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Environmental Technology
Verification Program
Advanced Monitoring
Systems Center
Test/QA Plan for Long-Term
Deployment of Multi-Parameter
Water Quality Probes/Sondes
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TEST/QA PLAN
FOR
LONG-TERM DEPLOYMENT OF
MULTI-PARAMETER WATER QUALITY PROBES/SONDES
May 13, 2002
Prepared by
Battelle
505 King Avenue
Columbus, OH 43201-2693
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Approval of ETV Advanced Monitoring Systems Center
"Test/QA Plan for Long-Term Deployment of
Multi-Parameter Water Quality Probes/Sondes"
Version 1.0
May 13,2002
Name
Company
Date
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DISTRIBUTION LIST
Ms. Elizabeth A. Betz
U.S. Environmental Protection Agency
National Exposure Research Laboratory
MD-44
Research Triangle Park, NC 27711
Mr. Robert Fuerst
U.S. Environmental Protection Agency
National Exposure Research Laboratory
MD-46
Research Triangle Park, NC 27711
Ms. Elizabeth Hunike
Quality Assurance Specialist
U.S. Environmental Protection Agency
National Exposure Research Laboratory
ERC Annex, MD-46
Research Triangle Park, NC 27711
Dr. Geoffrey Scott
Chief, Marine Ecotoxicology Branch
NOAA/NOS Center for Coastal Environmental
Health & Biomolecular Research
219 Ft. Johnson Road
Charleston, SC 29412
Dr. Paul Pennington
Research Specialist II
NOAA/NOS Center for Coastal Environmental
Health & Biomolecular Research
219 Ft. Johnson Road
Charleston, SC 29412
Dr. Alan Lewitus
South Carolina Department of Natural Resources-MRRI
217 Fort Johnston Road
Charleston, SC 29422
in
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Mr. Ron Chandler
YSI, Inc.
1700/1735 Brannum Lane
P.O. Box 279
Yellow Springs, OH 45387
Terry Dickey
Hydrolab Corporation
8700 Cameron Road
Suite 100
Austin, Texas 78754-3908
Mr. Regis Cook
General Oceanics
1925N.W. 163 Street
Miami, FL 33169
Ms. Pam Millet
Horiba Sales Engineer
17671 Armstrong Avenue
Irvine, CA 92614
Mr. Jeffrey D. Myers
Battelle
505 King Avenue
Columbus, OH 43201
Ms. Karen Riggs
Battelle
505 King Avenue
Columbus, OH 43201
Dr. Thomas Kelly
Battelle
505 King Avenue
Columbus, OH 43201
Mr. Charles Lawrie
Battelle
505 King Avenue
Columbus, OH 43201
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Mr. Zachary Willenberg
Battelle
505 King Avenue
Columbus, OH 43201
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TABLE OF CONTENTS
1. Introduction 1
1.1 ETV Background 1
1.2 Test Objective 1
1.3 Test Applicability 2
2. Technology Description 3
3. Verification Approach 3
3.1 Scope of Testing 3
3.2 Experimental Design 4
3.3 Reference Testing 6
3.4 Test Location 6
3.5 Roles and Responsibilities 7
3.5.1 Battelle 7
3.5.2 Vendors 11
3.5.3 EPA 11
3.5.4 Test Facility 12
4. Test Procedures 13
4.1 Site Selection 13
4.2 Multi-Parameter Water Probe Deployment 15
4.3 Mesocosm Testing 15
4.4 Saltwater Testing 17
4.5 Freshwater Testing 18
4.6 Multi-Parameter Water Probe Calibration 21
4.7 Reference Methods 21
4.7.1 pH 21
4.7.2 Turbidity 22
4.7.3 Dissolved Oxygen 22
4.7.4 Nitrate 22
4.7.5 Chlorophyll A 22
4.7.6 Conductivity 22
4.7.7 Temperature 23
5. Material and Equipment 23
5.1 Reagents 23
5.2 Sampling Equipment and Handling 23
5.3 Reference Equipment 24
VI
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6. Quality Assurance/Quality Control 24
6.1 Calibration 24
6.2 Field Quality Control 25
6.3 Sample Custody 26
6.4 Audits 26
6.4.1 Performance Evaluation Audits 26
6.4.2 Technical Systems Audit 29
6.4.3 Data Quality Audits 29
6.4.4 Assessment Reports 30
6.5 Corrective Action 30
7. Data Handling and Reporting 30
7.1 Documentation and Records 30
7.2 Data Review 31
7.3 Statistical Procedures 33
7.3.1 Pre- and Postcalibration Results 33
7.3.2 Relative Bias 33
7.3.3 Precision 34
7.3.4 Linearity 35
7.3.5 Inter-UnitReproducibility 35
7.4 Reporting 35
8. Health and Safety 36
9. References 36
Appendices
Appendix A SOPs and EPA Test Methods A-l
Appendix B Data Sheet/Chain of Custody B-l
Vll
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List of Figures
Figure 1. Mesocosm Tank 5
Figure 2. Organization Chart for Multi-Parameter Water Probe
Verification 8
Figure 3. Major Bodies of Water Leading into the Testing Area 14
List of Tables
Table 1. Schedule for the Multi-Parameter Water Probe Test 6
Table 2. Expected Ranges of Water Characteristics at the Planned Test Sites 14
Table 3. Schedule for Mesocosm Sample Collection 16
Table 4. Sample Analysis Location 17
Table 5. Schedule of Reference Method Sample Events on Each Day of Testing
at the Charleston Harbor Site 19
Table 6. Schedule of Reference Method Sample Events on Each Day of Testing
at the Lake Edmunds Site 20
Table 7. Maximum Holding Time 24
Table 8. Replicate Analysis Results 25
Table 9. Expected Values for Field Blanks 26
Table 10. Summary of Performance Evaluation Audits 27
Table 11. Summary of Data Recording Process 32
Vlll
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ACRONYMS
AMS Advanced Monitoring Systems
CCEHBR Center for Coastal Environmental Health and Biomolecular Research
DO dissolved oxygen
EPA United States Environmental Protection Agency
ETV Environmental Technology Verification
NIST National Institute of Standards and Technology
NOAA National Oceanic and Atmospheric Administration
QA quality assurance
QMP Quality Management Plan
SOP standard operating procedure
TSA technical systems audit
IX
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1. INTRODUCTION
1.1 ETV Background
This test/quality assurance (QA) plan provides detailed procedures for a verification test
of multi-parameter water quality probes/sondes that continuously measure water quality
parameters. The verification test will be conducted under the auspices of the U.S. Environmental
Protection Agency (EPA) through its Environmental Technology Verification (ETV) program.
