light

between 630 and 665 nm may interfere in the accurate

measurement of the method analytes. An absorbance

measurement is made at 750 nm to assess turbidity in the
Revision 1.2 September 1997 446.0-4

sample. This value is subtracted from the sample's step required to determine pheopigment-corrected chl a

absorbance at 665, 664, 647 and 630 nm. A 750 nm

absorbance value that is > .005 AU indicates a poorly

clarified solution. This is usually remedied by further

centrifugation or filtration of the sample prior to analysis.

4.2 The relative amounts of chlorophyll a, b and c1 +

c2 vary with the taxonomic composition of the

phytoplankton. Due to the spectral overlap of the

chlorophylls and pheo a, over- or underestimation of the

pigments is inevitable in solutions containing all of these

pigments.

Chl a is overestimated by the trichromatic equation of

Jeffrey and Humphrey when pheo a is present (Figure 1).

Lorenzen's modified monochromatic equation only

slightly overestimates chl a in the presence of chl b

(Figure 2). The degree of error in the measurement of extraction solvent (Table 1). Precision improves with

any pigment is directly related to the concentration of the increasing steeping periods. A drawback to prolonged

interfering pigment. Knowledge of the taxonomic steeping periods, however, is the extraction of interfering

composition of the sample, proper storage and good pigments. For example, if the primary pigment of interest

sample handling technique (to prevent degradation of chl is chl a, extended steeping periods may extract more of

a) can aid in determining whether to report trichromatic or

pheopigment-corrected chl a. If no such knowledge

exists, it is advisable to obtain values for all of the significant factor in the recovery of chl b and pheo a from

pigments and to compare the chl a results in light of the

apparent concentrations of the other pigments. detectable quantities, but not a significant factor in the

Obviously, if the chl a values vary widely, sound recovery of chl a. Chl b and pheo a are mutual

judgement must be used in deciding which pigments, chl interferents so that an actual increase in the recovery of

b and chl c + c , or pheo a, are in greatest abundance chl b leads to a slight apparent increase in pheo a. 1 2

relative to each other and to chl a. The method of

standard additions, explained in most analytical chemistry

textbooks, is recommended when greater accuracy is

required. 

Accuracy of chl b measurements is highly dependent

upon the concentration of chl a and pheo a. In pure

(16)

solutions of chl a and b, underestimation of chl b is

observed with increasing concentrations of chl a (Figure

3). Using the method of standard additions, the same

phenomenon was confirmed to occur in natural samples.

The underestimation of chl b is due in part to the spectral

component of chl a that is subtracted from chl b as chl c1

+ c2in the trichromatic equation. Chl a concentrations

that range from 4 to 10 times the concentration of chl b

lead to 13% to 38% underestimation of chl b. The highest

chl b:chl a ratio likely to occur in nature is 1:1.

Pheo a:chl a ratios rarely exceed 1:1. Pheo a is

overestimated in the presence of certain carotenoids(16)

and when chl b is converted to pheo b in the acidification

and pheo a. The rate of conversion of chl b to pheo b,

however, is slower than that of chl a to pheo a. It is

important, therefore, to allow the minimum time required

for conversion of chl a to pheo a before measuring

absorbance at 665 nm. Ninety seconds is recommended

by this method.

When a phytoplankton sample's composition is known

(i.e., green algae, diatoms, dinoflagellates) Jeffrey and

Humphrey's dichromatic equations for chl a, b, and c1 +

c2 are more accurate than the trichromatic equations

used here. 

(1)

4.3 Precision and recovery for any of the pigments is

related to efficient maceration of the filtered sample and

to the steeping period of the macerated filter in the

the other pigments but not necessarily more chl a.

Statistical analysis revealed steeping period to be a

a mixed assemblage containing these pigments in

4.4 Sample extracts must be clarified by

centrifugation prior to analysis.

4.5 All photosynthetic pigments are light and

temperature sensitive. Work must be performed in

subdued light and all standards, QC materials, and

filtered samples must be stored in the dark at -20 or

 -70 C to prevent rapid degradation. o

5.0 Safety

5.1 Each chemical used in this method should be

regarded as a potential health hazard and handled with

caution and respect. Each laboratory is responsible for

maintaining a current awareness file of Occupational

Safety and Health Administration (OSHA) regulations

regarding the safe handling of the chemicals specified in

this method. A file of MSDS also should be made

(17-20)

available to all personnel involved in the chemical

analysis.
 446.0-5 Revision 1.2 September 1997

5.2 The grinding of filters during the extraction step of 6.10.5 Disposable Pasteur type pipets or medicine

this method should be conducted in a fume hood due to droppers.

the volatilization of acetone by the tissue grinder.

6.0 Apparatus and Equipment

6.1 Spectrophotometer -- Visible, multiwavelength,

with a bandpass (resolution) not to exceed 2 nm. 

6.2 Centrifuge, capable of 675 g.

6.3 Tissue grinder, Teflon pestle (50 mm X 20 mm)

with grooves in the tip with 1/4" stainless steel rod long

enough to chuck onto a suitable drive motor and 30-mL

capacity round-bottomed, glass grinding tube.

6.4 Filters, glass fiber, 47-mm, or 25-mm, nominal

pore size of 0.7 μm unless otherwise justified by data

quality objectives. Whatman GF/F filters were used in 7.1 Acetone, HPLC grade, (CASRN 67-64-1).

this work.

6.5 Petri dishes, plastic, 50 X 9-mm, or some other 1.19), (CASRN 7647-01-0).

solid container for transporting and storing sampled

filters. 7.3 Chl a free of chl b and chl b substantially free of

6.6 Aluminum foil.

6.7 Laboratory tissues.

6.8 Tweezers or flat-tipped forceps.

6.9 Vacuum pump or source capable of maintaining

a vacuum up to 6 in. Hg (20 KPa).

6.10 Labware -- All reusable labware (glass,

polyethylene, Teflon, etc.) that comes in contact with

chlorophyll solutions should be clean and acid free. An

acceptable cleaning procedure is soaking for 4 h in

laboratory grade detergent and water, rinsing with tap

water, distilled deionized water and acetone.

6.10.1 Assorted Class A calibrated pipets.

6.10.2 Graduated cylinders, 500-mL and 1-L.

6.10.3 Volumetric flasks, Class A calibrated, 25-mL, 50-

mL, 100-mL and 1-L capacity.

6.10.4 Glass rods.

6.10.6 Glass cells for the spectrophotometer, 1, 2, 5 or

10 cms in length. If using multiple cells, they must be

matched.

6.10.7 Filtration apparatus consisting of 1 or 2-L filtration

flask, 47-mm fritted glass disk base and a glass filter

tower.

6.10.8 Centrifuge tubes, polypropylene or glass, 15-mL

capacity with nonpigmented screw-caps.

6.10.9 Polyethylene squirt bottles.

7.0 Reagents and Standards

7.2 Hydrochloric acid (HCl), concentrated (sp. gr.

chl a may be obtained from a commercial supplier such

as Sigma Chemical (St. Louis, MO). 

7.4 Water -- ASTM Type I water (ASTM D1193) is

required. Suitable water may be obtained by passing

distilled water through a mixed bed of anion and cation

exchange resins.

7.5 0.1 N HCl Solution -- Add 8.5 mL of

concentrated HCl to approximately 500 mL water and

dilute to 1 L.

7.6 Aqueous Acetone Solution -- 90% acetone/10%

ASTM Type I water. Carefully measure 100 mL of the

water into the 1-L graduated cylinder. Transfer to a 1-L

flask or storage bottle. Measure 900 mL of acetone into

the graduated cylinder and transfer to the flask or bottle

containing the water. Mix, label and store.

7.7 Chlorophyll Stock Standard Solution (SSS) --

Chl a (MW = 893.5) and chl b (MW = 907.5) from a

commercial supplier is shipped in amber glass ampules

that have been flame sealed. The dry standards must be

stored at -20EC in the dark. Tap the ampule until all the

dried pigment is in the bottom of the ampule. In subdued

light, carefully break the tip off the ampule. Transfer the

entire contents of the ampule into a 25-mL volumetric
Revision 1.2 September 1997 446.0-6

flask. Dilute to volume with 90% acetone, label the flask Prior to drawing a subsample from the water sample

and wrap with aluminum foil to protect from light. Pheo a container, thoroughly but gently agitate the container to

may be prepared by the mild acidification of chl a (to .003

N HCl) followed by a 1:1 molar neutralization with a base

such as dilute sodium hydroxide solution. When stored in

a light- and air-tight container at -20 C, the SSS is stable o

for at least six months. All dilutions of the SSS must be

determined spectrophotometrically using the equations in

Sect. 12. 

7.8 Laboratory Reagent Blank (LRB) -- A blank

filter that is extracted and analyzed just as a sample filter.

The LRB should be the last filter extracted of a sample

set. It is used to assess possible contamination of the

reagents or apparatus.

7.9 Quality Control Sample (QCS) -- Since there

are no commercially available QCSs, dilutions of a stock

standard may be used.

8.0 Sample Collection, Preservation and

Storage

8.1 Water Sample Collection -- Water may be

obtained by a pump or grab sampler. Data quality

objectives will determine the depth and frequency at

(21)

which samples are taken. Healthy phytoplankton,

however, are generally obtained from the photic zone

(depth at which the illumination level is 1% of surface

illumination). Enough water should be collected to

concentrate phytoplankton on at least three filters.

Filtration volume size will depend on the particulate load

of the water. Four liters may be required for open ocean

water where phytoplankton density is usually low,

whereas 1 L or less is generally sufficient for lake, bay or

estuary water. All apparatus should be clean and acidfree. Filtering should be performed in subdued light as

soon as possible after sampling since algal populations,

thus chlorophyll a concentration, can change in a

relatively short period of time. Aboard ship filtration is

highly recommended.

Assemble the filtration apparatus and attach the vacuum

source with vacuum gauge and regulator. Vacuum

filtration should not exceed 6 in. Hg (20 kPa). Higher

filtration pressures or excessively long filtration times (>10

min) may damage cells and result in loss of chlorophyll.

Care must be taken not to overload the filters. Do not

increase the vacuum during filtration.

suspend the particulates (stir or invert several times).

Pour the subsample into a graduated cylinder and

accurately measure the volume. Pour the subsample into

the filter tower of the filtration apparatus and apply a

vacuum (not to exceed 20 kPa). Typically, a sufficient

volume has been filtered when a visible green or brown

color is apparent on the filter. Do not suck the filter dry

with the vacuum; instead slowly release the vacuum as

the final volume approaches the level of the filter and

completely release the vacuum as the last bit of water is

pulled through the filter. Remove the filter from the fritted

base with tweezers, fold once with the particulate matter

inside, lightly blot the filter with a tissue to remove excess

moisture and place it in the petri dish or other suitable

container. If the filter will not be immediately extracted,

wrap the container with aluminum foil to protect the

phytoplankton from light and store the filter at -20 C or o

-70 C. Short term storage (2 to 4 h) on ice is acceptable, o

but samples should be stored at -20EC as soon as

possible.

8.2 Preservation -- Sampled filters should be stored

frozen (-20 C or -70 C) in the dark until extraction. o o

8.3 Holding Time -- Filters can be stored frozen at

-20 C for as long as 31⁄2 weeks without significant loss of o

chl a. 

(22)

9.0 Quality Control

9.1 Each Laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of laboratory capability and the continued

analysis of laboratory reagent blanks, field replicates and

QC samples as a continuing check on performance. The

laboratory is required to maintain performance records

that define the quality of the data generated.

