United Stetee Environment*! Monitoring September 1991
Environmental Protection Syeteme Laboratory
Agency Cincinnati, OH 45268
Reteerch end Development
AN INTERIM MANUAL OF
METHODS FOR THE DETERMINATION
OF NUTRIENTS IN ESTUARINE
AND COASTAL WATERS
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AN INTERIM MANUAL OF
METHODS FOR THE DETERMINATION
OF NUTRIENTS
IN ESTUARINE AND COASTAL WATERS
PROJECT OFFICER
LARRY LOBRING, CHIEF
INORGANIC CHEMISTRY BRANCH
CHEMISTRY RESEARCH DIVISION
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This interim manual has been reviewed by the Environmental Monitoring
Systems 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.
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FOREWORD
trtvironmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The Environmental
Monitoring Systems Laboratory - Cincinnati (EMSL-Cincinnati) conducts research
to:
o Develop and evaluate analytical methods to identify and measure the
concentration of chemical pollutants in marine and estuarine waters,
drinking waters, surface waters, groundwaters, wastewaters,
sediments, sludges, and solid wastes.
o Investigate methods for the identification and measurement of
viruses, bacteria and other microbiological organisms in aqueous
samples aid to determine the responses of aquatic organisms to water
quality.
o 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 water, surface water,
groundwater, wastewater, sediment and solid waste.
o 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
bioltjgical indicators.
This EMSL-Cincinnati publication, "Interim Manual of Methods -for the
Determination of Nutrients in Estuarine and Coastal Waters", was prepared as a
new initiative to gather together under a single cover a compendium of
validated laboratory analytical methods for the determination of nutrients in
the marine environment, fcTe -are pleased to provide this manua! and believe
that it will be of considerable value to many public and private laboratories
involved in marine studies for regulatory or other reasons.
Thomas A. Clark, Director
Environmental Monitoring Systems
Laboratory - Cincinnati
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ABSTRACT
This interim manual currently consists of two analytical methods for the
determination of nutrient analytes in marine water samples. The method for
low level ortho-phosphate is based on conventional antimony-phospho-molybdate
complex colorimetry. The applicable concentration range is approximately
0.0006 to 0.05 mg P/L. The nitrate method is based on cadmium reduction of
the nitrate to nitrite- The nitrite originally present and the reduced nitrate
are then determined col&rimetrically by diazotizing with sulfanilamide and
coupling mith N-l-naphthylelhylenediamine dihydrochloride. The applicable
concentration range is approximately 0.0002 to 0,07 mg N/L. These methods
have been multilaboratory tested and contain a statistical summary of the
results obtained in those studies. Methods for additional nutrient analytes
are currently being validated in a similar fashion and will be added to this
manual when completed. This interim manual is considered a growing document
that will eventually expand in scope to include organic and additional
inorganic analytes.
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TABLE OF CONTENTS
Method
Number Title Revision Date Page
Disclaimer. . . ii
Foreword iii
Abstract iv
Acknowledgment vi
Introduction and General Comments . \
353.4 Determination of Nitrite+ 1.1 6/91 3
Uitrate in Estuarine and
Coastal Waters by Automated
Colorimetric Analysis
365.5 Determination of Ortho-Phosphate K2 3/91 21
in Estuarine and Coastal Waters
by Automated Colorimetric Analysis
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ACKNOWLEDGMENT
This methods manual was prepared by the Inorganic Chemistry Branch of the
Chemistry Research Division, Environmental Monitoring Systems Laboratory -
Cincinnati (EMSL-CincinnatiJ. Preparation of the individual manuscripts and
coordination of the multilaboratory studies was performed by the staff of the
University of Maryland System, Chesapeake Biological Laboratory with special
thanks going to Carl Zinwiermann and Carolyn Keefe. Additional efforts by Jim
Longbottom EMSL-Cincinnati and Ken Edgell, The Bionetics Corporation in the
preparation and distribution of quality control samples and statistical
evaluation of the data are very much appreciated.
The overall USEPA effort to standardize analytical methods for use in the
marine environment was identified as a need and championed by the USEPA
Regions. The staff at Region 2 and Region 3 were, and continue to be,
Instrumental in identifying resources for this project and providing insight
from the regional perspective. We appreciate the regional efforts and expect
to call upon them on a continuing basis.
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INTRODUCTION
An Integral component of the role of the U.S Environmental Protection
Agency (USEPA) in assessing and protecting the quality of the environment is
the provision of means for monitoring environmental quality. In keeping with
this role, USEPA develops and disseminates analytical methods for measuring
chemical and physical parameters affecting this most important resource,
including contaminants which may have potential adverse effects upon the
health of our environment. This interim manual provides initially two
analytical methods for nutrient analytes which have been identified by the
USEPA Regions as the highest priority. Additional methods and revisions of
these methods will be made available in regular updates of this manual.
Additional methods for the quantitation of metals in the marine environment
may be found in EPA/600/4-91/010 "Methods for the Determination of Metals in
Environmental Samples", June, 1991.
GENERAL COMMENTS
The methods in this manual are not intended to be specific for any single
USEPA regulation, compliance monitoring program, or specific study. In the
past, manuals have been developed and published that respond to specific
regulations, such as the Safe Drinking Water Act (SDWA) or to special studies
such as the Environmental Monitoring and Assessment Program (EMAP) Near
Coastal Demonstration Project. These methods are, however, available for
incorporation into regulatory programs that require the measurement of
nutrients in marine waters.
The quality assurance sections are uniform and contain minimum
requirements for operating a reliable monitoring program: initial
demonstration of performance, routine analyses of reagent blanks, analyses of
fortified reagent blanks and fortified matrix samples, and analyses of quality
control (QC) samples. Other QC practices are recommended and may be adopted
to meet thr particular needs of monitoring programs e.g., analyses of field
reagent blanks, instrument control samples and performance evaluation samples.
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METHOD 353.4
DETERMKATIOH OF NITRiTE+N URATE IN ESTUARINE AND
COASTAL WATERS BY AUTOMATED COLORIMETRIC ANALYSIS
Carl F. Zimmermann
Carolyn M. Keefe
University of Maryland System
Center for Environmental and Estuarine Studies
Chesapeake Biological Laboratory
Solomons, MD 20688-0038
REVISION 1.1
June 1991
Adapted by
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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METHOD 353.4
DETERMINATION OF NITRITE+NITRATE IN ESTUARINE
AND COASTAL WATERS BY AUTOMATED COLORIMETRIC ANALYSIS
1. SCOPE AND APPLICATION
1.1 This method provides a procedure for the determination of low level
nitrite+nitrate concentrations normally found in estuarine and/or
coastal waters using the cadmium reduction technique/1' Nitrate
concentrations are obtained by subtracting nitrite values, which
have been previously analyzed, from the nitrite+nitrate values.
1.2 A statistically determined method detection limit {HDL} of 0.0002 mg
N/L has been determined by one laboratory.*2* The method is linear
to 0.070 mg N/L at a standard calibration setting of 9.0 on an
AutoAnalyzer II system. Where higher concentrations are
encountered, the method is also linear at less sensitive standard
calibration settings.