The purpose of the ETV program is to provide objective and quality-assured performance data
on environmental technologies, so that users, developers, regulators, and consultants can make
informed purchase and application decisions about these technologies. ETV verification does
not imply approval, certification, or designation by EPA, but rather provides a quantitative
assessment of the performance of a technology under specified test conditions.
The verification test will be coordinated by Battelle, of Columbus, Ohio, which is EPA's
partner in the ETV Advanced Monitoring Systems (AMS) Center. The scope of the AMS Center
covers verification of monitoring technologies for contaminants and natural species in air, water,
and soil. In performing the verification test, Battelle will follow the procedures specified in this
test/QA plan and will comply with the data quality requirements in the "Quality Management
Plan for the ETV Advanced Monitoring Systems Center" (QMP).1
1.2 Test Objective
The purpose of verification tests generated from this test/QA plan is to evaluate the
performance of multi-parameter water probes under realistic operating conditions. Specifically,
these probes will be deployed in a location or locations similar to those that would be used by
members of the water monitoring community, and the probes' performance will be evaluated by
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comparing pre- and postcalibration results and their measurements with standard reference
measurements. This test/QA plan calls for probes to be deployed in laboratory, freshwater, and
saltwater environments near Charleston, South Carolina, for a 2 ^-month field test in which the
probes will be operated continuously for periods up to 30 days. (Different locations and test
periods may be accommodated with this plan, if appropriate for the water probes being tested.)
During this time, water quality parameters such as turbidity, chlorophyll A, nitrate, conductivity,
temperature, dissolved oxygen (DO), and pH will be measured both by the monitors (when
applicable) and by reference methods. In the laboratory environment, these parameters will be
controlled, while in the freshwater and saltwater phases of the verification, these parameters will
not be controlled. During each phase, assessments of performance will be based upon com-
parisons to the reference results, and include determinations of accuracy, precision, linearity,
and inter-unit reproducibility.
1.3 Test Applicability
This test/QA plan is applicable to the verification testing of probes that operate
unattended in lakes; rivers; coastal areas; estuaries; bays; and other fresh, salt, or brackish
bodies of water and that continuously measure one or more water quality parameters, such as
turbidity, chlorophyll A, nitrate, conductivity, temperature, DO, or pH. In accordance with the
intent of the ETV program, the probes to be tested are commercially available and not
developmental products or prototypes. No enhancements of a commercially available product
can be used. This includes using any special anti-fouling coating or paints that are not part of
the standard product.
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2. TECHNOLOGY DESCRIPTION
The probes to be tested typically consist of a sensor or sensors in a rugged housing at the
end of a tethered line. The probes are portable and usually must be tethered to a buoy, dock,
piling, or similar structure. While some may be capable of wireless transmission of data, many
probes require that stored data be physically downloaded by the user.
The multi-parameter water probes that may be verified under this testing protocol must
be able to undergo the testing explained in Chapter 4. In general, probes must be able to
measure two or more parameters listed in Section 1.3 of the test/QA plan, in both salt and
freshwater. It must be deployable, in the sense that the probe must be able to make the water
quality measurements without the assistance or intervention of an operator. A probe must be
able to store the measured water quality values for a minimum of two weeks at an hourly
sampling rate and must be able to sample at a depths between 1 and 15 feet.
3. VERIFICATION APPROACH
3.1 Scope of Testing
The objective of this test is to establish the performance capabilities of multi-parameter
water probes under operating conditions that are realistic in terms of type of water body, depth,
duration of unattended operation, etc., as well as in a laboratory or controlled setting. To achieve
this goal, this verification test will involve three phases. In the first phase, the probes will be
tested in a saltwater location. The second phase of verification will take place at a freshwater
location. In each of these two phases, the probes will monitor the naturally occurring levels of
each parameter. These longer phases, of 30 sampling days each, will be used to determine how
well the monitors compare with the reference methods while being continuously deployed in a
field setting. The third phase will be in a laboratory or controlled environment. During this
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week-long phase, the probes will be tested over target parameter ranges that are partially
controlled. The turbidity and conductivity will be adjusted while recording the response of the
probes. In all tests, two units of each probe will be operated side by side to make inter-unit
comparisons.
3.2 Experimental Design
The test is designed to assess the performance of multi-parameter water probes relative to
reference methods that may consist of using either a grab sample and laboratory analysis or
another real-time monitor. This test will be closely coordinated with the National Oceanic and
Atmospheric Administration (NOAA) through the Center for Coastal Environmental Health and
Biomolecular Research (CCEHBR). The test will be performed at or near CCEHBR facilities in
Charleston, South Carolina. The approach to the verification test is summarized below, and the
statistical methodology for establishing performance parameters is described in Section 7.3. This
test will be in three phases, with each phase occurring in a different type of water body.
The first phase of the test will occur at a saltwater site at CCEHBR and will last
approximately one month. The CCEHBR campus has direct access to Charleston Harbor, which
is a tidally dominated body of water that receives some riverine input, with salinities ranging
from 20 to 35 parts per thousand. The South Carolina Department of Natural Resources has
several piers and docks than can be used to deploy the instruments. Also, other areas in close
proximity can be used if the instruments need to be deployed away from dock and boat activity.
Many types of land use in the area surrounding Charleston Harbor can affect overall water
quality, including residential, industrial, urban, and dredge spoil.