9.2 Initial Demonstration of Performance

(Mandatory)

9.2.1 The initial demonstration of performance is used

to characterize instrument performance (IDLs and LDRs)

and laboratory performance (MDLs and analyses of

QCSs) prior to sample analyses.

9.2.2 Standard Reference Material (SRM) 930e

(National Institute of Standards and Technology,
 446.0-7 Revision 1.2 September 1997

Gaithersburg, MD) or other suitable spectrophotometric value is not within the confidence limits established by

filter standards that test wavelength accuracy must be project data quality objectives, then the determinative step

analyzed yearly and the results compared to the of this method is unacceptable. The source of the

instrument manufacturer's specifications. If wavelength problem must be identified and corrected before

accuracy is not within manufacturer's specifications, continuing analyses.

identify and repair the problem.

9.2.3 Linear Dynamic Range (LDR) -- The LDR should this method for the first time should demonstrate

be determined by analyzing a minimum of 5 standard proficiency in the extraction of sampled filters (Sect. 11.1).

solutions ranging in concentration from 1 to 15 mg/L. Twenty to thirty natural samples should be obtained using

Perform the linear regression of absorbance response (at the procedure outlined in Sect. 8.1 of this method. Sets

pigment's wavelength maximum) vs. concentration and of 10 or more samples should be extracted and analyzed

obtain the constants m and b, where m is the slope and b according to Sect. 11.2. The percent relative standard

is the y-intercept. Incrementally analyze standards of

higher concentration until the measured absorbance 15% for samples that are at least 10X the IDL. 

response, R, of a standard no longer yields a calculated

concentration, Cc, that is ± 10% of the known

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

c

concentration and absorbance response defines the

upper limit of the LDR for your instrument. Absorbance

responses for samples should be well below the upper

limit of the LDR, ideally between .1 and 1.0 AU. 

9.2.4 Instrumental Detection Limit (IDL) -- Zero the step properly, perform the following QC procedure:

spectrophotometer with a solution of 90% acetone. Pure

pigment in 90% acetone should be serially diluted until it Prepare 100 mL of a 2.0 ppm chl a solution in 90%

yields a response at the selected wavelength between

.005 and .008 AU.

9.2.5 Estimated Detection Limit (EDL) -- At least seven

natural phytoplankton samples known to contain the

pigments of interest should be collected, extracted and

analyzed according to the procedures in Sects. 8 and 11,

using clean glassware and apparatus. The concentration

of the pigment of interest should be between 2 and 5

times the IDL. Dilution or spiking of the sample extract

solution to the appropriate concentration may be

necessary. Inaccuracies occur in the measurement of

chlorophylls b and c1 + c2 when the chl a concentration is

greater than ~5X the concentration of the accessory

pigment. Perform all calculations to obtain concentration

values in mg/L in the extract solution. Calculate the EDL

as follows :

(23)

 EDL = (3) X (S)

S = Standard deviation of the replicate analyses.

9.2.6 Quality Control Sample (QCS) -- When beginning

to use this method, on a quarterly basis or as required to

meet data quality needs, verify instrument performance

with the analysis of a QCS (Sect. 7.9). If the determined

9.2.7 Extraction Proficiency -- Personnel performing

deviation (%RSD) of trichromatic chl a should not exceed

9.2.8 Corrected Chl a -- Multilaboratory testing of this

method revealed that many analysts do not adequately

mix the acidified sample when determining the corrected

chl a. The problem manifests itself by highly erratic

 pheo a results, high %RSDs for correctetd chl a and poor

agreement between corrected and uncorrected chl a. To

determine if a new analyst is performing the acidification

acetone. The new analyst should analyze 5-10 separate

aliquots, using carefully rinsed cuvettes, according to

instructions in Section 11.2. Process the results

according to Section 12 and calculate separate means

and %RSDs for corrected and uncorrected chl a. If the

means differ by more than 10%, then the stock chl a has

probably degraded and fresh stock should be prepared.

The %RSD for corrected chl a should not exceed 5%. If

the %RSD exceeds 5%, repeat the procedure until

acceptable results are obtained.

 

9.3 Assessing Laboratory Performance

(Mandatory)

9.3.1 Laboratory Reagent Blank (LRB) -- The

laboratory must analyze at least one blank filter with each

sample batch. The LRB should be the last filter

extracted. LRB data are used to assess contamination

from the laboratory environment. LRB values that exceed

the IDL indicate contamination from the laboratory

environment. When LRB values constitute 10% or more

of the analyte level determined in a sample, fresh

samples or field replicates must be analyzed after the

contamination has been corrected and acceptable LRB

values have been obtained.
Revision 1.2 September 1997 446.0-8

10.0 Calibration and Standardization

10.1 Daily calibration of the spectrophotometer is not

required when using the equations discussed in this

method. It is extremely important, therefore, to perform

regular checks on instrument performance. By analyzing

a standard reference material such as SRM 930e

(National Institute of Standards and Technology,

Gaithersburg, MD) at least quarterly, wavelength

accuracy can be compared to instrument manufacturer's

specifications. Filter kits that allow stray light, bandpass

and linearity to be evaluated are also commercially

available. Although highly recommended, such kits are

not required for this method if the LDR is determined for

the pigment of interest and QCSs are routinely analyzed.

10.2 Allow the instrument to warm up for at least 30

min. Use a 90% acetone solution to zero the instrument

at all of the selected wavelengths. 750 nm, 664 nm, 647

nm and 630 nm are used for the determination of chl a,

chl b and chl c1 + c2. 750 nm, 665 nm and 664 nm are

used for the determination of pheopigment-corrected chl

a and pheo a. The instrument is now ready to analyze

samples.

11.0 Procedure

11.1 Extraction of Filter Samples

11.1.1 For convenience, a 10-mL final extraction volume

is described in the following procedure. A larger

extraction volume may be necessary if using a lowvolume 10-cm cell. On the other hand, a smaller

extraction volume can be used to obtain a concentration

factor. The filter residue retains 2-3 mL of solution after

centrifugation and a 1-cm cell requires approximately 3

mL of solution so that a recommended minimum

extraction volume is 6 mL.

11.1.2 If sampled filters have been frozen, remove them

from the freezer but keep them in the dark. Set up the

tissue grinder and have on hand laboratory tissues and

squirt bottles containing water and acetone. Workspace

lighting should be the minimum that is necessary to read

instructions and operate instrumentation. Remove a filter

from its container and place it in the glass grinding tube.

The filter may be torn into smaller pieces to facilitate

extraction. Push it to the bottom of the tube with a glass

rod. With a volumetric pipet, add 4 mL of the aqueous

acetone solution (Sect. 7.6) to the grinding tube. After the

filter has been converted to a slurry, grind the filter for

approximately 1 min at 500 rpm. (NOTE: Although

grinding is required, care must be taken not to overheat

the sample. Good judgement and common sense will

help you in deciding when the sample has been

sufficiently macerated.) Pour the slurry into a 15-mL

screw-cap centrifuge tube and, using a 6-mL volumetric

pipet, rinse the pestle and the grinding tube with the

aqueous acetone. Add the rinse to the centrifuge tube

containing the filter slurry. Cap the tube and shake it

vigorously. Place it in the dark before proceeding to the

next filter extraction. Before placing another filter in the

grinding tube, use the acetone and water squirt bottles to

thoroughly rinse the pestle, grinding tube and glass rod.

To reduce the volume of reagent grade solvents used for

rinsing between extractions, thoroughly rinse the grinding

tube and glass rod with tap water prior to a final rinse with

ASTM Type I water and acetone. The last rinse should

be with acetone. Use a clean tissue to remove any filter

residue that adheres to the pestle or to the steel rod of the

pestle. Proceed to the next filter and repeat the steps

above. The last filter extracted should be a blank. The

entire extraction with transferring and rinsing takes

approximately 5 min. Approximately 500 mL of acetone

and water waste are generated per 20 samples from the

rinsing of glassware and apparatus. 

11.1.3 Shake each tube vigorously again before placing

them to steep in the dark at 4 C. Samples should be

o

allowed to steep for a minimum of 2 h but not to exceed

24 h. Tubes should be shaken at least once, preferably

two to three times, during the steeping period to allow the

extraction solution to have maximum contact with the filter

slurry.

11.1.4 After steeping is complete, centrifuge samples for

15 min at 675 g or for 5 min at 1000 g. 

11.2 Sample Analysis

11.2.1 The instrument must be zeroed on a 90%

acetone solution as described in Sect. 10.2. In subdued

lighting, pour or pipet the supernatant of the extracted

sample into the glass spectrophotometer cell. If the

absorbance at 750 nm exceeds .005 AU, the sample

must be recentrifuged or filtered through a glass fiber

filter (syringe filter is recommended). The volume of

sample required in the instrument's cell must be known if

the pheopigment-corrected chl a and pheo a will be

determined so that acidification to the correct acid

concentration can be performed. For example, a cell that
 446.0-9 Revision 1.2 September 1997

holds 3 mL of extraction solution requires .09 mL of the .1 C = 26.7(Abs 664 - Abs 665 ) 

N HCl solution to obtain an acid concentration of .003 N.

Measure the sample's absorbance at the selected

wavelengths for chl a, chl b and chl c1 + c2. Dilute and

reanalyze the sample if the signal at the selected

wavelength is >90% of the signal previously determined

as the upper limit of the LDR. If pheopigment-corrected

chl a and pheo a will be determined, acidify the sample in

the cell to .003 N HCl using the .1 N HCl solution. Use a

disposable Pasteur type pipet to thoroughly mix the

sample by aspirating and dispensing the sample into the

cuvette, keeping the pipet tip below the surface of the

liquid to avoid aerating the sample, wait 90 sec and

measure the sample's absorbance at 750 and 665 nm.

NOTE: Proper mixing of the acidified sample is critical for

accurate and precise results.

12.0 Data Analysis and Calculations

12.1 Jeffrey and Humphrey's Trichromatic

Equations -- Subtract the absorbance value at 750 nm

from the absorbance values at 664, 647 and 630 nm.

Calculate the concentrations (mg/L) of chl a, b, and c1 +

c2 in the extract solution by inserting the 750 nm-corrected

absorbance values into the following equations:

C = 11.85 (Abs 664) - 1.54 (Abs 647) - .08 (Abs 630) E,a 

C = 21.03 (Abs 647) - 5.43 (Abs 664) - 2.66 (Abs 630) E,b

C = 24.52 (Abs 630) - 7.60 (Abs 647) - 1.67 (Abs 664) E,c 

where:

CE,a = concentration (mg/L) of chlorophyll a in the

extraction solution analyzed,

C = concentration (mg/L) of chlorophyll b in the extract E,b

solution.

CE,c = concentration (mg/L) of chlorophyll c 1 + c 2 in the

extract solution analyzed.

12.2 Lorenzen's Pheopigment-corrected Chl a and

Pheo a -- Subtract the absorbance values at 750 nm from

the absorbance values at 664 and 665 nm. Calculate the

concentrations (mg/L) in the extract solution, CE, by

inserting the 750 nm corrected absorbance values into

the following equations:

E,a b a

P = 26.7 [1.7 X (Abs 665 ) - (Abs 664 )] E,a a b

where,

C = concentration (mg/L) of chlorophyll a in the extract E,a 

solution measurted,

PE,a = concentration (mg/L) of pheophytin a in the

extraction measured.