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, matrix interferences and procedures
for their correction. A minimum of six months experience under
experienced supervision is recommended.
2. SUMMARY OF METHOD
2.3 An automated colorimetric method for the analysis of low level
nitrite+nitrate concentrations will be described. Filtered samples
are passed through a granulated copper cadmium column to reduce
nitrate to nitrite. The nitrite originally prese: t and the reduced
nitrate are then determined by diazotizing with sulfanilamide and
coupling with N-l-naphthylethylenediaraine dihydrochloride to form a
colored azo dye. The color produced is proportional to the
nitrite+nitrate concentration present in the sample. Nitrate is
obtained by subtracting nitrite values which have been previously
analyzed without the cadmium reduction column from the
nitrite+nitrate values.
3. DEFINITIONS
3.1 Calibration Standard - A solution prepared from the stock standard
solution which is used to calibrate the instrument response with
respect to analyte concentration. One of the standards in the
standard curve.
3.2 Dissolved - Material that will pass through a 0.45 /im membrane
filter assembly.
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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 the method is within accepted
control limits. This is basically a standard prepared in reagent
water which is analyzed as a sample.
3.4 Laboratory Reagent Blank (IRB) - 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.
3.5 Linear Calibration Range - The concentration range over which the
analytical working curve remains linear.
3.6 Method Detection Limit (MDL) - The minimum concentration of an
analyte that can be identified, measured, and reported with 39%
confidence that the analyte concentration is greater than zero.
3.7 Refractive Index - The difference in light intensity due to the
differences between the index of refraction of light in
seawater/estuarine water and deionized distilled water.
3.8 Stock Standard Solution - A concentrated solution of method analyte
prepared in the laboratory using assayed reference compounds or
purchased from a reputable commercial source.
4. INTERFERENCES
4.1 Metal ions may produce a positive error if present in sufficient
concentrations. The presence of large concentrations of sulfide
and/or sulfate will cause a loss of sensitivity to the
copper-cadmium column. (5,<°
4.2 Sample turbidity should be removed by filtration prior to analysis.
4.3 Refractive Index and "Salt Error" interferences should be corrected
for when analyzing estuarine/coastal samples {Sects. 12.2 and 12.3).
5. SAFETY
5.1 Water samples collected from the estuarine and/or ocean environments
are most often not at all hazardous. The individual who collects
samples should use proper technique, however.
5.2 Good laboratory technique should be used when preparing reagents. A
lab coat, safety goggles and gloves should be worn when preparing
the reagents; particularly the copper sulfate, and color reagent.
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5.3 Proper care needs to be demonstrated when operating any scientific
instrument.
6. APPARATUS AND EQUIPMENT
6.1 Continuous flow automated analytical system consisting of:
6.1.1 Sampler.
6.1.2 Manifold or Analytical Cartridge.
6.1.3 Proportioning pump.
6.1.4 Colorimeter equipped with 1.5 X 50 mm tubular flow cell and
550 nm filter.
6.1.5 Phototube: which can be used for 500-550 nm range.
6.1.6 Recorder or computer based data system.
6.2 Nitrogen-free glassware: All glassware used in the determination
must be low in residual nitrate to avoid sample/reagent
contamination. Washing with 10% HC1 and thoroughly rinsing with
reagent water has been found to be effective.
7. REAGENTS AND CONSUMABLE MAfERIALS
7.1 Stock Reagent Solutions
7.1.1 Ammonium Chloride Reagent—Dissolve 10.0 g of ammonium
chloride (NH4CL, CAS RN 12125-02-9) in one liter of reagent
water. Adjust to pH 8.5 by adding three to four NaOH (CAS
RN 1310-73-2) pellets as necessary. Add 5 drops of 2%
copper sulfate solution (Sect. 7.1.3). No addition of EDTA
is necessary. This reagent is stable for one week if kept
refrigerated.
7.1.2 Color keagent—Combine 1500 mL reagent water, 200.0 mL
concentrated phosphoric acid (H3P04, CAS RN 7664-38-2), 20.0
g sulfanilamide (CAS RN 63-74-1), and 1.0 g N-l
napthylethylenediamine dihydrochloride (CAS RN 1465-25-4).
Dilute to 2000 mL with reagent water. Add 2.0 mL BRIJ-35
(Bran & Luebbe, Elmsford, N.Y.). Store at 4*C in the dark.
This should be prepared every six weeks.
7.1.3 Copper Sulfate—Dissolve 2.0 g of copper sulfate
(CuS04.5H,0, ,CAS RN 7758-98-7) in 90.0 mL of reagent water.
Bring up to 100 mL with reagent water.
7.1.4 Refractive Reagent—Combine 100 mL of concentrated
phosphoric acid (H3P04) to 800 mL reagent water. Dilute to
1000 mL with reagent water. Add 1.0 mL BRIJ-35.
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7.1.5 Stock Nitrate Solution—Dissolve 0.721 g of pre-dried (60°C
for 1 hour) potassium nitrate (KNO,, CAS RN 7758-09-0) in
reagent water and dilute to 1000 ml. 1.0 mL « 0.100 mg N.
The stability of this stock standard is approximately 3
months, if kept refrigerated.
7.1.6 Stock Nitrite Solution—Dissolve 0.493 g pre-dried (60°C for
1 hour) sodium nitrite (NaN02, CAS RN 7632-00-0) in reagent
water and dilute to 1000 mL. 1.0 mL - 0.100 mg N. The
stability of this stock standard is approximately 3 months,
if kept refrigerated.
7.1.7 Reagent Water—Type 1 reagent grade water equal to or
exceeding standards established by American Society of
Testing Materials (ASTM) should be used in the preparation
of reagents and standards. Reverse osmosis systems or
distilling units which produce 18 megohm water are two
examples of acceptable water sources.
7.1.8 Low Nutrient Seawater—Obtain natural low nutrient seawater
(36 ppt salinity; <0.0002 mg N/L) or dissolve 31 g
analytical reagent grade sodium chloride, NaCl (CAS
7647-14-5); 10 g analytical reagent grade magnesium sulfate,
MgS04 (CAS 10034-99-8); and 0.05 g analytical reagent grade
sodium bicarbonate, NaHC03 (CAS 144-55-8), in 1 liter of
reagent water.
7.2 Cadmium Preparation—The following description of cadmium and the
column preparation is to be used as a general guideline. It is
recognized that between institution differences in the preparation,
shape, size and configuration of cadmium columns exist. All have
the capability of obtaining excellent results. The ultimate goal is
to obtain 100% reduction of nitrate to nitrite.
Use good quality cadmium (CAS RN 7440-43-9) filings. Depending on
the reductor column shape and size, cadmium filings should generally
be less than 0.5 mm but greater than 0.3 mm for glass columns and in
the 25-60 mesh size range for columns prepared by using flexible
tubing.
New cadmium filings should be rinsed with diethyl ether to remove
dirt and grease.
Approximately 10 grams of this cadmium is then treated with 50 mL of
6N HC1 in a 150 mL beaker. Swirl VERY CAREFULLY for one minute.