The second phase of the test will occur at a freshwater site and last approximately one
month. The site is a five-acre freshwater pond named Lake Edmunds approximately one mile
from the CCEHBR facilities, located on the property of a NOAA staff member (Dr. Peter Key).
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The third phase will take place over a one-week period at the CCEHBR's Mesocosm
Facility. This facility contains modular estuarine mesocosms, consisting of a 300-liter tank
containing elevated sediment trays and stream channels. Each sediment tray is arranged so that
an elevated salt marsh surface is formed. The sediment trays contain sediment, salt marsh
vegetation, and benthic communities. Stream channels contain phytoplankton, zooplankton, and
endemic macrofaunal species. Another component of the mesocosm is a reservoir or sump that
provides tidal water to the system through a pump system
controlled by a timer. Twice daily, seawater is pumped up
into the mesocosm tank from the sump to simulate a flood
tide. After six hours of flooding tide, the seawater is
allowed to drain back into the sump, simulating an ebb
tide for another six hours. Mesocosms used for this test
can be classified as "tidal" or "estuarine." Figure 1 shows
a single mesocosm tank.
The proposed schedule for the various testing
activities is given in Table 1. In each phase, individual
vendor's probes will be positioned as near each other as
possible. This will be done so that, for each vendor,
inter-unit comparisons can be made. In addition, the
probes from different vendors will be placed near each
other so that parameters such as photosynthesis and
mixing are as similar as possible.
Figure 1. Mesocosm Tank
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Table 1. Schedule for the Multi-parameter Water Probe Test
Activity
Vendor setup for saltwater site
Begin saltwater test
End saltwater test
Vendor setup for freshwater test
Begin freshwater test
End freshwater test
Vendor setup for mesocosm test
Begin mesocosm test
End mesocosm test
Vendor removal of equipment
Date
June 10
June 17
July 18
July 19
July 29
August 30
September 4
September 9
September 13
September 16
3.3 Reference Testing
During this verification test, various analytical methods will be used to monitor
turbidity, chlorophyll A, nitrate, conductivity, temperature, DO, and pH. Temperature, pH, DO,
and conductivity will be monitored in real time with devices that are collocated with the probes
being verified. Turbidity, chlorophyll A, and nitrate concentrations will be measured using
laboratory analysis of collected samples. Turbidity will be measured using a Hach Ratio XR
turbidity meter, chlorophyll A will be measured using a Turner 10-AU fluorometer, and nitrate
will be measured colorimetrically using a Lachat Instruments QuikChem autoanalyzer.
3.4 Test Location
CCEHBR meets the requirements of a test facility for this verification. Specifically, a test
facility must be capable of providing a secure and realistic location for deploying the multi-
parameter water probes, must have standard operating procedures (SOPs) or written methods in
place for the reference measurements, have trained personnel capable of performing these
activities according to those SOPs and must have documented QA procedures in place.
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Documentation of the staff training, SOPs, and other pertinent materials will be provided to
Battelle prior to test initiation.
3.5 Roles and Responsibilities
The verification test will be coordinated and supervised by Battelle personnel. Staff from
the CCEHBR test facility will participate in this test by operating the reference equipment,
collecting the water samples, downloading the data from the multi-parameter water probes, and
informing Battelle staff of any problems encountered. Vendor representatives will install,
maintain, and operate their respective technologies throughout the test unless they give written
consent for CCEHBR or Battelle staff to carry out these activities. QA oversight will be
provided by the Battelle Quality Manager, and the EPA ETV Quality Manager at her discretion.
The chart shown in Figure 2 shows the organization of responsibilities for Battelle, the vendor
companies, EPA, and the test facility. The specific responsibilities of these individuals are
detailed below.
3.5.1 Battelle
Mr. Jeffrey Myers, the Battelle Verification Test Coordinator will have the overall
responsibility for ensuring that the technical, schedule, and cost goals established for the
verification test are met. The Verification Test Coordinator will
Prepare the draft test/QA plan, verification reports, and verification statements
Revise the draft test/QA plan, verification reports, and verification statements in
response to reviewers' comments
Coordinate distribution of the final test/QA plan, verification reports, and
verification statements
Coordinate testing, measurement parameters, and schedules at the testing site
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Battelle
Quality Manager
C. Lawrie
Battelle
Management
Battelle AMS
Center Manager
K. Riggs
Battelle
Verification
Testing Leader
T. Kelly
Test Site
Management
Battelle
Verification
Test Coordinator
J. Myers
CCEHBR
Testing
Staff
Battelle
Testing
Staff
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EPA Center
Manager
R. Fuerst
EPA ETV
Quality Manager
E. Betz
Multi-Probe
Vendor
Representatives
Figure 2. Organization Chart for Multi-Parameter Water Probe Verification
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Ensure that all quality procedures specified in the test/QA plan and in the QMP
are followed
Respond to any issues raised in assessment reports and audits, including
instituting corrective action as necessary
Serve as the primary point of contact for vendor and test facility representatives
Establish a budget for the verification test and monitor staff effort to ensure that
the budget is not exceeded
Ensure that confidentiality of proprietary vendor technology and information is
maintained
Coordinate with sample analysis laboratory to ensure timely reporting of results.
Dr. Thomas J. Kelly, the Verification Testing Leader for the AMS Center will provide
technical guidance, oversee various stages of the verification test, and
Support the Verification Test Coordinator in preparing the test/QA plan and
organizing the testing and budgeting for the verification activities
Review the draft test/QA plan
Review the draft verification reports and statements
Ensure that confidentiality of proprietary vendor technology and information is
maintained.
Battelle's AMS Center Manager, Ms. Karen Riggs, will
Review the draft test/QA plan
Review the draft verification reports and statements
Ensure that necessary Battelle resources, including staff and facilities, are
committed to the verification test
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Support the Verification Test Coordinator in responding to any issues raised in
assessment reports and audits
• Maintain communication with EPA's AMS Center and Quality Managers.