Abs 664b = sample absorbance at 664 nm (minus

absorbance at 750 nm) measured before acidification,

and 

Abs 665a = sample absorbance at 665 nm (minus

absorbance at 750 nm) measured after acidification. 

12.3 Calculate the conentration of pigment in the

whole water sample using the following generalized

equation:

Cs = CE (a,b, or c) X extract volume (L) X DF

sample volume (L) X cell length (cm)

where:

C = concentration (mg/L) of pigment in the whole water s

sample.

C = concentration (mg/l) of pigment in extract E(a,b,or c)

measured in the cuvette.

.

extract volume = volume (L) of extract (before any

dilutions), typically 0.0104).

DF = any dilution factors.

sample volume = volume (L) of whole water sample that

was filtered, and 

cell length = optical path length (cm) of cuvette used

(typically 1 cm).

For example, calculate the conentration of chlorophyll a

in the whole water sample as:
Cs,b

'

CE,a X extract volume (L) DF

sample volume (L) X cell length (cm)

Revision 1.2 September 1997 446.0-10

12.4 LRB and QCS data should be reported with each median DLs over labs was then determined and is

sample data set. reported in Table 6. It is referred to as the pooled-EDL

13.0 Method Performance

13.1 Single Laboratory Performance

13.1.1 Replicate analyses were performed on low level

dilutions of the pure pigments in 90% acetone. The

results, contained in Table 2, give an indication of the

variability not attributable to sampling and extraction or

pigment interferences.

13.1.2 The IDLs and S-EDLs for the method analytes

are reported in Table 3. 

13.1.3 Precision (%RSD) for replicate analyses of two

distinct mixed assemblages are contained in Table 4.

13.1.4 Three QCS ampules were obtained from the

USEPA, analyzed and compared to the reference values

in Table 5. (NOTE: The USEPA no longer provides

pigment QCSs.)

13.2 Multilaboratory Testing - A Multilaboratory

validation and comparison study of EPA Methods 445.0,

446.0 and 447.0 for chlorophyll a was conducted in 1996

by Research Triangle Institute, Research Triangle park,

N.C. (EPA Contract No. 68-C5-0011). There were 24

volunteer participants in the spectrophotometric methods

component that returned data. The primary goals of the

study were to determine detection limits and to assess

precision and bias (as percent recovery) for select

unialgal species, and natural seawater.

13.2.1 The term, pooled-estimated detection limit (pEDL), is used in this method to distinguish it from the EPA

defined method detection limit (MDL). An EPA MDL

determination is not possible nor practical for a natural

water or pure species sample due to known spectral

interferences and to the fact that it is impossible to

prepare solutions of known concentrations that

incorporate all sources of error (sample collection,

filtration, processing). The statistical approach used to

determine the p-EDL was an adaptation of the Clayton,

et. al. method that does not assume error variances

24

across concentration and controls for Type II error. The

statistical approach used involved calculating an

estimated DL for each lab that had the desired Type I and

Type II error rates (0.01 and 0.05, respectively). The

(p-EDL).

Solutions of pure chlorophyll a in 90% acetone were

prepared at three concentrations (0.11, 0.2, and 1.6 ppm)

and shipped with blank glass fiber filters to participating

laboratories. Analysts were instructed to spike the filters

in duplicate with a given volume of solution and to

process the spiked filters according to the method. The

results from these data were used to determine a pooled

EDL (p-EDL) for each method. Results (in ppm) are

given in Table 6. The standard fluorometric and HPLC

methods gave the lowest p-EDLs while the

spectrophotometric (monochromatic equations) gave the

highest p-EDLs.

13.2.2 To address precision and bias in chlorophyll a

determination for different algal species three pure

uniagal cultures (amphidinium, dunnnaliella and

phaeodactylum) were cultured and grown in the

laboratory. Four different “concentrations” of each

species were prepared by filtering varying volumes of the

algae. The filters were frozen and shipped to participant

labs. Analysts were instructed to extract and analyze the

filters according to the respectiave methods. The “true”

concentration was assigned by taking the average of the

HPLC results for the highest concentration algae sample

since chlorophyll a is separatead from other interfereing

pigments prior to determination. Pooled precision data

(%RSD) are presented in Tables 7-9 and accuracy data

(as percent recovery) are presented in Table 10. No

significant differences in precision were observed across

conentrations for any of the species. It should be noted

that there was considerable lab-to-lab variation (as

exhibited by the min and max recoveries in Table 10) and

in this case the median is a better measurement of

central tendency than the mean.

In summary, the mean and median concentrations

determined for Amphidinium carterae (class

dinophyceae) are similar for all methods. No method

consistently exhibited high or low values relative to the

other methods. The only concentration trend observed

was that the spectrophotometric method-trichromatic
 446.0-11 Revision 1.2 September 1997

equations (SP-T) showed a slight percent increase in

recovery with increasing algae filtration volume.

For Dunaliella tertiolecti (class chlorophyceae) and

Phaeodactylum tricornutum (class bacillariophyceae)

there was generally good agreement between the

fluorometric and the spectrophotometric methods,

however, the HPLC method yielded lower recoveries with

increasing algae filtration volume for both species. No

definitive explanation can be offered at this time for this

phenomenon. A possible explanation for the

Phaeodactylum is that it contained significant amounts of

chlorophylide a which is determined as chlorophyll a in

the fluorometric and spectrophotometric methods. The

conventional fluorometric method (FL-STD) showed a

slight decrease in chlorophyll a recovery with increasing

Dunaliella filtration volume. The spectrophotometrictrichromatic equations (SP-T) showed a slight increase in

chlorophyll a recovery with increasing Dunaliella filtration

volume. The fluorometric and tahe spectrophotometric

methods both showed a slight decrease in chlorophyll a

recovery with increasing Phaeodactylum filtration volume.

Results for the natural seawater sample are presented in

Table 11. Only one filtration volume (100 mL) was

provided in duplicate to partaicpant labs.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

USEPA has established a preferred hierarchy of

environmental management techniques that places

pollution prevention as the management option of first

choice. Whenever feasible, laboratory personnel should

use pollution prevention techniques to address their waste

generation (e.g., Sect. 11.1.1). When wastes cannot be

feasibly reduced at the source, the Agency recommends

recycling as the next best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research

institutions, consult Less is Better: Laboratory Chemical

Management for Waste Reduction, available from the

American Chemical Society's Department of Government

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

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

15.0 Waste Management

15.1 The U.S. Environmental Protection Agency

requires that laboratory waste management practices be

conducted consistent with all applicable rules and

regulations. The Agency urges laboratories to protect the

air, water, and land by minimizing and controlling all

releases from hoods and bench operations, complying

with the letter and spirit of any sewer discharge permits

and regulations, and by complying with all solid and

hazardous waste regulations, particularly the hazardous

waste identification rules and land disposal restrictions.

For further information on waste management consult

The Waste Management Manual for Laboratory

Personnel, available from the American Chemical Society

at the address listed in the Sect. 14.2.

16.0 References

1. Jeffrey, S.W. and G.F. Humphrey, "New

Spectrophotometric Equations for Determining

Chlorophylls a, b, c1 + c2 in Higher Plants, Algae

and Natural Phytoplankton," Biochem. Physiol.

Pflanzen. Bd, 167, (1975), S. pp. 191-4.

2. Lorenzen, C.J., "Determination of Chlorophyll

and Pheo-Pigments: Spectrophotometric

Equations," Limnol. Oceanogr., 12 (1967), pp.

343-6.

3. Holm-Hansen, O., "Chlorophyll a determination:

improvements in methodology," OIKOS, 30

(1978), pp. 438-447.

4. Wright, S.W. and J.D. Shearer, "Rapid extraction

and HPLC of chlorophylls and carotenoids from

marine phytoplankton," J. Chrom., 294 (1984),

pp. 281-295.

5. Bowles, N.D., H.W. Paerl, and J. Tucker,

"Effective solvents and extraction periods

employed in phytoplankton carotenoid and

chlorophyll determination," Can. J. Fish. Aquat.

Sci., 42 (1985) pp. 1127-1131.

6. Shoaf, W.T. and B.W. Lium, "Improved extraction

of chlorophyll a and b from algae using dimethyl

sulfoxide," Limnol. and Oceanogr., 21(6) (1976)

pp. 926-928.
Revision 1.2 September 1997 446.0-12

7. Mantoura, R.F.C. and C.A. Llewellyn, "The rapid determinations by spectrophotometric,

determination of algal chlorophyll and carotenoid fluorometric, spectrofluorometric and HPLC

pigments and their breakdown products in methods," Marine Microbial Food Webs, 4(2),

natural waters by reverse-phase high

performance liquid chromatography," Anal.

Chim. Acta., 151 (1983) pp. 297-314. 16. Sartory, D.P., "The determination of algal

8. Yentsch, C.S. and D.W. Menzel, "A method for

the determination of phytoplankton chlorophyll

and phaeophytin by fluorescence," Deep Sea

Res., 10 (1963), pp. 221-231.

9. Strickland, J.D.H. and T.R. Parsons, A Practical

Handbook of Seawater Analysis, Bull. Fish. Res.

Board Can., 1972, No.167, p. 201.

10. USEPA Method 445.0, "In vitro determination of

chlorophyll a and pheophytin a in marine and

freshwater phytoplankton by fluorescence,"

Methods for the Determination of Chemical

Substances in Marine and Estuarine

Environmental Samples, EPA/600/R-92/121.

11. Wright, S.W., S.W. Jeffrey, R.F.C. Mantoura,

C.A. Llewellyn, T. Bjornland, D. Repeta, and N.

Welschmeyer, "Improved HPLC method for the

analysis of chlorophylls and carotenoids from

marine phytoplankton," Mar. Ecol. Prog. Ser.,

77:183.

12. Brown, L.M., B.T. Hargrave, and M.D.

MacKinnon, "Analysis of chlorophyll a in

sediments by high-pressure liquid

chromatography," Can. J. Fish. Aquat. Sci., 38

(1981) pp. 205-214.

13. Bidigare, R.R., M.C. Kennicutt, II, and J.M.

Brooks, "Rapid determination of chlorophylls and

their degradation products by HPLC," Limnol.

Oceanogr., 30(2) (1985) pp. 432-435.

14. Minguez-Mosquera, M.I., B. Gandul-Rojas, A.

Montano-Asquerino, and J. Garrido-Fernandez,

"Determination of chlorophylls and carotenoids

by HPLC during olive lactic fermentation," J.

Chrom., 585 (1991) pp. 259-266.

15. Neveux.J., D. Delmas, J.C. Romano, P. Algarra,

L. Ignatiades, A. Herbland, P. Morand, A. Neori,

D. Bonin, J. Barbe, A. Sukenik and T. Berman,

"Comparison of chlorophyll and pheopigment

(1990) pp. 217-238. 

chlorophyllous pigments by high performance

liquid chromatography and spectrophotometry,"

Water Research, 19(5), (1985), pp. 605-10.

17. Carcinogens - Working With Carcinogens,

Department of Health, Education and Welfare,

Public Health Service, Center for Disease

Control, National Institute for Occupational Safety

and Health, Publication No. 77-206, 1977.

18. "OSHA Safety and Health Standards, General

Industry," (29 CFR 1910), Occupational Safety

and Health Administration, OSHA 2206, revised

January 1976.