Carefully decant the HC1 and thoroughly rinse (at least 10 times)
with reagent water. Decant the reagent water and add a 50 mL
portion of 2% (w/v) copper sulfate solution (Sect. 7.1.3). While
swirling, brown flakes of colloidal copper will appear and the blue
color of the solution will fade. Decant and repeat this sequence
until the blue color does not fade and a brown colloidal precipitate
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forms. From this point on the Cu-Cd filings should not come in
contact with air.
Wash the filings thoroughly with reagent water until all blue color
is gone and the supernatant is free of particulate matter (usually a
minimum of 10 rinses is necessary). The filings are now ready to be
packed into the column.
7.2-1 Column Preparation—Fill the reductor column with ammonium
chloride reagent (Sect. 7.1.1) and transfer the prepared
cadmium filings to the column using a Pasteur pipette or
employ some other method which avoids contact of the Cd
particles with air. One end of the reductor column should
be plugged with glass wool. Column shape and size varies
with users. Some examples include a 22 cm length of 0.110*
ID tubing, a 35 cm length of 0.090" ID tubing or a 3.5"
length of glass tubing.
When the entire column is fairly well packed with granules,
insert another glass wool plug at the top of the column and
with reagents pumping through the system, attach the
column. Remember to have no air bubbles in the valve
(Figure 1) and to attach the column to the intake side of
the valve first.
Check for good flow characteristics (regular bubble pattern)
after the addition of air bubbles beyond the column. If the
column is packed too tightly, an inconsistent flow pattern
will be evident.
Prior to sample analysis, condition the column by pumping
through the sample line approximately 1 mg N (nitrate)/L for
five minutes followed by 1 mg N (nitrite)/L for 10 minutes.
7.2.2 Secondary Nitrate Solution—Dilute 1.0 mL of stock nitrate
solution (Sect. 7.1.5) to 100 mL with reagent water. 1.0 mL
of this solution = 0.001 mg N. Refrigerate and store no
longer than a few days.
7.2.3 Prepare a series of standards by diluting suitable volumes
of Secondary Nitrate Solution (Sect. 7.2.2) 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 Nutrient
Seawater (Sect. 7.1.8) diluted to that salinity and that the
Sampler Wash Solution also be Low Nutrient Seawater (Sect.
7.1.8) diluted to that salinity. 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 (Sect.
12.2). The following dilutions, brought up to 100 mL with
reagent water, are suggested.
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mL of Standard
Nitrate Solution (7.2.2) Cone.,
up to 100 ml reagent water nq N/L
0.5 0.005
1.0 0.010
2.0 0.020
4.0 0.040
6.0 0.060
7.2.4 Saline Nitrate Standards—When analyzing samples of varying
salinities, it is also recommended that standards be
prepared in a series of salinities 1n order to quantify the
"salt error/ the shift in the colorimetric response of
nitrate due to the change in the ionic strength of the
solution. The following dilutions prepared in 100 mL
volumetric flasks, brought up to volume with reagent water,
are suggested.
mL of Low
mL of Standard
Salinity
Nutrient
Nitrate
Cone.,
(ODtl
Seawater f7.1.81
Solution (7.2.2)
mg N/L
0
0
6.0
0.060
9
25
6.0
0.060
18
50
6.0
0.060
27
75
6.0
0.060
34
94
6.0
0.060
7.2.5 Secondary Nitrite Solution—Dilute 1.0 ml of stock nitrite
solution (Sect. 7.1.6) to 100 mL with reagent water. 1.0 mL
of this solution * 0.001 mg N. Refrigerate and store no
longer than a few days.
7.2.6 Working Nitrite Solution—One working standard needs to be
prepared which will act as a check on the reduction capa-
bility of the cadmium column. Therefore, 6.0 mL of (Sect.
7.2.5) up to 100 mL will yield a concentration of 0.060 mg
N/L.
8. 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 and submersible pump systems. The dissolved
fraction is defined as that fraction which passes through a 0.45 tm
pore size filter.
8.1.1 A hydrocast encompasses a series of sampling bottles
(Niskin, Nansen, Go-Flo or equivalent) which are attached at
fixed intervals to a hydro wire. These bottles are sent
through the water column "open" and are then closed either
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electronically or via a "messenger" when the bottles have
reached the desired depth.
8.1.2 The submersible pump system requires a weighted hose being
sent to the desired depth in the water column and water then
being 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: Samples should be analyzed as quickly as
possible. If the samples are to be analyzed within 24 hours of
collection, then refrigeration at 4°C is acceptable. A widely
accepted method of preservation within the oceanographic/estuarine
community for samples requiring longer storage is freezing at -20ttC.
A maximum two month limit of storing these frozen estuarine and
coastal samples has been recommended. Differences between nitrate
samples analyzed immediately on board ship and analyses of frozen
samples were not significant after several d^ys/5* Studies'6, '
recommended freezing inorganic nitrogen samples for a maximum ten
days and that the observed differences between immediate analysis
and that of frozen samples had no practical effect. Freezing
estuarine water samples for nutrient analyses is now an accepted
form of preservation in the Chesapeake Bay Monitoring Program and
the Long Island Sound Study, two National Estuary Program efforts.
8.3 Sample Storage: Long-term storage of frozen samples should be in
clearly labelled polyethylene bottles or polystyrene cups compatible
with the analytical system's automatic sampler (Sect. 6.1.1).
9. CALIBRATION AND STANDARDIZATION
9.1 Calibration (Refer to Sect. 10.2.3).
9.2 Internal standardization (Refer to Sects. 1.2, 6.1.2, 6.1.4, and
6.1.5)
10. QUALITY CONTROL
10.1 A formal quality control (QC) program is strongly recommended. The
minimum requirements of this program should consist of an initial
demonstration of laboratory capability, the continued analysis of
unknowns on an irregular basis as a continuing check on performance,
and an internal program of laboratory duplicates, spikes and
fortified samples which are used as a check of precision and
recovery.
10.2 Initial Demonstration of Performance
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10.2.1 The initial demonstration of performance is used to
characterize instrument performance (method detection limits
and linear calibration ranges).
10.2.2 MDLs should be established for all analytes, using a low
level estuarine water sample. To determine MDL values,
analyze seven replicate aliquots of 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:
HDL - 3(S)
where S ¦ the standard deviation of the replicate
analyses.
MDLs should be determined every six months or whenever a
significant change in background or instrument response
occurs.
10.2.3 Linear calibration ranges - Standard curves should be
analyzed through several standard calibration settings
(calibrated sensitivity settings). In so doing, where
higher concentrations are encountered, the method will also
have been checked for linearity using sets of standards of
higher concentrations.
10.3 Assessing Laboratory Performance
10.3.1 Laboratory Reagent Blank (LRB) - A laboratory should analyze
at least one reagent blank (Sect. 3.5) with each set of
samples. Reagent blank data are used to assess
contamination from the laboratory environment and should an
analyte value in the reagent blank exceed the MDL, then
laboratory or reagent contamination should be suspected.