Ensure that confidentiality of proprietary vendor technology and information is
maintained.
Battelle's Quality Manager for this verification test, Mr. Charles Lawrie, will
Review the draft test/QA plan
Conduct a technical systems audit (ISA) once during the verification test
Audit at least 10% of the verification data
Prepare and distribute an assessment report for each audit
Verify implementation of any necessary corrective action
Issue a stop work order if self-audits indicate that data quality is being
compromised or if proper safety practices are not followed; notify the Battelle
AMS Center Manager if a stop work order is issued.
Provide a summary of the audit activities and results for the verification reports
Review the draft verification reports and statements
Have overall responsibility for ensuring that the test/QA plan and ETV QMP are
followed
Ensure that Battelle management is informed if persistent quality problems are
not corrected
• Interface with EPA's ETV Quality Manager.
Ensure that confidentiality of proprietary vendor technology and information is
maintained.
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3.5.2 Vendors
Vendors will
Review the draft test/QA plan and provide comments and recommendations
Approve the revised test/QA plan
Work with Battelle to commit to a specific schedule for the verification test
Provide duplicate commercial-ready monitors for testing
Provide an on-site operator(s) throughout the verification test period to install the
monitors and maintain them during testing, unless written consent is given for
Battelle or CCEHBR staff to perform those responsibilities.
Remove monitors and other related equipment from the test facility upon
completing the verification test
Review and comment upon their respective draft verification reports and
statements.
3.5.3 EPA
EPA's responsibilities in the AMS Center are based on the requirements stated in the
QAMP for the AMS Center.(2) The roles of the specific EPA staff are as follows:
EPA's ETV Quality Manager, Ms. Elizabeth Betz, will
Review the draft test/QA plan
Perform, at her option, one external ISA during the verification test
Notify the Battelle AMS Center Manager to facilitate a stop work order if an
external audit indicates that data quality is being compromised
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Prepare and distribute an assessment report summarizing results of the external
audit, if performed
Review draft verification reports and statements.
Ensure that confidentiality of proprietary vendor technology and information is
maintained.
EPA's AMS Center Manager, Mr. Robert Fuerst, will
Review the draft test/QA plan
Approve the final test/QA plan
Ensure that confidentiality of proprietary vendor technology and information is
maintained
Approve the final verification reports
Review the draft verification statements.
Ensure that confidentiality of proprietary vendor technology and information is
maintained.
3.5.4 Test Facility
Dr. Paul Pennington, Research Specialist at the CCEHBR test facility, will
Assist in developing the test/QA plan for the verification test
Allow access to the facility to vendor, Battelle, and EPA representatives during
the field test periods
Provide necessary safety instructions to Battelle, EPA, and vendor personnel for
operations at the test facility
Select a secure location for each of the three testing phases
Assist vendors in installing the probes at each location
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Perform sample collections and analyses as detailed in the test procedures section
ofthetest/QAplan
• Perform reference measurements
Provide all test data to Battelle electronically, in mutually agreed upon format
Provide EPA and Battelle staff access to and /or copies of appropriate QA
documentation of test equipment and procedures (e.g., SOPs, calibration data)
Provide information regarding education and experience of each researcher
involved in the verification
Assist in Battelle's reporting of the test facility's QA/quality control results
Review portions of the draft verification reports to assure accurate descriptions of
the test facility operations and to provide technical insight on verification results.
4. TEST PROCEDURES
4.1 Site Selection
Below are the general procedures to be followed at each of the test sites. Three test sites
will be used for this verification in an attempt to expose the probes to as wide a range of
conditions as possible while conducting an efficient test. The site selection process requires that
several important criteria be met. First, the three sites must include one controlled, one saltwater
(or brackish), and one freshwater location. The sites must allow for collocation of numerous
probes because each vendor will provide duplicate probes for the test. The sites must be
accessible daily so that timely water collections can be made; and the site must, to the extent
possible, be free from interference from the public. A secure facility is not required, but is
preferred. For this verification, the three locations chosen are the mesocosm site at the
CCEHBR facility in Charleston, the Charleston Harbor, and Lake Edmunds. Figure 3 shows a
map of South Carolina and a close-up map showing the testing sites. The CCEHBR was selected
with the understanding that its facilities are under federal jurisdiction and, therefore, staff
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involved in the test may be subject to
safety/security constraints that have not been
identified in this test/QA plan.
The sites at or near the CCEHBR facility
were selected for several reasons. First it was
beneficial to involve a major user (NOAA) of the
multi-parameter water probes in the test to allow a
broader verification test than would be possible
using only Battelle facilities. Second, CCEHBR
has secure, nearby sites available for all three
phases of the test (mesocosm, freshwater, and
saltwater), which allows resources to be devoted
to testing rather than to building infrastructure for
the test. Finally, these sites offer a useful variation
of water conditions for testing. Typical ranges for
the target parameters to be monitored are given in
Table 2.
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Figure 3. Major Bodies of Water Leading
into the Test Area
Table 2. Expected Ranges of Water Characteristics at the Planned Test Sites
Parameter
pH
Turbidity
DO
Conductivity
Temperature
Nitrate
Chlorophyll A
Salinity
Mesocosm
Low
7.5
0.1 NTU
2.0 mg/L
0.0
15C
0.1 mg/L
5ug/L
Oppt
High
8.3
10 NTU
10.00 mg/L
36mS/cm2
35C
Img/L
60 ug/L
20ppt
Bay
Low
7.5
-
2.5 mg/L
-
-
0.1 mg/L
5 ug/L
20ppt
High
8.3
-
8.0 mg/L
-
-
Img/L
60 ug/L
30ppt
Lake Edmunds
Low
7.0
-
-
-
-
0.1 mg/L
5 ug/L
Oppt
High
8.0
-
-
-
-
Img/L
60 ug/L
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4.2 Multi-Parameter Water Probe Deployment
Probes will be set up in the 300-liter mesocosm tank at the Mesocosm Facility and
prepared for a one-week test. Because of space considerations, more than one mesocosm tank
may be used; but, in all cases, each probe will be provided with water from the same source, and
each individual vendor's probes will be collocated within the same tank so that inter-unit
reproducibility can be evaluated.