19. Safety in Academic Chemistry Laboratories,

American Chemical Society publication,

Committee on Chemical Safety, 3rd Edition,

1979.

20. "Proposed OSHA Safety and Health Standards,

Laboratories," Occupational Safety and Health

Administration, Federal Register, July 24, 1986.

21. Marshall, C.T., A. Morin and R.H. Peters,

"Estimates of Mean Chlorophyll-a concentration:

Precision, Accuracy and Sampling design," Wat.

Res. Bull., 24(5), (1988), pp. 1027-1034.

22. Weber, C.I., L.A. Fay, G.B. Collins, D.E. Rathke,

and J. Tobin, "A Review of Methods for the

Analysis of Chlorophyll in Periphyton and

Plankton of Marine and Freshwater Systems,"

work funded by the Ohio Sea Grant Program,

Ohio State University. Grant No.NA84AA-D00079, 1986, 54 pp.

23. Code of Federal Regulations 40, Ch.1,

Pt.136, Appendix B.

24. Clayton, C.A., J.W. Hines and P.D. Elkins,

“Detection limits within specified assurance

probabilities.” Analytical Chemistry, 59(1987),

pp. 2506-2514. 
 446.0-13 Revision 1.2 September 1997

17.0 Tables, Diagrams, Flowcharts, and Validation Data

FIGURE 1 - The effect of pheo a on calculated pigment

 concentrations.
Revision 1.2 September 1997 446.0-14

FIGURE 2 - The effect of Chl b on pheopigment - corrected Chl a.
 446.0-15 Revision 1.2 September 1997

FIGURE 3 - The underestimation of Chl b with increasing concentrations of Chl a.
Revision 1.2 September 1997 446.0-16

TABLE 1. COMPARISON OF PRECISION AND RECOVERY OF PIGMENTS FOR 4 h AND 24 h STEEPING PERIODS

 chl a chl b chl c1+c2 pheo a corr a

 4h 24h 4h 24h 4h 24h 4h 24h 4h 24h

 

N 6 6 6 6 6 6 6 6 6 6

SD 1.22 0.88 0.42 0.21 0.44 0.37 1.08 1.23 1.46 1.04

Mean 26.14 25.73 0.49 1.72 5.87 5.26 1.38 2.88 24.47 23.29

%RSD 24.67 3.40 6.35 12.00 7.43 7.04 78.35 42.62 5.97 4.47

 

N - Number of samples

SD - Standard deviation

Mean - Concentration in natural water, mg/L

%RSD - Percent relative standard deviation
 446.0-17 Revision 1.2 September 1997

TABLE 2. REPLICATE ANALYSES OF PURE PIGMENTS AT LOW CONCENTRATIONS

 Modified

 Trichromatic Equations Monochromatic Equations 

chl a chl b chl a chl b

 N 7 7 N 7 6

SD .000612 .009792 SD .010091 .011990

Mean .102 mg/L .109 mg/L Mean .103 mg/L .171 mg/L

%RSD .60 8.9 %RSD 9.8 7.0

TABLE 3. INSTRUMENTAL AND METHOD DETECTION LIMITS

 INSTRUMENTAL DETECTION LIMITS1

 (Concentrations in mg/L)

 Modified 

 Trichromatic Equations Monochromatic Equation

chl a .080 pheo a .085 

chl b .093 

 S-ESTIMATED DETECTION LIMITS1

 (Concentrations in mg/L)

 Modified

 Trichromatic Equations Monochromatic Equation

chl a .037 chl a .053 

2 2

chl b .070 pheo a .076 2 2

chl c1 + c2.0873

 

 Determinations made using a 1-cm path length cell. 1

 Mixed assemblage samples from San Francisco Bay. 2

 Predominantly diatoms from Raritan Bay. 3
Revision 1.2 September 1997 446.0-18

TABLE 4. ANALYSES OF NATURAL SAMPLES

 

SAN FRANCISCO BAY 

 Modified

 Trichromatic Equations Monochromatic Equations

 chl a chl b chl c1+c2 pheo a corr a

 

N 7 7 7 7 7

SD 0.0118 0.0062 0.0096 0.0244 0.0168 

Mean 0.2097 0.04271 0.03561 0.0806 0.1582 

%RSD 5.62 14.50 26.82 30.21 0.64 

RARITAN BAY

 Modified

 Trichromatic Equations Monochromatic Equations

 chl a chl b chl c1+c2 pheo a corr a

 

N 7 7 7 7 7

SD 0.0732 0.0223 0.0277 0.0697 0.0521

Mean 1.4484 0.0914 0.2867 0.1720 1.3045 

%RSD 5.06 24.43 9.65 40.53 3.99 

 

Mean concentrations (mg/L) reported in final extraction volume of 10 mL. Samples were macerated and allowed to steep for

approximately 24 h.

N - Number of samples

SD - Standard deviation

Mean - Concentration in natural water

%RSD - Percent relative standard deviation
 446.0-19 Revision 1.2 September 1997

TABLE 5. ANALYSES OF USEPA QC SAMPLES

Ampule 1 (3 separate ampules, chl a only)

 Modified

 Trichromatic Equations Monochromatic Equations

 Mean Reference %RSD Mean Reference %RSD

chl a 2.54 mg/L 2.59 .61 chl a 2.56 mg/L 2.70 .8

pheo a ND

 

 ND - None detected

Ampule 2 (3 separate ampules, all method pigments)

 Modified

 Trichromatic Equations Monochromatic Equations

 Mean Reference %RSD Mean Reference %RSD

chl a 4.87 mg/L 4.86 .1 chl a 3.70 mg/L 3.76 2.3

chl b 1.12 mg/L 1.02 1.3 pheo a 1.79 mg/L 1.70 4.4

chl c1 + c2.29 mg/L .37 4.9

 
Revision 1.2 September 1997 446.0-20

TABLE 6. POOLED ESTIMATED DETECTION LIMITS FOR CHLOROPHYLL A METHODS(1)

_____________________________________________________________

Method N p-EDL (mg/L) (2) (3) (4)

FL -Mod 8 0.096 (5)

FL - Std 9 0.082

HPLC 4 0.081

SP-M 15 0.229

SP-T 15 0.104

_____________________________________________________________

(1) See Section 13.2.1 for a description of the statistical approach used to determine p-EDLs. 

(2) FL-Mod = fluorometric method using special interference filters.

 FL-Std = conventional fluorometric method with pheophytin a correction.

 HPLC = EPA method 447.0

 SP-M = EPA method 446.0, monochromatic equation.

 SP-T = EPA method 446.0, trichromatic equations.

(3) N = number of labs whose data was used.

(4) The p-EDL was determined with p = 0.01 and q (type II error rate) = 0.05.

(5) Due to the large dilutions required to analyze the solutions by fluorometry, the fluormetric p-EDLs are

 unrealistically high.
 446.0-21 Revision 1.2 September 1997

TABLE 7. POOLED PRECISION FOR AMPHIDINIUM CARTERAE SAMPLES

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

SP-M 5 17 0.068 0.026 37.8

 10 19 0.139 0.037 26.6

 50 19 0.679 0.150 22.1

100 19 1.366 0.205 15

SP-T 5 16 0.059 0.021 35.1

 10 18 0.130 0.027 20.8

 50 18 0.720 0.102 14.2

100 18 1.408 0.175 12.4

___________________________________________________________________________________

(1) SP-M = Pheophytin a - corrected chlorophyll a method using monochromatic equations.

 SP-T = Trichromatic equations method.

 

(2) N = Number of volunteer labs whose data was used.
Revision 1.2 September 1997 446.0-22

TABLE 8. POOLED PRECISION FOR DUNALIELLA TERTIOLECTI SAMPLES

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

SP-M 5 19 0.166 0.043 26.0

 10 19 0.344 0.083 24.0

 50 19 1.709 0.213 12.5

100 19 3.268 0.631 19.3

SP-T 5 18 0.161 0.030 18.4

 10 18 0.339 0.058 17.1

 50 18 1.809 0.190 10.5

100 18 3.500 0.524 15.0

___________________________________________________________________________________

(1) SP-M = Pheophytin a corrected chlorophyll a method using monochromatic equations.

 SP-T = Trichromatic equationss method. 

(2) N = number of volunteer labs whose data was used.
 446.0-23 Revision 1.2 September 1997

TABLE 9. POOLED PRECISION FOR PHAEODACTYLUM TRICORNUTUM SAMPLES

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

SP-M 5 19 0.223 0.054 24.1

 10 19 0.456 0.091 19.9

 50 19 2.042 0.454 22.2

100 19 4.083 0.694 17.0

SP-T 5 18 0.224 0.031 14.0

 10 18 0.465 0.077 16.5

 50 18 2.223 0.217 9.7

100 18 4.422 0.317 7.2

___________________________________________________________________________________

(1) SP-M = Pheophytin a corrected chorophyll a method using monochromatic equations.

(2) N = number of volunteer labs whose data was used.
Revision 1.2 September 1997 446.0-24

TABLE 10. MINIMUM, MEDIAN, AND MAXIMUM PERCENT RECOVERIES BY GENERA, METHOD, AND

 CONCENTRATION LEVEL

Species Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 Level 4

Amphidinium Minimum FL-MOD 70 73 75 76

FL-STD 66 91 91 90

HPLC 82 85 87 88

SP-M 36 48 68 64

SP-T 21 63 71 70

Median FL-MOD 105 112 105 104

FL-STD 109 107 111 109

HPLC 102 106 112 105

SP-M 99 101 101 101

SP-T 95 96 106 107

Maximum FL-MOD 121 126 143 146

FL-STD 156 154 148 148

HPLC 284 210 131 116

SP-M 141 133 126 125

SP-T 115 116 119 117

Dunaliella Minimum FL-MOD 162 159 157 156

FL-STD 179 171 165 164

HPLC 165 109 64 41

SP-M 120 188 167 164

SP-T 167 169 166 165

Median FL-MOD 206 246 227 223

FL-STD 250 228 224 210

HPLC 252 177 89 80

SP-M 240 247 247 243
Table 10 cont’d

 446.0-25 Revision 1.2 September 1997

Species

Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 Level 4

SP-T 225 244 256 256

Dunaliella Maximum FL-MOD 295 277 287 288

FL-STD 439 385 276 261

HPLC 392 273 172 154

SP-M 342 316 296 293

SP-T 291 283 283 283

Phaeodactylum Minimum FL-MOD 216 183 157 154

FL-STD 189 220 223 219

HPLC 150 119 84 75

SP-M 161 138 156 160

SP-T 203 195 216 244

Median FL-MOD 292 285 250 245

FL-STD 296 263 254 254

HPLC 225 203 114 90

SP-M 287 274 254 253

SP-T 286 281 277 274

Maximum FL-MOD 357 337 320 318

FL-STD 371 415 415 334

HPLC 394 289 182 139

SP-M 446 344 330 328

SP-T 357 316 318 299
Revision 1.2 September 1997 446.0-26

TABLE 11. CHLOROPHYLL A CONCENTRATIONS IN mg/L DETERMINED IN FILTERED SEAWATER

 SAMPLES

Method Con.(1) No. Obs. No. Labs Mean Std. Dev. RSD(%) Minimum Median Maxium

FL-MOD 100 14 7 1.418 0.425 30.0 0.675 1.455 2.060

FL-STD 100 15 8 1.576 0.237 15.0 1.151 1.541 1.977

HPLC 100 10 5 1.384 0.213 15.4 1.080 1.410 1.680

SP-M 100 38 19 1.499 0.219 14.6 0.945 1.533 1.922

SP-T 100 36 18 1.636 0.160 9.8 1.250 1.650 1.948

All Methods 100 113 57 1.533 0.251 16.4 0.657 1.579 2.060

(1) Con = mLs of seawater filtered.
447.0-1

Method 447.0

Determination of Chlorophylls a and b and Identification 

of Other Pigments of Interest in Marine and Freshwater

Algae Using High Performance Liquid Chromatography 

with Visible Wavelength Detection

Elizabeth J. Arar

Version 1.0

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Version 1.0 September 1997 447.0-2

METHOD 447.0

DETERMINATION OF CHLOROPHYLLS a AND b AND IDENTIFICATION 

OF OTHER PIGMENTS OF INTEREST IN MARINE AND FRESHWATER

ALGAE USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 

WITH VISIBLE WAVELENGTH DETECTION

1.0 Scope and Application

1.1 This method provides a procedure for

determination of chlorophylls a (chl a) and b (chl b) found

in marine and freshwater phytoplankton. Reversedphase high performance liquid chromatography (HPLC)

with detection at 440 nm is used to separate the pigments

from a complex pigment mixture and measure them in

the sub-microgram range. For additional reference, other

taxonomically important yet commercially unavailable

pigments of interest are identified by retention time.