10.3.2 Laboratory Fortified Blank (LFB) - A laboratory should
analyze at least one fortified blank (Sect. 3.4) with each
batch of samples. Calculate accuracy as percent recovery.
If the recovery of an analyte falls or rises in a consistent
pattern, then the source of the problem should be identified
and resolved before continuing the analyses. This is
basically analyzing standards as sample and is an excellent
check on the overall performance of the entire analytical
system.
10.4 Assessing Analyte Recovery - Laboratory Fortified Sample Matrix
10.4.1 A laboratory should add a known amount of analyte to a
minimum of 5% of the routine samples or one sample per
sample set, whichever is greater. The analyte concentration
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should be high enough to be seen above the original
concentration of the sample and should be at least four
times greater than the MDL.
10.4.2 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 LFB's. Percent recoveries may be calculated using
the following equation:
R = ¦C*~C) x 100
where, R - percent recovery
Cs = actual fortified sample concentration
(background + concentrated addition)
C = Sample background concentration
s = Concentrated addition to the sample
10.4.3 If the recovery of the analyte falls outside the designated
range, but the laboratory performance for that analyte is in
control, ttfe fortified sample should be prepared again and
reanalyzed. If the result is the same after reanalysis, the
recovery problem encountered with the fortified sample is
judged to be matrix related, not system related.
10.5 Precision
10.5.1 A single laboratory analyzed four filtered samples collected
from the Chesapeake Bay. Seven replicates of each sample
were processed and analyzed. The results are as follows:
Concentration Standard Deviation
Sample fmq N/D fma N/L)
PROCEDURE
1 0.1491 0.0009
2 0.0053 0.0012
3 1.3543 0.0127
4 0.0064 0.0004
11.1 If samples have not been freshly collected and are frozen, thaw the
samples to room temperature.
11.2 Set up manifold as shown in Figure 2.
11.3 Allow both colorimeter and recorder to warm up for 30 minutes.
Obtain a steady baseline with reagent water pumping through the
system, add reagents to the sample stream and after the reagent
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baseline is steady; note that rise (reagent baseline), and adjust
baseline.
For analysis of samples with a narrow,salinity range, it is
advisable to use low nutrient seawater as wash water in the sampler
in place of deionized water. For samples with a large salinity
range, it is suggested that deionized water and procedures (Sects.
12.2 and 12.3) be employed.
11.4 A good sampling rate is approximately 40 samples/hr. with a 9:1
sample:wash ratio.
11.5 Place standards (prepared in reagent water~[Sect. 7.2.3] and saline
water—[Sect. 7.2.4], and the working nitrite standard—[Sect.
7.2.6]) in Sampler in order of decreasing concentration. Complete
filling the sampler tray with samples, blanks, internal standards
and other quality control samples.
11.6 Commence analysis.
11.6.1 If the peak height of the 0.060 mg N/L nitrate standard
prepared in deionized water (Sect. 7.2.3) is less than 90%
of the peak height of the 0.060 mg N/L nitrite standard
(Sect. 7.2.6), halt analyses and prepare a new cadmium
reduction column (Sect. 7.2.1).
11.6.2 If a low concentration sample peak follows a high
concentration sample peak, a certain amount of carry over
can be expected. It is recommended that if there is not a
clearly defined low concentration peak, that the sample be
reanalyzed at the end of the sample run.
CALCULATIONS
12.1 Concentrations of nitrite+nitrate are calculated from the linear
regression obtained from the standard curve in which the
concentrations of the standards are entered as the independent
variable and their corresponding peak heights are the dependent
variable.
12.2 Refractive Index Correction For Estuarine/Coastal Systems
12.2.1 The absorbance peak obtained by an automated system for
nitrate in a seawater sample (when compared to a reagent
[deionized] water baseline) represents the sum of
absorbances from at least four sources: (1) the light
changes due to the differences in the index of refraction of
the seawater and reagent water; (2) reaction products (e.g.,
precipitates) of 8RIJ-35 and the seawater; (3) the
absorbance of colored substances dissolved in the sample;
and (4) reaction products of the nitrite and the nitrate
(reduced to nitrite by the cadmium column) in the sample
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with the color reagent.(8) The first three sources of color
are corrected for by the refractive index correction
described here.
12.2.2 Obtain a second set of peak heights for all samples and
standards with Refractive Reagent (Sect. 7.1.4) being pumped
through the system in place of Color Reagent (Sect. 7.1.2).
All other reagents remain the same. 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.
12.2.3 Subtract the refractive index peak heights from the heights
obtained for the nitrate determination.
12.2.4 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. First analyze a set of nitrate standards (Sect.
7.2.3) with Color Reagent (Sect. 7.1.2) and obtain a linear
regression from the standard curve (Sect. 12.1). For each
sample, the apparent nitrate concentration due to refractive
index is then calculated from its peak height obtained with
Refractive Reagent (Sect. 7.1.4) and the regression of
nitrate standards obtained with Color Reagent (Sect. 7.1.2)
for each sample. Its salinity is entered as the independent
variable (X variable) and its apparent nitrate due to its
refractive index in that colorimeter is entered as the
dependent variable (Y variable). The resulting regression
allows the operator to subtract an apparent nitrate
concentration when the salinity is known, as long as other
matrix effects (Sects. 12.2.2-2 and 12.2.2-3) remain
unchanged. Thus, the operator would.not be required to
obtain refractive index peak heights for all s.;nples after a
large data set has been found to yield consistent apparent
nitrate concentrations due to salinity. An example of
typical results from one laboratory follows:
Salinity fppt)
Apparent nitrate conc. due
to refractive index (ma N/L)
1
6
10
22
0.0001
0.0004
0.0007
0.0015
12.2.5 An example of a typical equation is:
mg N/L apparent N03 = 0.000069 X Salinity (ppt)
where 0.000069 is the slope of the line
14
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12.3 Correction for Salt Error in Estuarine/Coastal Samples
12.3.1 When calculating concentrations of samples of varying
salinities from standards prepared in reagent water, it is
necessary to first correct for Refractive Index errors
(Sect. 12.2), then correct for the "Salt Error" alteration
in color development due to the ionic strength of the
samples.
12.3.2 Plot the salinity of the saline standards as the independent
variable (X variable) and the apparent concentration cf
nitrate (mg N/L) from the peak height corrected for
refractive index (Sect. 12.2) calculated from the regression
of standards in reagent water (Sects. 7.2.3 and 12.1) as the
dependent variable (Y variable) for all 0.060 mg N/L
standards. The resulting regression allows the operator to
correct the concentrations of the samples of known salinity
for the color enhancement due to Salt Error. An example of
typical results from orie laboratory fallows:
Peak height of Uncorrected
0.060 mg N/L mg N/L calculated
standard after from regression
Salinity correction for of standards
(opt) refractive index in reagent water
0 85 Q.0600
9 87 0.0614
16 89 0.0628
27 92 0.0649
34 94 0.0663
12.3.3 An example of a typical equation tD correct for "Salt Error"
is:
Corrected mg N/L = Uncorrected ma N/L X 0.0600
(Salinity X 0.000187) + 0.060
where 0.0533 Is the concentration of nitrate standard (Sect.