The saltwater test will take place on a portion of the Charleston Harbor located on the
CCEHBR campus. The probes will be set up for a 30-day test. Each of the probes will be located
within the same area, moored to the piling of the pier and accessible to CCEHBR staff for daily
observation, reference measurements, and water sample collection.
The freshwater phase of the verification test will occur in Lake Edmunds on James
Island, located approximately one mile from the CCEHBR facility.
Each vendor will be responsible for the initial setup of the probe at each test location
unless written permission is given to CCEHBR or Battelle to set up the probe. Vendors may set
up at the first site while training the appropriate Battelle or CCEHBR staff so that, during the
next two deployments, the probes may be redeployed without vendor staff members present.
4.3 Mesocosm Testing
Mesocosm testing will be performed according to the schedule shown in Table 3. The
mesocosms fill and drain with water daily, simulating a tide. Water samples will be collected at
four intervals during each test day, spaced evenly throughout the normal operating hours of the
facility (nominally 6 a.m. to 6 p.m.). During this period, the mesocosms will be manipulated to
introduce variations in the measured parameters. The turbidity of the systems will be varied by
operating a pump near the sediment trays to suspend additional solids in the water. Conductivity
will be varied by adding freshwater to the saltwater during one of the fill-and-drain cycles.
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Nitrate will be varied by spiking the mesocosms with an appropriate amount of chemical during
the fill cycle. Temperature, pH, and DO will be allowed to vary naturally, with any variations
driven by natural forces and the changes in the other test parameters (for example nutrient
spiking is likely to vary the corresponding chlorophyll A concentrations). The parameters will
be varied over the ranges specified in Table 2 and monitored by the multi-parameter probes
undergoing testing. During this period, each of the collected samples will be analyzed using a
reference method for comparison. Three replicate samples will be collected from each tank per
sampling event and each replicate analyzed for the parameters shown in Table 4. The average
value of the three replicates will be reported as the reference value, along with the standard
deviation.
Table 4. Sample Analysis Location
pH
Turbidity
DO
Chlorophyll A
Conductivity
Temperature
Nitrate
Analysis Location
on site
laboratory
on site
laboratory
on site
on site
laboratory
4.4 Saltwater Testing
The saltwater test will occur at the Charleston Harbor site. This portion of the
verification test will last for 30 days, during which time the probes will monitor the naturally
occurring range of the target parameters, while samples for simultaneous reference
measurements will be collected during each sampling event. Sample collection times will be
rotated among the morning, afternoon, and evening throughout the test. In addition, two periods
of intense sampling will occur at the beginning (Days 1 and 2) and the end (Days 29 and 30) of
the sampling period, during which time samples will be taken at 30-minute intervals for eight
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hours. For the first 15 days, the probes will be deployed to a depth of one to two feet. For the
last 15 days, the probes will be deployed to a depth of 15 feet. At this site, samples for
laboratory reference measurements will be taken using aNiskin sampling device, which allows a
sample to be taken at depth. Three replicates will be taken per sampling event and each replicate
analyzed. Temperature measurements will be taken at depth using a thermocouple on the end of
a five-meter pole. The average value of the three replicates will be used as the reference value.
Table 5 shows the recommended sampling times and numbers of sample events throughout the
test period. The probes will be deployed by tethering them to the side of a bulkhead already
located in the harbor. The probes from an individual vendor will be attached to the bulkhead so
that they are as close to each other as possible and near the probes from the additional vendors
participating in the test. If possible, the probes from each of the vendors will be located at the
corners of a one-meter square frame, from which the probes will be hung.
4.5 Freshwater Testing
Freshwater testing will be done at Lake Edmunds. Because this site is more shallow than
Charleston Harbor, only one depth will be used; however, the same sample collection schedule
will be followed. This portion of the verification test will last for 30 testing days, during which
time the probes will monitor the naturally occurring target parameters, while simultaneous
reference measurements will be collected during each sampling event, again rotating among
collection times. Two periods of intense sampling also will occur at the beginning (Days 1 and
2) and the end (Days 29 and 30) of the sampling period, during which time samples will be
taken at 30-minute intervals for eight hours. Three replicates will be taken per sampling event
and each replicate analyzed. The average value of the three replicates will be used as the
reference value. Table 6 shows the recommended sampling times and numbers of samples to be
collected throughout the test period.
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Table 5. Schedule of Reference Method Sample Events on Each Day of Testing at the
Charleston Harbor Site
Sampling Day
Morning
Afternoon
Evening
Total Sampling
Events
Shallow Deployment
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
6
6
1"
r
1
6
6
lb
1
1"
r
4
4
1
r
ib
ib
16
16
3
0
0
0
2
0
0
0
3
0
0
0
3
Deep Deployment
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1
lb
r
i"
6
6
i"
i
r
i"
r
6
6
i
ib
ib
4
4
2
0
0
3
0
0
2
1
0
1
1
0
2
16
16
Sample to be split into a laboratory replicate
Field blank taken simultaneously
0 Field spike taken simultaneously
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Table 6. Schedule of Reference Method Sample Events on Each Day of Testing at the Lake
Edmunds Site
Sampling Day
Morning
Afternoon
Evening
Total Sampling
Events
Shallow Deployment
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
6
6
1"
1
r
i
ib
i"
6
6
6
6
lb
1
1"
r
i"
i
i
r
i
i
6
6
4
4
1
r
ib
ib
i
ib
r
ib
4
4
16
16
3
0
0
0
2
0
0
0
3
0
0
0
3
2
0
0
3
0
0
2
1
0
3
0
0
2
16
16
* Sample to be split into a laboratory replicate
b Field blank taken simultaneously
0 Field spike taken simultaneously
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Lake Edmunds is shallow; and, therefore, the probes can be deployed by driving large
posts into the bottom of the pond and tethering the instruments onto the posts with cable ties.