1.2 This method differs from previous descriptions of

HPLC methods in several respects. Quality

assurance/quality control measures are described in

Sect. 9.0, sample collection and extraction procedures

are described in Sect. 8.0 and reference chromatograms

of pure pigments and reference algae are provided.

This method has also been evaluated in a multilaboratory

study along with EPA Methods 445.0 and 446.0.

Estimated detection limits, precision and bias are reported

in Section 13. 

 

Chemical Abstracts Service

Analyte Registry Number (CASRN)

 

Chlorophyll a 479-61-8

Chlorophyll b 519-62-0

 

1.3 Instrumental detection limits (IDLs) of 0.7 ng chl

a, and 0.4 ng chl b in pure solutions of 90% acetone were

determined by this laboratory. Method detection limit

(MDL) determinations were made by analyzing seven

replicate unialgal samples containing the chl a and b.

Single-laboratory MDLs were chl a - 7 ng and chl b - 4 ng.

A multilaboratory estimated detection limit (EDL) (in mg/L

of extract is reported in Section 13.

1.4 Most taxonomically important pigments are not

commercially available, therefore, a laboratory must be

willing to extract and purify pigments from pure algal

cultures to quantify and qualitatively identify these very

important pigments. This method contains

chromatographic information of select pure pigments

found either in marine or freshwater algae. The

information is included to aid the analyst in qualitatively

identifying individual pigments and possibly algal species

in natural samples.

1.5 This method uses 90% acetone as the extraction

solvent because of its efficiency for extracting chl a from

most types of algae. (NOTE: There is evidence that

certain chlorophylls and carotenoids are more thoroughly

extracted with methanol or dimethyl sulfoxide.) Using (1-3) (4)

high performance liquid chromatography (HPLC),

Mantoura and Llewellyn found that methanol led to the (5)

formation of chl a derivative products, whereas 90%

acetone did not. Bowles, et al.(3) found that for chl a 90%

acetone was an effective solvent when the steeping

period was optimized for the predominant species

present.) 

1.6 One of the limitations of visible wavelength

detection is low sensitivity. It may be preferable to use

fluorometry or HPLC with fluorometric detection if

(6-8) (913)

high volumes of water (>4 L) must be filtered to obtain

detectable quantities of chl a or b. 

1.7 This method is for use by analysts experienced in

handling photosynthetic pigments and in the operation of

HPLC or by analysts under the close supervision of such

qualified persons.

2.0 Summary of Method

2.1 The HPLC is calibrated with a chl a and b

solution that has been spectrophotometrically quantified
 447.0-3 Version 1.0 September 1997

according to EPA Method 446. Chlorophyll-containing reagents, or apparatus. For this method the LRB is a

phytoplankton in a measured volume of sample water are blank filter that has been extracted as a sample.

concentrated by filtration at low vacuum through a glass

fiber filter. The pigments are extracted from the 3.6 Material Safety Data Sheet (MSDS) -- Written

phytoplankton into 90% acetone with the aid of a information provided by vendors concerning a chemical's

mechanical tissue grinder and allowed to steep for a toxicity, health hazards, physical properties, fire, and

minimum of 2 h, but not exceeding 24 h, to ensure reactivity data including storage, spill, and handling

thorough extraction of the pigments. The filter slurry is precautions.

centrifuged at 675 g for 15 min (or at 1000 g for 5 min) to

clarify the solution. An aliquot of the supernatant is 3.7 Method Detection Limit (MDL) -- The minimum

filtered through a 0.45 μm syringe filter and 50 to 200 μL

is injected onto a reversed-phase column. Following

separation using a ternary gradient, concentrations are

reported in μg/L (ppb) or mg/L (ppm) in the whole water

sample. This method is based on the HPLC work of

Wright, et. al.(9)

3.0 Definitions

3.1 Calibration Standard (CAL) -- A solution

prepared from dilution of a stock standard solution. The

CAL solution is used to calibrate the instrument response

with respect to analyte concentration or mass.

3.2 Calibration Check Standard (CALCHK) -- A

mid-point calibration solution that is analyzed periodically

in a sample set to verify that the instrument response to

the analyte has not changed during the course of

analysis.

 

3.3 Field Replicates -- Separate samples collected

at the same time and placed under identical

circumstances and treated exactly the same throughout

field and laboratory procedures. Analyses of field

replicates give a measure of the precision associated with

sample collection, preservation and storage, as well as

with laboratory procedures.

3.4 Instrument Detection Limit (IDL) -- The

minimum quantity of analyte or the concentration

equivalent that gives an analyte signal equal to three

times the standard deviation of a background signal at the

selected wavelength, mass, retention time, absorbance

line, etc. 

3.5 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water or other blank matrices that are treated

exactly as a sample including exposure to all glassware,

equipment, solvents, reagents, internal standards, and

surrogates that are used with other samples. The LRB is

used to determine if method analytes or other

interferences are present in the laboratory environment,

concentration of an analyte that can be identified,

measured and reported with 99% confidence that the

analyte concentration is greater than zero.

3.8 Quality Control Sample (QCS) -- A solution of

method analytes of known concentrations that is used to

fortify an aliquot of LRB or sample matrix. Ideally, the

QCS is obtained from a source external to the laboratory

and different from the source of calibration standards. It

is used to check laboratory performance with externally

prepared test materials.

4.0 Interferences

4.1 Any compound extracted from the filter or

acquired from laboratory contamination that absorbs light

at 440 nm may interfere in the accurate measurement of

the method analytes. 

4.2 Proper storage and good sample handling

technique are critical in preventing degradation of the

pigments.

4.3 Precision and recovery for any of the pigments is

related to efficient extraction, i.e. efficient maceration of

the filtered sample and to the steeping period of the

macerated filter in the extraction solvent. Precision

improves with increasing steeping periods, however, a

drawback to prolonged steeping periods is the possibility

of pigment degradation. The extracted sample must be

kept cold and in the dark to minimize degradation. 

4.4 Sample extracts must be clarified by filtration

through a 0.45 μm filter prior to analysis by HPLC to

prevent column fouling.

4.5 All photosynthetic pigments are light and

temperature sensitive. Work must be performed in

subdued light and all standards, QC materials, and

filtered samples must be stored in the dark at -20 C or o

 -70 C to prevent rapid degradation. o
Version 1.0 September 1997 447.0-4

5.0 Safety

5.1 Each chemical used in this method should be

regarded as a potential health hazard and handled with

caution and respect. Each laboratory is responsible for

maintaining a current awareness file of Occupational

Safety and Health Administration (OSHA) regulations

regarding the safe handling of the chemicals specified in

this method. A file of MSDS also should be made

(15-18)

available to all personnel involved in the chemical

analysis.

5.2 The grinding of filters during the extraction step of

this method should be conducted in a fume hood due to

the volatilization of acetone by the tissue grinder.

6.0 Apparatus and Equipment

6.1 Centrifuge, capable of 675 g.

6.2 Tissue grinder, Teflon pestle (50 mm X 20 mm)

with grooves in the tip with 1/4" stainless steel rod long

enough to chuck onto a suitable drive motor and 30-mL

capacity round-bottomed, glass grinding tube.

6.3 Filters, glass fiber, 47-mm or 25-mm nominal

pore size of 0.7 μm unless otherwise justified by data

quality objectives. Whatman GF/F filters were used in this

work.

6.4 Petri dishes, plastic, 50 X 9-mm, or some other

solid container for transporting and storing sampled

filters.

6.5 Aluminum foil.

6.6 Laboratory tissues.

6.7 Tweezers or flat-tipped forceps.

6.8 Vacuum pump or source capable of maintaining

a vacuum up to 6 in. Hg (20 KPa).

6.9 Labware -- All reusable labware (glass,

polyethylene, Teflon, etc.) that comes in contact with

chlorophyll solutions should be clean and acid free. An

acceptable cleaning procedure is soaking for 4 h in

laboratory grade detergent and water, rinsing with tap

water, distilled deionized water and acetone.

6.9.1 Assorted Class A calibrated pipets.

6.9.2 Graduated cylinders, 500-mL and 1-L.

6.9.3 Volumetric flasks, Class A calibrated, 10-mL, 25-

mL, 50-mL, 100-mL and 1-L capacity.

6.9.4 Glass rods or spatulas.

6.9.5 Pasteur Type pipets or medicine droppers.

6.9.6 Filtration apparatus consisting of 1 or 2-L filtration

flask, 47-mm fritted glass disk base and a glass filter

tower.

6.9.7 Centrifuge tubes, polypropylene or glass, 15-mL

capacity with nonpigmented screw-caps.

6.9.8 Polyethylene squirt bottles.

6.9.9 Amber 2-mL HPLC autosampler vials with screw

or clamp caps.

6.9.10 Glass syringe, 1 or 2-mL capacity.

6.9.11 HPLC compatible, low-volume, acetone resistant

glass fiber or PTFE syringe filters.

6.10 Liquid Chromatograph

6.10.1 This method uses a ternary gradient thus

requiring a programmable gradient pump with at least

three pump inlets for the three different mobile phases

required. A Dionex Model 4500 chromatograph equipped

with a gradient pump, UV/VIS detector (cell path length,

6 mm, volume 9 μL) and PC data analysis (Dionex AI450

software, Version 3.32) system was used to generate

data for this method. Tubing was made of polyether ether

ketone (PEEK). A Dionex degas module was used to

sparge all eluents with helium. 

6.10.2 Helium or other inert gas for degassing the

mobile phases OR other means of degassing such as

sonication under vacuum.

6.10.3 Sample loops of various sizes (50-200 μL).

6.10.4 Guard Column -- A short column containing the

same packing material as the analytical column placed

before the analytical column to protect it from fouling by

small particles. The guard column can be replaced

periodically if it is noticed that system back pressure has

increased over time.
 447.0-5 Version 1.0 September 1997

6.10.5 Analytical Column -- A C18 reversed-phase the graduated cylinder and transfer to the flask or bottle

column with end capping. A J.T. Baker 4.6 mm X 250 containing the water. Mix, label and store.

mm, 5 μm pore size column was used to generate the

data in this method.