7.2.4) present in each saline standard; salinity of the
sample is in ppt; 0.000187 is the slope of the regression
equation (Sect. 12.3.1); and 0.060 is the y intercept of the
regression equation (Sect. 12.3.1}.
12.4 Results should be reported in mg N/L, fig N/L or jig at N/L.
Table 1. describes these various units and conversions normally used
in estuarine/coastal nutrient analyses.
PRECISION AND ACCURACY
Not Yet Determined - See Attachment 1.
15
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REFERENCES
1. Wood, E.D., F.A.G. Armstrong and F.A. Richards. 1967. Determination
of Nitrate in Seawater by Cadmium-Copper Reduction to Nitrite. J.
Mar. Biol. Assoc. U.K. 47: 23.
2. 40 CFR, [part] 136 Appendix B. Definition and Procedure for the
Determination of the Method Detection Limit. Revision 1.11.
3. U.S. EPA. 1974. Methods for Chemical Analysis of Water and Wastes.
Methods Development and Quality Assurance Research Laboratory.
National Environmental Research Center. Cincinnati, Ohio 45268.
4. Grasshoff, K., M. Ehrhardt and K. Kremling. 1983. Methods of
Seawater Analysis. Verlag Chemie, Federal Republic of Germany.
419 pp.
5. 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.
6. Thayer, G.W. 1970. Comparison of Two Storage Methods for the
Analysis of Nitrogen and Phosphorus Fractions in Estuarine Water.
Ches. Sci. 11:3, 155-158.
7. 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. 32pp.
8. Loder, T.C. and P.M. Glibert. 1977. Blank and Salinity Corrections
for Automated Nutrient Analysis of Estuarine and Seawaters. 7th
Technic n International Congress: 48-56, Tarrytown, N.Y.
9. Froelich, P.N. and M.E.Q. Pilson. 1978. Systematic Absorbance
Errors with Technicon AutoAnalyzer II Colorimeters. Water Research
12:599-603.
10. ADDITIONAL BIBLIOGRAPHY
10.1 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. Qua!. 5:1
42-46.
16
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Table 1. Commonly used terminology to describe concentration.
mg N/L = ppm (parts per million)
Hq N/L » ppb (parts per billion)
fig at N/L - mq N/L
0.014
fig at N/L - ua N/L
14
17
-------
Figure 1. Reduction column valve assembly
4
I
1
Valve Assembly
Column
Not drawn to scale
18
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Figure 2. MANIFOLD CONFIGURATION FOR NITRITE+NITRATE
To Sample Wash Receptacle
GRN/GRN (Water) 2.0 mL/min
o
o
o
o
o
Debubbler
5 Turn Coll
OOP
Cadmium
Reduction
Column
Color Reagent
22 Turn Coil
Waste
COLORIMETER
550 nm filters
3LK/BLK (Air) 0.32 mL/min
WHT/WHT 0.6 mL/min
BLK/BLK (Air) 0.32 mL/min
YEL/YEL (NH4CI) 1.2 mL/min
Sampler
40/hr.
9:1
BLK/BLK (Sample) 0.32 mL/min
BLK/BLK 0.32 mL/min
GRY/GRY (From F/C)
1.0 mL/min
50 x 1.5 mm ID F/C 199-B021-01 Phototube
19
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ATTACHMENT 1
EMSl wwNITRATE JULY 1991
NURirE-tMITAATE IN ESTUARIME/CQASTAL WATERS
LAB 0 1 3 4 5 6 7 8 10 11 12 13
SAMPLE A.A H3 P04
HPEL UHH AfWL CBL MEM. ECU USEFA VIHS MEL MOSS LAND HRSOHSL
Uttw 0.0000 0.0000 0.0018 0.0000 -0.0001 0.0019 0.0000 0.0000 - 0.0001 0.0000 0.0000
RWS 1 0.0005 0.0032 0.0136 0.0111 0.0106 0.0001 0.0123 0.0080 0.0110 0.0108 0.0137
sws 2 0.0072 0.0068 0.0110 0.0090 0.0090 O.OOB3 0.0109 0.0055 0.0089 0.0094 0.0088
AUS ] 0.0210 0.0188 0.0253 0.0220 0.0216 0.0186 0.0253 . 0.0172 0.0223 0.0230 0.0249
RUS 4 0.0264 0.0245 0.0309 0.0277 0.0270 0.0244 0.0296 0.0224 0.0279 0.0284 0.0325
BUS 5 0.0024 O.OOU 0.0051 0.0044 0.0042 0.0056 0.0054 0.0049 0.0045 0.0048 0.0038
BUS 6 0.0010 0.0020 0.0036 0.0032 0.0032 0.0053 0.0049 0.0025 0.0033 0.0036 0.0043
RWS 7 0.0429 0.0405 0.0452 0.0437 0.0434 0.0418 0.0474 0.0598 0.0440 0.0460 0.0489
RUS « 0.0548 0.0531 0.0574 0.0552 0.0570 0.0537 0.0559 0.0483 0.0553 0.0577 0.0606
OC 1 0.0152 0.0160 0.0169 0.0171 0.0164 0.0156 0.0167 0.0135 0.0171 0.0175 0.0155
USS 0.0000 0.0079 0.0006 0.0050 0.0013 0.0076 0.0000 0.0031 0.0006 0.0070 0.0023
SSS ? 0.0063 0.0177 0.0249 0.0119 0.0195 0.0134 0.0617 0.0376 0.0265 0.0185 0.0153
SSS 10 0.0036 0.0158 0.0235 0.0098 0.0165 0.0126 0.0584 0.0170 0.0251 0.0156 0.0105
SSS 11 0.0183 0.0230 0.0210 0.0254 0.0300 0.0226 0.0693 0.0326 . 0.0342 0.027V 0.0176
SSS 12 0.0251 0.0306 0.0208 0.0300 0.0357 0.0291 0.0709 0.0232 0.0349 0.0331 0.0238
SSS 15 0.0006 0.0082 O.OOS2 0.0080 0.0119 0.0103 0.0305 0.0061 0.0106 0.0110 0.0096
SSS 14 Q.0000 0.0040 0.0046 0.0062 0.0057 0.0096 0.0288 0.0039 0.0045 0.0103 0.0053
SSS 15 0.0408 0.0441 0.0318 0.0491 0.0463 0.0444 0.0326 0.0392 0.0316 0.0488 0.0288
SSS 16 0.0536 0.0586 0.0378 0.0573 0.0609 0.0560 0.0410 0.0530 0.0377 0.0589 0.0370
OC 2 • 0.0253 0.0179 0.0181 0.0167 0.0148 0.0087 0.0129 0.0190 0.0183 0.0153
, J, UCS 1 0.0000 0.0031 0.0013 0.0010 0.0027 0.0014 0.0000 -0.0004 0.0008 0.0014 0.0021
o nl \7 0.0424 0.0455 0.0429 0.0445 0.0454 0.0450 0.04T5 Q.CiOZ 0.0412 0.0498 0.0(10
CD I IB 0.0542 0.05-80 0.0425 0.05SB 0.06T3 0.0457 O.OSIO 0.05U 0.0513 0.G614 0.0490
OC 5 0.0151 0.(167 0.0165 0.0174 0.01W 0.0159 0.01 BO 0.0129 0.0184 O.Om 0.0153
UCt 2 0.0008 0.001B 0.0074 0.0016 0.0007 0.0010 0.0000 0.0052 0.0005 O.OOI5 0.002B
CB2 19 0.0116 0.00B9 0.0165 0.0122 0.0113 0.0079 0.0096 0.0122 0.0089 0.0112 0.0099
CB2 20 0.0096 0.0089 0.0147 0.0106 0.0087 0.0064 0.0005 0.0116 O.OOT4 0.0094 0.0156
OC 4 0.0155 0.0139 0.0173 0.0171 0.0168 0.0159 0.0182 0,0141 0.0193 • 0.0153
UC8 3 0.0174 0.0203 0.0178 0.0209 0.0224 0.0116 0.0189 0.0160 0.0175 0.0182 0.0111
C83 21 0.0397 0.0452 0.0373 0.0426 0.0682 0.0340 0.0400 0.0356 0.0355 0.0434 0.0294
CSS 22 0.0457 0.0525 0.0422 0.0461 0.0732 0.0395 0.0430 0.0432 0.0404 0.0485 0.0346
AC 5 0.0156 0.0192 0.0171 0.0176 0.01A9 0.0150 0.0184 0,0133 0.0194 0.0172 0.0139
• HOT RUN
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Method 365.5
DETERMINATION OF ORTHO-PHOSPHATE 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 20688-0038
REVISION 1.2
March 1991
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
21