While wearing appropriate gear, the testers will be able to wade into the pond and force the
posts into the bottom with a sledgehammer. This method has been used often at the same site,
and there have been no problems in the past while using other instrumentation in the ponds.
Sample collection at the freshwater site will be done without entering the water to limit errors
induced by disturbing the water during sampling.
4.6 Multi-Parameter Water Probe Calibration
Pre- and postcalibration of the multi-parameter water probes will be done for each
measured parameter according to that vendor's instruction manual. This calibration will use
NIST-traceable standards when applicable. Vendors may choose to supply the necessary
calibration solutions and devices specific to the probe being verified.
4.7 Reference Methods
Reference measurements taken during this verification test will be presented, along with
the data obtained during the pre- and postcalibration results.
4.7.1 pH
A National Institute of Standards and Technology (NIST)-traceable handheld pH meter
from Oakton will be operated according to the manufacturer's instructions.
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4.7.2 Turbidity
A Hach Ratio XR turbidity meter will be operated according to the manufacturer's
instructions.
4.7.3 Dissolved Oxygen
DO will be measured using a NIST-traceable commercially available probe, operated
according to the manufacturer's instructions.
4.7.4 Nitrate
Nitrate concentrations will be determined colorimetrically using a Lachat Instruments
QuikChem autoanalyzer operated according to the manufacturer's instructions. The University
of South Carolina uses QuikChem® Method 31-107-04-1-D(3) for this determination. The
method is included in Appendix A.
4.7.5 Chlorophyll A
Chlorophyll A concentrations will be determined using a fluorescence technique on a
Turner 10-AU fluorometer operated according to the manufacturer's instructions. The method
for this determination is the SOP used at CCEHBR. This SOP, based on EPA Method 445.0, is
included in Appendix A.
4.7.6 Conductivity
A NIST-traceable handheld Orion conductivity meter (mS) will be operated according to
the manufacturer's instructions.
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4.7.7 Temperature
A NIST-traceable handheld thermocouple and readout will be used to monitor the water
temperature (°C). This thermocouple will be used according to the manufacturer's instructions.
5. Material and Equipment
5.1 Reagents
Reagents to be used include distilled deionized water (for field blanks), a Hach Ratio XR
turbidity standard from Advanced Polymer Systems (level, purity, etc.), a chlorophyll A
standard from Sigma (C6144), a nitrate standard, and preservation reagents, as specified in the
methods in Appendix A.
5.2 Sampling Equipment and Handling
Sampling equipment will consist of 0.5- or 1-L sample containers (glass bottles) and the
Niskin sampling device being provided by CCEHBR, along with all sample storage equipment.
The recommended maximum sample holding time is given in Table 7.
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Table 7. Maximum Holding Time
pH
Turbidity
DO
Chlorophyll A
Conductivity
Temperature
Nitrate
Holding Time
none3
24 hours
none
1 week
none
none
2 weeks
"Sample analysis performed immediately after sample collection.
5.3 Reference Equipment
Reference equipment includes a handheld pH meter (Oakton), turbidity meter (Hach
Ratio XR), autoanalyzer (Lachat Instruments QuikChem 8000), fluorometer (Turner 10-AU),
handheld conductivity meter, handheld thermocouple, and a DO meter.
6. QUALITY ASSURANCE/QUALITY CONTROL
6.1 Calibration
Both the on-line and laboratory reference instrumentation to be used in this verification
test will be calibrated by the CCEHBR test facility according to the SOPs and schedules in place
at the test facility. Documentation of these calibration results will be provided to Battelle. The
conductivity, DO, and pH meters will be calibrated before each sampling event. The auto-
analyzers, turbidity meter, and fluorometer used to measure nitrate, turbidity, and chlorophyll A
will be calibrated at each sample analysis period. The thermocouple will be calibrated in the six
months prior to the test completion date.
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6.2 Field Quality Control
To ensure that the sample collection and analysis procedures are properly controlled, a
field blank and a laboratory replicate sample will be taken at the times shown in Tables 5 and 6.
The field blank will be a container of deionized water taken to the field and then brought back to
the laboratory. It will be analyzed in the same manner as the collected samples. The laboratory
replicate sample will be collected once each week during a regular sampling period. These
replicate samples will simply be the field sample split into two separate samples (containers) and
analyzed by the same methods. The results from the replicate analysis should be within the
accuracy reported in Table 8. The expected values for the field blanks are given in Table 9. In
addition, sample spikes will be taken in distilled water on the schedule shown in Tables 5 and 6.
Sample spikes will be taken for only nitrate. The nitrate spike will be at 0.5mg/L.
Table 8. Replicate Analysis Results
pH
Turbidity
DO
Chlorophyll A
Conductivity
Temperature
Nitrate
Accuracy (±)
0.1
5NTU
5%
5%
5%
1°C
10%
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Table 9. Expected Values for Field Blanks
Turbidity
Chlorophyll A
Nitrate
Expected Maximum
1NTU3
3 x average of three
blank filters
5 us at N/LC
"NTU = nephelometric turbidity unit
b at P/L = atoms of phosphorus per liter
c at N/L = atoms of nitrogen per liter
6.3 Sample Custody
Transportation for sample collection at the Lake Edmunds site will be provided by
CCEHBR, and collected samples will be transported to the laboratory in an ice-filled cooler. All
samples will be accompanied by the sample collection sheet and chain-of-custody form included
in Appendix B.
6.4 Audits
Independent of test facility and EPA QA activities, Battelle will be responsible for
ensuring that the following audits are conducted as part of this verification test.
6.4.1 Performance Evaluation Audits
A performance evaluation audit will be conducted to assess the quality of the reference
measurements made in this verification test. Each type of reference measurement will be com-
pared with an independent probe or a NIST-traceable standard that is independent of those used
during the testing. The acceptance criteria for the results of this audit are noted below. This audit
will be performed once during the verification test. Table 10 gives a summary of the audits to be
performed.