6.10.6 A visible wavelength detector with a low volume

flow-through cell. Detection is at 440 nm.

6.10.7 A recorder, integrator or computer for recording

detector response as a function of time.

6.10.8 Although not required, an autosampler

(preferably refrigerated) is highly recommended.

7.0 Reagents and Standards

7.1 Acetone, HPLC grade, (CASRN 67-64-1). 

7.2 Methanol, HPLC grade, (CASRN 67-56-1).

Prepare ELUENT A, 80% (v/v) methanol/20% 0.5 M

ammonium acetate, by adding 800 mL of methanol and

200 mL of the 0.5 M ammonium acetate (Sect. 7.5) to an

eluent container.

7.3 Acetonitrile, HPLC grade, (CASRN 75-05-8).

Prepare ELUENT B, 90% (v/v) acetonitrile/10% water, by

adding 900 mL of acetonitrile and 100 mL of water (Sect.

7.7) to an eluent container.

7.4 Ethyl acetate, HPLC grade, (CASRN 141-78-6).

ELUENT C, 100% ethyl acetate.

7.5 Ammonium acetate, ACS grade (CASRN 631-

61-8). Prepare a 0.5 M solution by dissolving 38.54 g in

approximately 600 mL of water in a 1-L volumetric flask.

After the ammonium acetate has dissolved, dilute to

volume with water.

7.6 Chl a free of chl b and chl b substantially free of

chl a may be obtained from a commercial supplier such

as Sigma Chemical (St. Louis, MO). 

7.7 Water -- ASTM Type I water (ASTM D1193) is

required. Suitable water may be obtained by passing

distilled water through a mixed bed of anion and cation

exchange resins.

7.8 Aqueous Acetone Solution -- 90% acetone/10%

ASTM Type I water. Carefully measure 100 mL of the

water into the 1-L graduated cylinder. Transfer to a 1-L

flask or storage bottle. Measure 900 mL of acetone into

7.9 Chlorophyll Stock Standard Solution (SSS) --

Chl a (MW = 893.5) and chl b (MW = 907.5) from a

commercial supplier is shipped in amber glass ampules

that have been flame sealed. The dry standards must be

stored at -20 or -70EC in the dark. Tap the ampule until

all the dried pigment is in the bottom of the ampule. In

subdued light, carefully break the tip off the ampule.

Transfer the entire contents of the ampule into a 25-mL

volumetric flask. Dilute to volume with 90% acetone: (1

mg in 25 mL = 40 mg chl a/L) and (1 mg in 25 ml = 40

mg chl b/L), label the flasks and wrap with aluminum foil

to protect from light. When stored in a light- and air-tight

container at -20 or -70 C, the SSS is stable for at least six o

months. Dilutions of the SSS should always be confirmed

spectrophotometrically using EPA Method 446.

7.10 Laboratory Reagent Blank (LRB) -- A blank

filter that is extracted and analyzed just as a sample filter.

The LRB should be the last filter extracted of a sample

set. It is used to assess possible contamination of the

reagents or apparatus.

7.11 Quality Control Sample (QCS) -- Since there

are no commercially available QCSs, dilutions of a stock

standard of a different lot number from that used to

prepare calibration solutions may be used.

8.0 Sample Collection, Preservation and

Storage

8.1 Water Sample Collection -- Water may be

obtained by a pump or grab sampler. Data quality

objectives will determine the depth and frequency at

(21)

which samples are taken. Healthy phytoplankton,

however, are generally obtained from the photic zone

(region in which the illumination level is 1% of surface

illumination). Enough water should be collected to

concentrate phytoplankton on at least three filters so that

precision can be assessed. Filtration volume size will

depend on the particulate load of the water. Four liters

may be required for open ocean water where

phytoplankton density is usually low, whereas 1 L or less

is generally sufficient for lake, bay or estuary water. All

apparatus should be clean and acid-free. Filtering should

be performed in subdued light as soon as possible after

sampling since algal populations, thus pigment

concentrations, can change in relatively short periods of

time. Aboard ship filtration is highly recommended.
Version 1.0 September 1997 447.0-6

Assemble the filtration apparatus and attach the vacuum 9.2 Initial Demonstration of Performance

source with vacuum gauge and regulator. Vacuum (Mandatory)

filtration should not exceed 6 in. Hg (20 kPa). Higher

filtration pressures may damage cells and result in loss of 9.2.1 The initial demonstration of performance is used

chlorophyll. Care must be taken not to overload the to characterize instrument performance (IDLs) and

filters. Do not increase the vacuum during filtration. laboratory performance (MDLs, extraction proficiency,

Prior to drawing a subsample from the water sample

container, gently stir or invert the container several times 9.2.2 Instrumental Detection Limit (IDL) -- After a low

to suspend the particles. Pour the subsample into a level calibration (Sect. 10), prepare a standard solution

graduated cylinder and accurately measure the volume. that upon injection into the chromatograph yields an

Pour the subsample into the filter tower of the filtration absorbance of 0.002-0.010. If using an autosampler,

apparatus and apply a vacuum (not to exceed 20 kPa). variable volumes may be injected and the micrograms

Typically, a sufficient volume has been filtered when a

visible green or brown color is apparent on the filter. Do

not suck the filter dry with the vacuum; instead slowly

release the vacuum as the final volume approaches the

level of the filter and completely release the vacuum as

the last bit of water is pulled through the filter. Remove

the filter from the fritted base with tweezers, fold once

with the particulate matter inside, lightly blot the filter with

a tissue to remove excess moisture and place it in the

petri dish or other suitable container. If the filter will not

be immediately extracted, wrap the container with

aluminum foil to protect the phytoplankton from light and

store the filter at -20 C or -70 C. Short term storage (2 to o o

4 h) on ice is acceptable, but samples should be stored

at -20EC or -70 C as soon as possible. o

8.2 Preservation -- Sampled filters should be stored

frozen (-20 C or -70 C) in the dark until extraction. o o

8.3 Holding Time -- Filters can be stored frozen at 

-20 C for as long as 31⁄2 weeks without significant loss of o

chl a. 

(20)

9.0 Quality Control

9.1 Each Laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of laboratory capability and the continued

analysis of laboratory reagent blanks, field replicates,

QCSs, and CALCHKs as a continuing check on

performance. The laboratory is required to maintain

performance records that define the quality of the data

generated.

and analyses of QCSs) prior to sample analyses.

(μg) injected calculated by multiplying the known

concentration (μg/μL) of the standard by the volume

injected (μL). A practical starting point may be to inject

0.05 μg (that would be a 50 μL injection of a 1.0 mg/L

standard solution) and reduce or increase the mass

injected according to the resulting signal. Avoid injecting

really small volumes (< 10 μL). After the quantity of

pigment has been selected, make three injections and

calculate the IDL by multiplying the standard deviation of

the calculated mass by 3.

9.2.3 Method Detection Limit (MDL) -- At least seven

natural phytoplankton samples known to contain the

pigments of interest should be collected, extracted and

analyzed according to the procedures in Sects. 8 and 11,

using clean glassware and apparatus. Mass of the

pigment injected into the chromatograph should be 2 to

5 times the IDL. Dilution of the sample extract solution to

the appropriate concentration or reducing the volume of

sample injected may be necessary. Calculate the MDL

(in micrograms) as follows.(19)

 MDL = (t) X (S)

where, t = Student's t-value for n-1 degrees of freedom at

the 99% confidence level. t = 3.143 for six degrees of

freedom.

S = Standard deviation of the replicate analyses.

9.2.4 Quality Control Sample (QCS) -- When beginning

to use this method, on a quarterly basis or as required to

meet data quality needs, verify instrument performance

with the analysis of a QCS (Sect. 7.11). If the determined

value is not within +10% of the spectrophotometrically

determined value, then the instrument should be

recalibrated with fresh stock standard and the QCS

reanalyzed. If the redetermined value is still

unacceptable then the source of the problem must be

identified and corrected before continuing analyses.
 447.0-7 Version 1.0 September 1997

9.2.5 Extraction Proficiency -- Personnel performing 10.3 Program the pump with the following gradient:

this method for the first time should demonstrate

proficiency in the extraction of sampled filters (Sect. 11.1). Time Flow %1 %2 %3 Condition

Fifteen to twenty natural samples should be obtained 0.0 1.0 100 0 0 Injection

using the procedure outlined in Sect. 8.1 of this method. 2.0 1.0 0 100 0 Linear Gradient

Sets of 10 samples or more should be extracted and 2.6 1.0 0 90 10 Linear Gradient

analyzed according to Sect. 11. The percent relative 13.6 1.0 0 65 35 Linear Gradient

standard deviation (%RSD) should not exceed 15% for 20.0 1.0 0 31 69 Linear Gradient

samples that are at least 10X the IDL. 22.0 1.0 0 100 0 Linear Gradient

9.3 Assessing Laboratory Performance 30.0 1.0 100 0 0 Equilibration

(Mandatory)

9.3.1 Laboratory Reagent Blank (LRB) -- The

laboratory must analyze at least one blank filter with each 10.4 The first analysis is a blank 90% acetone solution

sample batch. The LRB should be the last filter followed by calibration. Calibrate with at least three

extracted. LRB data are used to assess contamination concentrations, covering no more than one order of

from the laboratory environment. LRB values that exceed magnitude, and bracketing the concentrations of

the IDL indicate contamination from the laboratory samples. If an autosampler is used, variable volumes

environment. If the LRB value constitutes 10% or more ranging from 10 - 100% of the sample injection loop

of the analyte level determined in a sample, fresh volume are injected to give a calibration of detector

samples or field replicates must be analyzed after the response versus mass of pigment. If doing manual

contamination has been corrected and acceptable LRB injections, variable solution concentrations are made and

values have been obtained. a fixed sample loop volume is injected for standards and

9.3.2 Calibration Check Standard (CALCHK) -- The versus mass or detector response versus concentration

laboratory must analyze one CALCHK for every ten

samples to verify calibration. If the CALCHK is not +10%

of the spectrophotometrically determined concentration,

then the instrument must be recalibrated. 

10.0 Calibration and Standardization

10.1 Allow the visible wavelength detector (440 nm) to

warm up for at least 15 min before calibration. Prepare

ELUENTS A - C and degas by sparging with an inert gas

for 10 minutes or sonicating under vacuum for 5 minutes.

Prime the pump for each eluent taking care to remove all

air that may be in the liquid lines. Equilibrate the column

for ten minutes with 100% of ELUENT A. 

10.2 Remove the SSS from the freezer and allow it to

come to room temperature. Add 1 mL of the SSS to a

10-mL volumetric flask and dilute to 10 mL with 90%

acetone. Prepare the chl a and b separately and

determine the concentrations according to EPA Method

446 using the monochromatic equations for chl a

determination. After the concentration of the SSS is

determined, add 1 mL of the chl a SSS plus 1 mL of the

chl b SSS to a separate 10-mL flask and dilute to volume.

Store the calibration standard in a light tight glass bottle.

 

25.0 1.0 100 0 0 Linear Gradient

Flow is in mL/min.

samples. Calibration can be either detector response

(mg/L or μg/L). Linearity across sensitivity settings of the

detector must be confirmed if samples are analyzed at a

different sensitivity settings from that of the calibration. 

10.5 Construct a calibration curve of analyte response

(area) versus concentration (mg/L in solution) or mass

(μg) of pigment and perform a linear regression to

determine the slope and y-intercept. A typical coefficient

of determination is > 0.99. 