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METHOD 365.5
DETERMINATION OF ORTHO-PHOSPHATE IN ESTUARINE AND
COASTAL WATERS BY AUTOMATED COLORIMETRIC ANALYSIS
1. SCOPE AND APPLICATION
1.1 This method provides a procedure for the determination of low level
ortho-phosphate concentrations normally found in estuarine and/or
coastal waters. It is basically the method of Murphy and Riley' *
adapted for automated segmented flow analysis'" in which the two
reagent solutions are added separately for greater reagent stability
and facility of sample separation.
1.2 A statistically determined method detection limit (MDL) of 0.0006 mg
P/l has been determined by one laboratory.4 5 The method is linear
to 0.050 mg P/l at a standard calibration setting of 9.0 on an
AutoAnalyzer II system. Where higher concentrations are
encountered, the method is also linear at lower standard calibration
settings.
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, matrix interferences and procedures
for their correction. A minimum of six months experience under
experienced supervision is recommended.
2. SUMMARY OF METHOD
2.1 An automated colorimetric method for the analysis of low level
ortho-phosphate concentrations will be described. Ammonium
molybdate and antimony potissium tartrate react in an acid medium
with dilute solutions of phosphorus to form an antimony-phospho-
molybdate complex. This complex is reduced to an intensely
blue-colored complex by ascorbic acid. The color produced is
proportional to the phosphate concentration present in the sample.
3. DEFINITIONS
3.1 Calibration Standard - A solution prepared from the stock standard
solution which is used to calibrate the instrument response with
respect to analyte concentration. One of the standards in the
standard curve.
3.2 Dissolved - Material that will pass through a 0.45 pun membrane
filter assembly.
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
22
-------
purpose is to determine whether the method is within accepted
control limits. This is basically a standard prepared in reagent
water which is analyzed as a sample.
3.4 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.
3.5 Linear Calibration Range - The concentration range over which the
analytical working curve remains linear.
3.6 Method Detection Limit - The minimum concentration of an analyte
that can be identified, measured, and reported with 99% confidence
that the analyte concentration is greater than zero.
3.7 Refractive Index - The difference in light intensity due to the
differences between the index of refraction of light in
seawater/estuarine water and deionized distilled water.
3.8 Stock Standard Solution - A concentrated solution of method analyte
prepared in the laboratory using assayed reference compounds or
purchased from a reputable commercial source.
4. INTERFERENCES
4.1 It is reported that the interference caused by copper, arsenate
and/or silicate is minimal to the ortho-phosphate determination
because of their extremely low concentrations normally found in
estuarine or coastal waters. High iron concentrations can cause
precipitation of and subsequent loss of phosphorus. Hydrogen
sulfide effects (samples collected from deep anoxic basins) can be
treated by simple dilution of the sample, .ince high sulfide
concentrations are most often associated with high phosphate
values.(4)
4.2 Mercuric chloride, used as a preservative, interferes.(5>
4.3 Sample turbidity should be removed by filtration prior to analysis.
4.4 Refractive Index interferences should be corrected for estuarine/
coastal samples (Sect. 12.2).
5. SAFETY
5.1 Water samples collected from the estuarine and or ocean environment
are most often not at all hazardous. The individual who collects
samples should use proper technique, however.
23
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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.
5.3 Proper care needs to be demonstrated when operating any scientific,
instrument.
6. APPARATUS AND EQUIPMENT
6.1 Continuous flow automated analytical system consisting of:
6.1.1 Sampler.
6.1.2 Manifold or Analytical Cartridge equipped with 37°C heating
bath.
6.1.3 Proportioning pump.
6.1.4 Colorimeter equipped with 1.5 X 50 mm tubular flow cell and
880 nm filter.
6.1.5 Phototube: which can be used for 600-900 nm range.
6.1.6 Recorder or computer based data system.
6.2 Phosphate-free glassware: All glassware used in the determination
must be low in residual phosphate to avoid sample/reagent
contamination. Mashing with 10% HC1 and thoroughly rinsing with
distilled/deionized water has been found to be effective.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Stock Reagent Solutions
7.1.1 Ammonium Molybdate Solution (40 g/L>— Dissolve 20.0 g of
ammonium molybdate tetrahydrate ((NHJ^0^.4^0, CAS RN
12027-67-7)) in approximately 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 3
months.
7.1.2 Antimony Potassium Tartrate Solution (3.0 g/L) — Dissolve
0.3 g of antimony potassium tartrate [(K(SbO)C4H,06 * 1/2
H20, CAS RN 11071-15-1] in approximately 90 ml of reagent
water and dilute to 100 mL. This reagent is stable for
approximately 3 months.
7.1.3 Ascorbic Acid Solution (18.0 g/L) — Dissolve 18.0 g of
ascorbic acid (C,H606, CAS RN 50-81-7) in approximately 800
mL of reagent water and dilute to one liter. Dispense
approximately 75 mL into clean poly bottles and freeze. The
stability of the frozen ascorbic acid is approximately 3
24
-------
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 Oodecyl
sulfate, CHjCCHj)llOSQjNa, CAS RN 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 it's stability is approximately three
weeks.
7.1.5 Sulfuric Acid Solution (4.9 N) -- Add 136 mL concentrated
sulfuric acid (H2S04, CAS RN 7664-93-9) to approximately 800
mL reagent water while cooling. After the solution is
cooled, dilute to one liter with reagent water.