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Table 10. Summary of Performance Evaluation Audits
Audited Parameter
pH
Turbidity
DO
Nitrate
Chlorophyll A
Conductivity
Temperature
Audit Procedure
Independent monitor
Independent turbidity standard
Independent monitor
Independent nitrate standard
Independent chlorophyll
standard
Independent monitor
Independent monitor
Acceptable Tolerance
±0.1 pH
±10%
±5%
±10%
±10%
±5%
±1°C
6.4.1.1 pH
The handheld pH meter from Oakton will be compared with another handheld pH meter
made by a different manufacturer and operated according to the manufacturer's instructions. A
tolerance of ±0.1 pH unit is expected.
6.4.1.2 Turbidity
The measurement of an independent turbidity standard will be compared using the Hach
turbidity meter. An agreement of within 10% in nephelometric turbidity units is expected.
6.4.1.3. Dissolved Oxygen
The DO measurement will be compared with a handheld DO monitor made by a different
manufacturer. Agreement within 5% is expected.
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6.4.1.4 Nitrate
A nitrate audit will be performed, using an independent nitrate standard, by delivering a
spiked sample to the Lachat Instruments QuikChem autoanalyzer. Agreement between the
results of this analysis and the spiked concentration is expected to be within 10%.
6.4.1.5 Chlorophyll A
A chlorophyll A audit will be performed, using an independent chlorophyll A standard,
by delivering a diluted standard to the Turner 10-AU fluorometer. Agreement between the
results of this analysis and the spiked concentration is expected to be within 10%
6.4.1.6 Conductivity
An independent handheld conductivity meter made by a different manufacturer will be
used to perform the conductivity audit. Agreement between the results of this meter and those of
the test reference meter is expected to be within 5%.
6.4.1.7 Temperature
A NIST-traceable mercury-in-glass thermometer will be used for the temperature
performance audit. The comparison will be done on a sample of collected water. An agreement
within ±1°C is expected.
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6.4.2 Technical Systems Audits
Battelle's Quality Manager will perform a ISA at least once during this verification test.
The purpose of this audit is to ensure that the verification test is being performed in accordance
with the AMS Center QMP(1), this test/QA plan, published reference methods, and any SOPs
used by the CCEHBR test facility. In this audit, the Battelle Quality Manager may review the
reference methods used, compare actual test procedures to those specified or referenced in this
plan, and review data acquisition and handling procedures. A TSA report will be prepared,
including a statement of findings and the actions taken to address any adverse findings. The
EPA ETV Quality Manager will receive a copy of Battelle's TSA report.
At EPA's discretion, EPA QA staff may also conduct an independent on-site TSA during
the verification test. The TSA findings will be communicated to testing staff at the time of the
audit and documented in a TSA report.
6.4.3 Data Quality Audits
Battelle's Quality Manager will audit at least 10% of the verification data acquired in the
verification test. The Battelle Quality Manager will trace the data from initial acquisition,
through reduction and statistical comparisons, to final reporting. All calculations performed on
the data undergoing audit will be checked.
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6.4.4 Assessment Reports
Each assessment and audit will be documented in accordance with Section 2.9.7 of the
QMP for the AMS Center.(1) Assessment reports will include the following:
•D Identification of any adverse findings or potential problems
•D Response to adverse findings or potential problems
•D Possible recommendations for resolving problems
•D Citation of any noteworthy practices that may be of use to others
• Confirmation that solutions have been implemented and are effective.
6.5 Corrective Action
The Battelle Quality Manager, during the course of any assessment or audit, will identify
to the technical staff performing experimental activities any immediate corrective action that
should be taken. If serious quality problems exist, the Battelle Quality Manager is authorized to
stop work. Once the assessment report has been prepared, the Verification Test Coordinator will
ensure that a response is provided for each adverse finding or potential problem and will
implement any necessary follow-up corrective action. The Battelle Quality Manager will ensure
that follow-up corrective action has been taken.
7. DATA HANDLING AND REPORTING
7.1 Documentation and Records
A variety of data will be acquired and recorded electronically and manually by either
Battelle or CCEHBR staff in this verification test. Operational information, required
maintenance, and results from the reference methods will generally be documented in a
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laboratory record book and on the data sheet/chain-of-custody form in Appendix B. In general,
the results from the multi-parameter water probes will be recorded electronically. The electronic
data stored on the probe will be collected by the field staff during each sampling event. Once
collected, this data will reside at the test facility until the entire test is finished. All of the
electronic raw data will then be transferred to Battelle Columbus where it will be permanently
stored with the study binder, along with the rest of the study data. Table 11 summarizes the
types of data to be recorded and the process for recording data. At the conclusion of the test,
CCEHBR will be provided with an electronic copy of the raw data generated during the
verification.
7.2 Data Review
Data generated by the test facility and vendors in the verification test will be provided to
Battelle and will be reviewed by the Verification Test Coordinator before they are used to
calculate, evaluate, or report verification results. All data are to be recorded directly in the
laboratory record book as soon as they are available. Records are to be written in ink, written
legibly, and have any corrections initialed by the person performing the correction. These data
will include electronic data, entries in laboratory record books, operating data from the test
facility, and equipment calibration records. The person performing the review will add his/her
initials and the date to a hard copy of the record being reviewed within two weeks of the
measurement. This hard copy will be placed in the files for this verification test by the
Verification Test Coordinator. In addition, data calculations performed by Battelle will be spot-
checked by Battelle technical staff to ensure that calculations are performed correctly.
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Table 11. Summary of Data Recording Process
Data to be
Recorded
Dates, times of test
events
Test parameters
Probe data
- digital display
- electronic output
Reference monitor
readings/reference
analytical results
Reference
calibration data
Performance
evaluation audit
results
Responsible
Party
CCEHBR
Battelle/
CCEHBR
CCEHBR
CCEHBR
CCEHBR
CCEHBR
Battelle
Where Recorded
Laboratory record
books/data sheets
Laboratory record
books/data sheets
Data sheets
Probe data
acquisition system
(data logger, PC,
laptop, etc.).