10.6 Calibration must be performed at least weekly

although it is not necessary to calibrate daily. Daily midpoint CALCHKs must yield calculated concentrations

+10% of the spectrophotometrically determined

concentration.

11.0 Procedure

11.1 Extraction of Filter Samples

11.1.1 For convenience, a 10-mL final extraction volume

is described in the following procedure. A smaller

extraction volume may be used to improve detection

limits. 
Version 1.0 September 1997 447.0-8

11.1.2 If sampled filters have been frozen, remove them 11.2.1 Draw into a clean syringe 2-3 times the injection

from the freezer but keep them in the dark. Set up the loop volume and inject into the chromatograph. If using

tissue grinder and have on hand laboratory tissues and an autosampler, load the sample tray, prepare a

wash bottles containing water and acetone. Workspace schedule and begin analysis. A typical analyses order

lighting should be the minimum that is necessary to read might be: (1) blank 90% acetone, (2) CALCHK, (3) 10

instructions and operate instrumentation. Remove a filter samples, (4) CALCHK, (5) QCS.

from its container and place it in the glass grinding tube.

You may also tear the filter into smaller pieces and push 11.2.2 If the calculated CALCHK is not +10 of the

them to the bottom of the tube with a glass rod. With a spectrophotometrically determined concentration then

volumetric pipet, add 3 mL of the aqueous acetone recalibrate with fresh calibration solutions.

solution (Sect. 7.6) to the grinding tube. Grind the filter

until it has become a slurry. (NOTE: Although grinding

is required, care must be taken not to overheat the

sample. Good judgement and common sense will help

you in deciding when the sample has been sufficiently

macerated.) Pour the slurry into a 15-mL screw-cap

centrifuge tube and, using a 7-mL volumetric pipet, rinse

the pestle and the grinding tube with the aqueous

acetone. Add the rinse to the centrifuge tube containing

the filter slurry. Cap the tube and shake it vigorously.

Place it in the dark before proceeding to the next filter

extraction. Before placing another filter in the grinding

tube, use the acetone and water squirt bottles to

thoroughly rinse the pestle, grinding tube and glass rod.

To reduce the volume of reagent grade solvents used for

rinsing between extractions, thoroughly rinse the grinding

tube and glass rod with tap water prior to a final rinse with

ASTM Type I water and acetone. The last rinse should

be with acetone. Use a clean tissue to remove any filter

residue that adheres to the pestle or to the steel rod of the

pestle. Proceed to the next filter and repeat the steps

above. The last filter extracted should be a blank. The

entire extraction with transferring and rinsing takes

approximately 5 min. Approximately 500 mL of acetone

and water waste are generated per 20 samples from the

rinsing of glassware and apparatus. 

11.1.3 Again, shake each tube vigorously before placing

them to steep in the dark at 4 C. Samples should be

o

allowed to steep for a minimum of 2 h but not to exceed

24 h. Tubes should be shaken at least once, preferably

two to three times, during the steeping period to allow the

extraction solution to have maximum contact with the filter

slurry.

11.1.4 After steeping is complete, centrifuge samples for

15 min at 675 g or for 5 min at 1000 g. Draw

approximately 1 mL into a glass syringe, attach a 0.45 μm

syringe filter, filter the extract into an amber autosampler

vial, cap and label the vial. Protect the filtered samples

from light and heat. If using a refrigerated autosampler,

chill to 10 C. o

 

11.2 Sample Analysis

12.0 Data Analysis and Calculations

12.1 From the chl a or b area response of the sample,

calculate the mass injected or concentration (CE) of the

solution that was analyzed using the calibration data.

Mass injected must be converted to concentration in

extract by dividing mass by volume injected (μL) and

multiplying by 1000 to give concentration in mg/L

(mg/L = μg/mL). Concentration of the natural water

sample may be reported in mg/L by the following

formula:

CE X extract volume (L) X DF 

sample volume (L)

where:

 CE = concentration (mg/L) of pigment in extract.

 DF = any dilution factors.

 L = liters.

12.2 LRB and QCS data should be reported with each

data set.

13.0 Method Performance

13.1 Single Laboratory Performance

13.1.1 An IDL was determined by preparing a mixed chl

a (0.703 ppm) and chl b (0.437 ppm) standard. The

injected mass yielded 0.004 AU for chl a (0.035 μg) and

0.003 AU for chl b (0.022 μg). Seven replicate 50 μL

injections were made and the standard deviation of the

calculated concentration was multiplied by three to

determine an IDL. The IDL determined for chl a was 0.76

ng and 0.44 ng for chl b. The %RSDs for chl a and chl b

was 0.45 and 0.67, respectively.

13.1.2 MDLs for chl a and chl b were determined by

spiking seven replicate filtered samples of Pycnacoccus,

extracting and processing according to this method. An
 447.0-9 Version 1.0 September 1997

injection volume of 100 μL yielded an MDL for chl a of 7.0

ng and 4.0 ng for chl b. The RSDs were 5.1% for chl b

and 4.7 % for chl a.

13.1.3 Recoveries of chl a and chl b from filtered

samples of phaeodactylum were determined by spiking

three filters with known amounts of the pigments,

extracting, processing and analyzing the extraction

solution according to the method, along with three

unspiked filtered samples (to determine the native levels

in the algae). The spiked levels were 1.1 ppm chl a and

0.53 ppm chl b in the 10 mL extraction volume. Chl a

was 87% recovered and chl b was 94% recovered.

13.1.4 Figures 1-7 are chromatograms of seven

reference unialga cultures processed according to this

method.

13.1.5 Table 1 is a list of pure pigments with retention

times obtained using this method. Purified pigments were

prepared under contract to EPA by Moss Landing Marine

Laboratory, Moss Landing, CA.

13.1.6 Table 2 contains single lab precision data for

seven reference algal suspensions.

13.2 Multilaboratory Testing - A Multilaboratory

validation and comparison study of EPA Methods 445.0,

446.0 and 447.0 for chlorophyll a was conducted in 1996

by Research Triangle Institute, Research Triangle Park,

N.C. (EPA Contract No. 68-C5-0011). There were 8

volunteer participants in the HPLC methods component

that returned data. The primary goals of the study were

to determine estimated detection limits and to assess

precision and bias (as percent recovery) for select

unialgal species, and natural seawater.

13.2.1 The term, pooled estimated detection limit (pEDL), is used in this method to distinguish it from the EPA

defined method detection limit (MDL). The statistical

approach used to determine the p-EDL was an adaptation

of the Clayton, et. al. method that does not assume

(21)

constant error variances across concentration and

controls for Type II error. The approach used involved

calculating an estimated DL for each lab that had the

desired Type I and Type II error rates (0.01 and 0.05,

respectively). The median DLs over labs was then

determined and is reported in Table 3. It is referred to as

Pooled-EDL (p-EDL).

The p-EDL was determined in the following manner.

Solutions of pure chlorophyll a in 90% acetone were

prepared at three concentrations (0.11, 0.2 and 1.6 ppm)

and shipped with blank glass fiber filters to participating

laboratories. Analysts were instructed to spike the filters

in duplicate with a given volume of solution and to

process the spiked filters according to the method. The

results from these data were used to determine a p-EDL

for each method. Results (in ppm) are given in Table 3.

The standard fluorometric and HPLC methods gave the

lowest p-EDLs while the spectrophotometric

(monochromatic equations) gave the highest p-EDLs.

13.2.2 To address precision and bias in chlorophyll a

determination for different algal species, three pure

unialgal cultures (Amphidinium, Dunaliella and

Phaeodactylum) were cultured and grown in the

laboratory. Four different “concentrations” of each

species were prepared by filtering varying volumes of the

algae. The filters were frozen and shipped to participant

labs. Analysts were instructed to extract and analyze the

filters according to the respective methods. The “true”

concentration was assigned by taking the average of the

HPLC results for the highest concentration algae sample

since chlorophyll a is separated from other interfering

pigments prior to determination . Pooled precision data

are presented in Tables 4-6 and accuracy data (as

percent recovery) are presented in Table 7. No

significant differences in precision (%RSD) were observed

across concentrations for any of the methods or species.

It should be noted that there was considerable lab-to-lab

variation (as exhibited by the min and max recoveries in

Table 7) and in this case the median is a better measure

of central tendency than the mean.

In summary, the mean and median concentrations

determined for Amphidinium carterae (class

dinophyceae) are similar for all methods. No method

consistently exhibited high or low values relative to the

other methods. The only concentration trend observed

was that the spectrophotometric method-trichromatic

equations (SP-T) showed a slight percent increase in

recovery with increasing algae filtration volume.

For Dunaliella tertiolecti (class chlorophyceae) and

Phaeodactylum tricornutum (class bacillariophyceae)

there was generally good agreement between the

fluorometric and the spectrophotometric methods,

however, the HPLC method yielded lower recoveries with

increasing algae filtration volume for both species. No

definitive explanation can be offered at this time for this

phenomenon. A possible explanation for the

Phaeodactylum is that it contained significant amounts of

chlorophyllide a which is determined as chlorophyll a in

the fluorometric and spectrophotometric methods. The

conventional fluorometric method (FL-STD) showed a

slight decrease in chlorophyll a recovery with increasing

Dunaliella filtration volume. The spectrophotometric-
Version 1.0 September 1997 447.0-10

trichromatic equations (SP-T) showed a slight increase in

chlorophyll a recovery with incresing dunaliella filtration

volume. The fluorometric and athe spectrophotometric

methods both showed a slight decrease in chlorophyll a

recovery with increasing Phaeodactylum filtration volume.

Results for the natural seawater sample are presented in

Table 8. Only one filtration volume (100 mL) was

provided in duplicate to Participant labs.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

USEPA has established a preferred hierarchy of

environmental management techniques that places

pollution prevention as the management option of first

choice. Whenever feasible, laboratory personnel should

use pollution prevention techniques to address their waste

generation (e.g., Sect. 11.1.2). When wastes cannot be

feasibly reduced at the source, the Agency recommends

recycling as the next best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research

institutions, consult Less is Better: Laboratory Chemical

Management for Waste Reduction, available from the

American Chemical Society's Department of Government

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

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

15.0 Waste Management

15.1 The U.S. Environmental Protection Agency

requires that laboratory waste management practices be

conducted consistent with all applicable rules and

regulations. The Agency urges laboratories to protect the

air, water, and land by minimizing and controlling all

releases from hoods and bench operations, complying

with the letter and spirit of any sewer discharge permits

and regulations, and by complying with all solid and

hazardous waste regulations, particularly the hazardous

waste identification rules and land disposal restrictions.

For further information on waste management consult

The Waste Management Manual for Laboratory

Personnel, available from the American Chemical Society

at the address listed in the Sect. 14.2.

16.0 References

1. Holm-Hansen, O., "Chlorophyll a determination:

improvements in methodology," OIKOS, 30

(1978), pp. 438-447.

2. Wright, S.W. and J.D. Shearer, "Rapid extraction

and HPLC of chlorophylls and carotenoids from

marine phytoplankton," J. Chrom., 294 (1984),

pp. 281-295.

3. Bowles, N.D., H.W. Paerl, and J. Tucker,

"Effective solvents and extraction periods

employed in phytoplankton carotenoid and

chlorophyll determination," Can. J. Fish. Aquat.

Sci., 42 (1985) pp. 1127-1131.