7.1.6 Stock Phosphorus Solution ~ Dissolve 0.439 g of pre-dried
(105 degree C for 1 hour) potassium phosphate, monobasic
(KH,P0,f CAS RN 7778-77-0) in deionized water and dilute to
1000 mL. 1.0 mL - 0.100 mg P. The stability of this stock
standard is approximately 3 months, if kept refrigerated.
7.1.7 Reagent Water — Type 1 reagent grade water equal to or
exceeding standards established by American Society of
Testing Materials (ASTM) should be used in the preparation
of reagents and standards. Reverse osmosis systems or
distilling units which produce 18 megohm water are two
examples of acceptable water sources.
7.1.8 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 (CAS
7647-14-5); 10 g analytical grade magnesium sulfate, MgSO,
(CAS 10034-99-8); and 0.05 g analytical reagent grade sodium
bicarbonate, NaHC03 (CAS 144-55-8), in 1 liter of deionized
water.
Working Reagents
7.2.1 Reagent A — Mix the following reagents in these proportions
for 142 mL of Reagent A: 100 mL of 4.9N H?SO, (Sect. 7.1.5),
30 mL of ammonium molybdate solution (Sect. 7.1.1), 10 ml of
antimony potassium tartrate solution (Secct. 7.1.2), and 2.0
mL of SLS solution (Sect. 7.1.4). Prepare daily.
7.2.2 Reagent B — Add approximately 0.5 mL of the SLS solution
(Sect. 7.1.4) to the 75 ml of ascorbic acid solution (Sect.
7.1.3). Stability is approximately 10 days if kept
refrigerated.
-------
7.2.3 Refractive Reagent A — Add 50 mL of 4.9 N H2S04 (Sect.
7.1.5) to 20 mL of reagent water. Add 1 mL of SLS (Sect.
7.1.4) to this solution. Prepare every few days.
7.2.4 Secondary Phosphorus Solution — Take 1.0 mL of Stock
Phosphorus Standard (Sect. 7.1.6) and dilute to 100 mL with
deionized water. 1.0 mL - 0.0010 mg P. Refrigerate and
keep no longer than 10 days.
7.2.5 Prepare a series of standards by diluting suitable volumes
of standard solutions (Sect. 7.2.4) to 100 mL with deionized
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. When analyzing samples of varying
salinities, it is recommended that the standard curve be
prepared in deionized water and refractive index corrections
be made to the sample concentrations (Sect. 12.2). The
following dilutions are suggested.
8. 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 and submersible pump systems. The dissolved
fraction is defined as that fraction which passes through a 0.45 /an
pore size filter.
8.1.1 A hydrocast encompasses a series of sampling bottles
(Niskin, Nansen, Go-Flo or equivalent) which are attached at
fixed intervals to a hydro wire. These bottles are sent
through the water column "open* and are .then closed either
electronically or via a "messenger" when the bottles have
reached the desired depth.
8.1.2 The submersible pump system requires a weighted hose being
sent to the desired depth in the water column and water then
being pumped from that depth to the deck of the ship for
processing.
mL of Standard
Phosphorus Solution (7.2.4^
Cone.
0.1
0.2
0.5
1.0
2.0
4,0
5.0
0.0010
0.0020
0.0050
0.0100
0.0200
0.0400
0.0500
26
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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: Samples should be analyzed as quickly as
possible. If the samples are to be analyzed within 24 hours of
collection, then refrigeration at 4"C is acceptable. A widely
accepted method of preservation within the oceanographic/estuarine
community for samples requiring longer storage is freezing at -20°C.
No change in soluble inorganic phosphate concentration was found in
river water samples after 12 weeks of freezing at -20°C.(6> A
maximum two month limit of storing frozen estuarine and coastal
samples has been recommendedc2) while others(8) recommended freezing
inorganic phosphorus samples for a maximum ten days and that the
observed differences between immediate analysis and that of frozen
samples had no practical effect.{9) Freezing estuarine water
samples for nutrient analyses is now an acccepted form of
preservation in the Chesapeake Bay Monitoring Program and the Long
Island Sound Study, two National Estuary Program efforts.
8.3 Sample Storage: Long-term storage of frozen samples should be in
clearly labelled polyethylene bottles or polystyrene cups compatible
with the analytical system's automatic sampler (Sect. 6.1.1).
9. CALIBRATION AND STANDARDIZATION
9.1 Calibration (Refer to Sect. 10.2.3).
9.2 Internal standardization (Refer to Sects. 1.2, 6.1.2, 6.1.4, and
6.1.5)
10. QUALITY CONTROL
10.1 A formal quality control (QC) program is strongly recommended. The
minimum requirements of this program should consist of an initial
demonstration of laboratory capability, the continued analysis of
unknowns on an irregular basis as a continuing check on performance,
and an internal program of laboratory duplicates, spikes and
fortified samples which are used as a check of precision and
recovery.
10.2 Initial Demonstration of Performance
10.2-1 The initial demonstration of performance is used to
characterize instrument performance (MDLs and linear
calibration ranges).
10.2.2 MDLs should be established for all analytes, using a low
level estuarine water sample. To determine MDL values,
analyze seven replicate aliquots of water and process
through the entire analytical method. Perform all
27
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calculations defined in the method and report the
concentration values in the appropriate units. Calculate
the MDL as follows:
HDL - 3(S)
where S » the standard deviation of the replicate analyses.
Method detection limits should be determined every six
months or whenever a significant change in background or
instrument response occurs.
10.2.3 Linear calibration ranges - Standard curves should be
analyzed through a number of standard calibration settings.
In so doing, where higher concentrations are encountered,
the method will also be linear for different sets of
standards at lower standard calibration settings.
10.3 Assessing Laboratory Performance
10.3.1 Laboratory Reagent Blank (LRB) - A laboratory should analyze
at least one reagent blank (Sect. 3.5) with each set of
samples. Reagent blank data are used to assess
contamination from the laboratory environment and should an
analyte value in the reagent blank exceed the MDL, then
laboratory or reagent contamination should be suspected.
10.3.2 Laboratory Fortified Blank (LFB) - A laboratory should
analyze at least one fortified blank (Sect. 3.4) with each
batch of samples. Calculate accuracy as percent recovery.
If the recovery of an analyte falls or rises in a consistent
pattern, then the source of the problem should be identified
and resolved before continuing the analyses. This is
basically analyzing standards as sample and is an excellent
check on the overall performance of the entire analytical
system.
10.4 Assessing Analyte Recovery - Laboratory Fortified Sample Matrix
10.4.1 A laboratory should add a known amount of analyte to a
minimum of 5% of the routine samples or one sample per
sample set, whichever is greater. The analyte concentration
should be high enough to be seen above the original
concentration of the sample and should not be less than four
times the MDL.
10.4.2 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 LFB's.
28
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Percent recoveries may be calculated using the following
equation:
(Cs-C)
R = x 100
where, R » percent recovery
Cs « actual fortified sample concentration
(background + concentrated addition)
C = Sample background concentration
s » Concentrated addition to the sample
10.4.3 If the recovery of the analyte falls outside the designated
range, but the laboratory performance for that analyte is in
control, the fortified sample should be prepared again and
reanalyzed. If the result is the same after reanalysis, the
recovery problem encountered with the fortified sample is
judged to be matrix related, not system related.
10.5 Precision
10.5:1 A single laboratory analyzed four filtered samples collected
from distinctly different areas of the Chesapeake Bay.
Seven replicates of each sample were processed and analyzed.
The results are as follows:
Concentration Standard Deviation
Sample (mo P/Ll (mq P/Ll
1 0.0484 0.0005
2 0.0033 0.0007
3 0.0112 0.0002
4 0.0011 0.0004
29
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11. PROCEDURE
11.1 If samples have not been freshly collected and are frozen, thaw the
samples to room temperature.
11.2 Set up manifold as shown in Figure 1.
11.3 Allow both colorimeter and recorder to warm up for 30 minutes.
Obtain a steady baseline with deionized water pumping through the
system, add reagents to the sample stream and after the reagent
baseline is steady; note that rise (reagent baseline), and adjust
baseline.
For analysis of samples with a narrow salinity range, it is
advisable to use low nutrient seawater as wash water in the sampler
in place of deionized water. For samples with a large salinity
range, it is suggested that deionized wash water and procedure Sect.
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 in Sampler in order of decreasing concentration.
Complete filling the sampler tray with samples, blanks, internal
standards and other quality control samples.
11.6 Commence analysis.
12. CALCULATIONS
12.1 Concentrations of ortho-phosphate are calculated from the linear
regression obtained from the standard curve in which the
concentrations of the standards are entered as the independent
variables and their corresponding peak heights are the dependent
variables.
12.2 Refractive Index Correction for Estuarine/Coastal Systems
12.2.1 Obtain a second set of peak heights for all samples and
standards with Refractive Reagent A {Sect. 7.2.3) being
pumped through the system in place of Reagent A (Sect.
7.2.1). Reagent B (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/10'
12.2.2. Subtract the refractive index peak heights from the heights
obtained for the ortho-phosphate determination. Calculate
the regression equation using the corrected standard peak
30
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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 ortho-phosphate
concentration due to refractive index is calculated from its
peak height obtained with Refractive Reagent A (Sect. 7.2.3)
and Reagent B (Sect. 7.2.2) and the regression of ortho-
phosphate standards obtained with ortho-phosphate Reagent A
(Sect. 7.2.1) and Reagent B (Sect. 7.2.2) for each sample.
Its salinity is entered as the independent variable (X
variable) and its apparent ortho-phosphate concentration due
to its refractive index in that colorimeter is entered as
the dependent variable (Y variable). The resulting
regression allows the operator to subtract an apparent
ortho-phosphate 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 ortho-phosphate
concentrations due to salinity. An example follows:
12.2.4 An example of a typical equation is:
mg P/L apparent P04 = 0.000087 X Salinity (ppt)
where 0.000087 is the slope of the line
12.3 Results should be reported in mg P/L, /ig P/L or (ig at P/L.
Table 1. describes these various units and conversions normally
used in estuarine/coastal nutrient analyses.
PRECISION AND ACCURACY
13.1 In a collaborative study involving ten laboratories, reagent water,
mixed-salinity Chesapeake Bay water, and Sargasso Sea water were
spiked and analyzed at three Youden pair concentrations in the range
of 5.0-95.9 M9/L using this method. Data summaries and regression
equations are provided in Table 2 that describe the mean recovery,
single-operator standard deviation and overall standard deviation
observed in this study.
Salinity (optl
Apparent ortho-phosphate
conc. due to refractive
index (ma P/L)
1
5
10
20
0.0002
0.0006
0.0009
0.0017
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13.2 In the same study, the laboratories also analyzed reagent water and
Sargasso Sea water fortified at 1.2 and 1.6 fig/L. The
interlaboratory estimate of the method detection limit (IMOL) was
estimated from the reagent water results to be 2.0 #ig/L.
REFERENCES
1. Murphy, 0. and J.P. Riley. 1962. A Modified Single Solution Method
for the Determination of Phosphate in Natural Haters. Analytica
Chim. Acta 27: 31-36.0.
2. Technicon Industrial Systems. 1973. Ortho-phosphate in Water and
Seawater. Industrial Method 155-71W. Technicon Industrial Systems,
Tarrytown, N.Y. 10591.
3. 40 CFR, [part] 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
pp.
5. USEPA. 1974. Methods for Chemical Analysis of Water and Wastes.
Methods Development and Quality Assurance Research Laboratory.
National Environmental Research Center. Cincinnati, Ohio 45268.
6. 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.
7. MacDortald, 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.
8. Thayer, G.W. 1970. Comparison of Two Storage Methods for the
Analysis of Nitrogen and Phosphorus Fractions in Estuarine Water.
Ches. Sci. 11:3, 155-158.
9. 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, YA., 23062. 32pp.
10. Froelich, P.N. and M.E.Q. Pilson. 1978. Systematic Absorbance
Errors with Technicon AutoAnalyzer II Colorimeters. Water Research
12: 599-603.
32
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Table 1. COMMONLY USED TERMINOLOGY TO DESCRIBE CONCENTRATION.
mg P/L » ppm (parts per million)
fig P/L « ppb (parts per billion)
Hq at P/L - mg P/L
0.031
ng at P/L - jig P/t.
31
33
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TABLE 2. SINGLE-ANALYST PRECISION, OVERALL PRECISION AND RECOVERY
WATER TYPE ORTHO-PHOSPHATE
0*g/L)
APPLICABLE CONC. RANGE (5.00 - 95.90)
REAGENT WATER
SINGLE-ANALYST PRECISION SR - 0.012X + 0.31
OVERALL PRECISION S - 0.030X + 0.41
MEAN RECOVERY X - 1.004C - 0.38
SARGASSO SEA WATER
SINGLE-ANALYST PRECISION SR - 0.026X + 0.29
OVERALL PRECISION S = 0.097X + 0.38
MEAN RECOVERY X = 1.068C - 0.74
CHESAPEAKE BAY WATER
SINGLE-ANALYST PRECISION SR =» 0.030X + 0.16
OVERALL PRECISION S = 0.066X + 0.07
MEAN RECOVERY X = 1.019C - 0.87
X = MEAN RECOVERY
C = TRUE VALUE FOR THE CONCENTRATION
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Figure 1. MANIFOLD CONFIGURATION FOR ORTHO-PHOSPHATE
To Sample Wash Receptacle
GRN/GRN (Water) 2.0 mL/min
37degree
Seating
bath
5 Turn Coils
mo noo
BLK/BLK (Air) 0.32 mL/min
YEL/YEL (Sample) 1.2 mL/min
Reagent B
Waste
COLORIMETER
880 nm filters
Sampler
40/hr.
9:1
ORN/WHT 0.23 mL/min
Reagent A
ORN/GRN 0.10 mL/min
ORN/ORN (From F/C)
0.42 mL/min
50 x 1.5 mm ID F/C 199-B021-04 Phototube
35
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