4. Shoaf, W.T. and B.W. Lium, "Improved extraction

of chlorophyll a and b from algae using dimethyl

sulfoxide," Limnol. and Oceanogr., 21(6) (1976)

pp. 926-928.

5. Mantoura, R.F.C. and C.A. Llewellyn, "The rapid

determination of algal chlorophyll and carotenoid

pigments and their breakdown products in

natural waters by reverse-phase high

performance liquid chromatography," Anal.

Chim. Acta., 151 (1983) pp. 297-314.

6. Yentsch, C.S. and D.W. Menzel, "A method for

the determination of phytoplankton chlorophyll

and phaeophytin by fluorescence," Deep Sea

Res., 10 (1963), pp. 221-231.

7. Strickland, J.D.H. and T.R. Parsons, A Practical

Handbook of Seawater Analysis, Bull. Fish. Res.

Board Can., 1972, No.167, p. 201.

8. USEPA Method 445.0, "In vitro determination of

chlorophyll a and pheophytin a in marine and

freshwater phytoplankton by fluorescence,"

Methods for the Determination of Chemical

Substances in Marine and Estuarine

Environmental Samples, EPA/600/R-92/121.

9. Wright, S.W., S.W. Jeffrey, R.F.C. Mantoura,

C.A. Llewellyn, T. Bjornland, D. Repeta, and N.

Welschmeyer, "Improved HPLC method for the

analysis of chlorophylls and carotenoids from

marine phytoplankton," Mar. Ecol. Prog. Ser.,

77:183.
 447.0-11 Version 1.0 September 1997

10. Brown, L.M., B.T. Hargrave, and M.D. 20. Weber, C.I., L.A.Fay, G.B. Collins, D.E. Rathke,

MacKinnon, "Analysis of chlorophyll a in and J. Tobin, “A Review of Methods for the

sediments by high-pressure liquid

chromatography," Can. J. Fish. Aquat. Sci., 38

(1981) pp. 205-214.

11. Bidigare, R.R., M.C. Kennicutt, II, and J.M.

Brooks, "Rapid determination of chlorophylls and

their degradation products by HPLC," Limnol.

Oceanogr., 30(2) (1985) pp. 432-435.

12. Minguez-Mosquera, M.I., B. Gandul-Rojas, A.

Montano-Asquerino, and J. Garrido-Fernandez,

"Determination of chlorophylls and carotenoids

by HPLC during olive lactic fermentation," J.

Chrom., 585 (1991) pp. 259-266.

13. Neveux.J., D. Delmas, J.C. Romano, P. Algarra,

L. Ignatiades, A. Herbland, P. Morand, A. Neori,

D. Bonin, J. Barbe, A. Sukenik and T. Berman,

"Comparison of chlorophyll and pheopigment

determinations by spectrophotometric,

fluorometric, spectrofluorometric and HPLC

methods," Marine Microbial Food Webs, 4(2),

(1990) pp. 217-238. 

14. Sartory, D.P., "The determination of algal

chlorophyllous pigments by high performance

liquid chromatography and spectrophotometry,"

Water Research, 19(5), (1985), pp. 605-10.

15. Carcinogens - Working With Carcinogens,

Department of Health, Education and Welfare,

Public Health Service, Center for Disease

Control, National Institute for Occupational Safety

and Health, Publication No. 77-206, 1977.

16. "OSHA Safety and Health Standards, General

Industry," (29 CFR 1910), Occupational Safety

and Health Administration, OSHA 2206, revised

January 1976.

17. Safety in Academic Chemistry Laboratories,

American Chemical Society publication,

Committee on Chemical Safety, 3rd Edition,

1979.

18. "Proposed OSHA Safety and Health Standards,

Laboratories," Occupational Safety and Health

Administration, Federal Register, July 24, 1986.

19. Code of Federal Regulations 40, Ch.1,

Pt.136, Appendix B.

Analysis of Chlorophyll in Periphyton and

Plankton of Marine and Freshwater Systems,”

Oho State University, Grant No. NA84AA-D00079, 1986, 54 pp.

21. Clayton, C.A., J.W. Hine, and P.D. Elkins,

“Detection Limits within Specified Assurance

Probabilities,” Analytical Chemistry, 59(1987), pp.

2506-2514.
Version 1.0 September 1997 447.0-12

17.0 Tables, Diagrams, Flowcharts, and Validation Data

Table 1. Pure Pigments and Retention Times

PIGMENT RETENTION TIME

19' butanoyloxyfucoxanthin 8.13

2,4-divinylpheoporphrin a5 8.60

Peridinin 8.69

Fucoxanthin 8.75

19' hexanoyloxyfucoxanthin 8.90

Neoxanthin 10.07

Chlorophyll C3 10.27

Chlorophyll C2 10.40

Prasinoxanthin 11.20

Violaxanthin 12.00

Diadinoxanthin 15.20

Chlorophyll b 15.60

Myxoxanthophyll 17.00

Aphanaxanthin 17.20

Chlorophyll a 17.80

Monadoxanthin 17.93

Lutein 18.00

Alloxanthin 18.07

Nostaxanthin 18.70

Diatoxanthin 19.07

Zeaxanthin 19.40
 447.0-13 Version 1.0 September 1997

Table 2. Single Lab Precision for Seven Pure Unialgal Cultures

Algae Chlorophyll a Chlorophyll b

Pycnacoccus provasolii N 3 3

(1)

Mean (mg/L) 2.15 1.47 (2)

STD DEV 0.114 0.065

% RSD 5.31 4.45

Rhodomonas salina N 3 3

(1)

Mean (mg/L) 4.0 ND(3)

STD DEV 0.014 ND

% RSD 0.28 ND

Selenastrum N 3 3

capricornitum

(1)

Mean (mg/L) 4.25 0.483

STD DEV 0.199 0.058

% RSD 4.68 12.01

Amphidinium carterae N 3 3

(1)

Mean (mg/L) 2.38 ND

STD DEV 0.176 ND

% RSD 7.40 ND

Dunaliella tertiolecti N 3 3

(1)

Mean (mg/L) 6.68 1.42

STD DEV 0.635 0.0412

% RSD 9.51 2.90

Emiliania huxleyi N 3 ND (1)

Mean (mg/L) 1.03 ND

STD DEV 0.008 ND

% RSD 0.79 ND

Phaeodactylum N 3 ND

tricornutum

(1)

Mean (mg/L) 1.09 ND

STD DEV 0.072 ND

% RSD 7.07 ND

(1) N = Number of filtered samples.

(2) Mean concentration in extract solution.

(3) ND = none detected.
Version 1.0 September 1997 447.0-14

TABLE 3. POOLED ESTIMATED DETECTION LIMITS FOR CHLOROPHYLL A METHODS(1)

_____________________________________________________________

Method N p-EDL (mg/L) (2) (3) (4)

FL -Mod 8 0.096 (5)

FL - Std 9 0.082

HPLC 4 0.081

SP-M 15 0.229

SP-T 15 0.104

_____________________________________________________________

(1) See Section 13.2.1 for a description of the statistical approach used to determine p-EDLs. 

(2) FL-Mod = fluorometric method using special interference filters.

 FL-Std = conventional fluorometric method with pheophytin a correction.

 HPLC = EPA method 447.0

 SP-M = EPA method 446.0, monochromatic equation.

 SP-T = EPA method 446.0, trichromatic equations.

(3) N = number of labs whose data was used.

(4) The p-EDL was determined with p = 0.01 and q (type II error rate) = 0.05.

(5) Due to the large dilutions required to analyze the solutions by fluorometry, the fluormetric p-EDLs are

 unrealistically high.
 447.0-15 Version 1.0 September 1997

TABLE 4. Measured Chlorophyll a (mg/L) in Dunaliella Samples

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

HPLC 5 5 0.172 0.064 36.8

 10 5 0.276 0.074 26.8

 50 5 0.757 0.344 45.4

100 5 1.420 0.672 47.3

___________________________________________________________________________________

(1) Not all participants labs followed the EPA method exactly.

 

(2) N = Number of volunteer labs whose data was used.
Version 1.0 September 1997 447.0-16

TABLE 5. Measured Chlorophyll a (mg/L) in Amphidinium Samples

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

HPLC 5 5 0.104 0.043 56.8

 10 5 0.172 0.083 37.5

 50 5 0.743 0.213 17.4

100 5 1.394 0.631 14.5

___________________________________________________________________________________

(1) Not all participants labs followed the EPA method exactly.

(2) N = number of volunteer labs whose data was used.
 447.0-17 Version 1.0 September 1997

TABLE 6. Measured Chlorophyll a in Phaeodactylum Samples

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

HPLC 5 5 0.193 0.074 38.4

 10 5 0.317 0.114 36.1

 50 5 1.024 0.340 33.2

100 5 1.525 0.487 29.9

___________________________________________________________________________________

(1) Not all participants labs followed the EPA method exactly.

(2) N = number of volunteer labs whose data was used.
Version 1.0 September 1997 447.0-18

TABLE 7. Minimum, Median, and Maximum Percent Recoveries by Genera, Method, and Concentration Level

Species Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 Level 4

Amphidinium Minimum FL-MOD 70 73 75 76

FL-STD 66 91 91 90

HPLC 82 85 87 88

SP-M 36 48 68 64

SP-T 21 63 71 70

Median FL-MOD 105 112 105 104

FL-STD 109 107 111 109

HPLC 102 106 112 105

SP-M 99 101 101 101

SP-T 95 96 106 107

Maximum FL-MOD 121 126 143 146

FL-STD 156 154 148 148

HPLC 284 210 131 116

SP-M 141 133 126 125

SP-T 115 116 119 117

Dunaliella Minimum FL-MOD 162 159 157 156

FL-STD 179 171 165 164

HPLC 165 109 64 41

SP-M 120 188 167 164

SP-T 167 169 166 165

Median FL-MOD 206 246 227 223

FL-STD 250 228 224 210

HPLC 252 177 89 80

SP-M 240 247 247 243
Table 7. Cont’d.

 447.0-19 Version 1.0 September 1997

Species Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 Level 4

SP-T 225 244 256 256

Dunaliella Maximum FL-MOD 295 277 287 288

FL-STD 439 385 276 261

HPLC 392 273 172 154

SP-M 342 316 296 293

SP-T 291 283 283 283

Phaeodactylum Minimum FL-MOD 216 183 157 154

FL-STD 189 220 223 219

HPLC 150 119 84 75

SP-M 161 138 156 160

SP-T 203 195 216 244

Median FL-MOD 292 285 250 245

FL-STD 296 263 254 254

HPLC 225 203 114 90

SP-M 287 274 254 253

SP-T 286 281 277 274

Maximum FL-MOD 357 337 320 318
Version 1.0 September 1997 447.0-20

Table 8. Chlorophyll a Concentrations in mg/L Determined in Filtered Seawater Samples

Method Con. No. Obs No. Labs Mean Std. Dev RSD (%) Minimum Median Maximum (1)

FL-MOD 100 14 7 1.418 0.425 30.0 0.675 1.455 2.060

FL-STD 100 15 8 1.576 0.237 15.0 1.151 1.541 1.977

HPLC 100 10 5 1.384 0.213 15.4 1.080 1.410 1.680

SP-M 100 38 19 1.499 0.219 14.6 0.945 1.533 1.922

SP-T 100 36 18 1.636 0.160 9.8 1.250 1.650 1.948

All Methods 100 113 57 1.533 0.251 16.4 0.657 1.579 2.060

(1) Con = mLs of seawater filtered.