Laboratory record
book/data sheets
or data
management
system, as
appropriate
Laboratory record
books/data
sheets/data
acquisition system
Laboratory record
books/data
sheets/DAS
How Often
Recorded
Start/end of test; at
each change of a test
parameter; at sample
collection.
Each sample
collection
Each sample
collection;
data downloaded at
least once per day
After each batch
sample collection;
data recorded after
reference method
performed
Whenever zero and
calibration checks
are done
At times of
performance
evaluation audits
Purpose of Data
Used to organize/check
test results; manually
incorporate data into
spreadsheets - stored in
study binder
Used to organize/check
test results; manually
incorporate data into
spreadsheets - stored in
study binder
Used to organize/check
test results; incorporate
data into electronic
spreadsheets - stored in
study binder
Used to organize/check
test results; manually
incorporate data into
spreadsheets - stored in
study binder
Document correct
performance of
reference methods
Test reference methods
with independent
standards/
measurements
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7.3 Statistical Procedures
7.3.1 Pre- and Postcalibration Results
A tabulation of the pre- and postcalibration results will be presented, where applicable,
for each of the measured parameters. The results will be expressed as percent change for a given
time period (days). If not prohibited by the vendor's typical operating instructions, a weekly
check of the calibration will be performed as well.
The results from the calibration checks will be summarized, and accuracy will be
determined each time the calibration check is conducted. This accuracy will be reported as a
percentage, calculated using the following equation:
A=l-(Cs-Cp)/Cs
Where Cs is the value of the standard and Cp is the value measured by the vendor's probe.
7.3.2 Relative Bias
Results from the multi-parameter water probes being verified will be compared to the
results obtained from the reference analyses. Water samples will be analyzed by both the
reference method and the probes being verified. The results for each sample will be recorded,
and the accuracy will be expressed in terms of the relative bias (8), as calculated from the
following equation:
CD-CR
B=^= xlOO (2)
CR
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where CP is the reading from the probe being verified, and CR is the average of the duplicate
reference measurements. This calculation will be performed for each reference sample analysis
for each of the eight target water parameters (Table 2). Readings of pH will be converted to H+
concentration, and temperature readings will be converted to absolute units prior to making this
calculation. Relative bias will be assessed independently for each analyzer provided by a single
vendor to determine inter-unit reproducibility.
7.3.3 Precision
The standard deviation (S) of the results for replicate measurements made during stable
operation at the mesocosm will be calculated and used as a measure of probe precision at each
sampling period:
n 1/2
s =
(3)
where n is the number of replicate samples, Ck is the concentration reported for the kth measure-
ment, and C is the average concentration of the replicate samples, i.e.,
%RSD = £lOO (4)
v~^
Precision will be calculated for each of the eight target water parameters. Probe precision will be
reported in terms of the percent relative standard deviation of the series of measurements.
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7.3.4 Linearity
For target water parameters with a sufficiently wide range of variation, linearity will be
assessed by linear regression, with the analyte concentration measured by the reference method
as independent variable and the reading from the analyzer being verified as dependent variable.
Linearity will be expressed in terms of the slope, intercept, and coefficient of determination (r2).
Linearity for pH will be assessed by converting pH results to FT concentration before compari-
son. Linearity will be assessed separately for each unit of each water probe being tested and for
each of the mesocosm, saltwater, and freshwater test sites.
7.3.5 Inter-Unit Reproducibility
The results obtained from identical units of each probe will be compiled independently
for each analyzer and compared to assess inter-unit reproducibility. The results will be inter-
preted using a t-test, or other appropriate comparison, to assess whether significant differences
exist between the units tested.
7.4. Reporting
The statistical comparisons that result from each of the tests described above will be
conducted separately for each of the probes being tested, and information on the additional
performance parameters will be compiled and reported. Separate verification reports will be
prepared, each addressing a technology provided by one commercial vendor. Each report will
show separate verification results from the duplicate probes undergoing testing, along with
calculations of the inter-unit reproducibility of the technology. For each test, the verification
report will present the test procedures and test data, as well as the results of the statistical
evaluation of those data.
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All interaction with the probes (such as during maintenance, cleaning, and calibration)
will be noted at the time of the test and reported. In addition, descriptions of the data-recording
procedures, consumables used, and required reagents will be presented in the report.
The verification report will briefly describe the ETV program, the AMS Center, and the
procedures used in verification testing. These sections will be common to each verification
report resulting from this verification test. The results of the verification test will then be stated
quantitatively, without comparison to any other technology tested or comment on the
acceptability of the technology's performance. The preparation of draft verification reports,
review of reports by vendors and others, revision of the reports, final approval, and distribution
of the reports will be conducted as stated in the "Generic Verification Protocol for the Advanced
Monitoring Systems Center."(4) Preparation, approval, and use of verification statements
summarizing the results of this test also will be subject to the requirements of that same
protocol.
8. HEALTH AND SAFETY
The CCEHBR test facility will provide appropriate safety instructions regarding
potential hazards during the verification testing to Battelle, EPA, and vendor staff, both at the
CCEHBR site and upon arrival at the test sites.
9. REFERENCES
1. "Quality Management Plan for the ETV Advanced Monitoring Systems Center," Version
3.0, Environmental Technology Verification Program, Battelle, Columbus, Ohio,
December 2001.
2. U.S. Environmental Protection Agency, "Environmental Technology Verification
Program Quality and Management Plan for the Pilot Period (1995-2000)," EPA-600/R-
98/064, Cincinnati, Ohio, May 1998.
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3. QuikChem® Method 31-115-01-1-H, "Determination of Orthophosphate by Flow
Injection Analysis," January 4, 2001.
4. QuikChem® Method 31-107-04-1-D, "Determination of Nitrate and/or Nitrite in
Brackish Waters by Flow Injection Analysis," November 20, 2000.
5. "Generic Verification Protocol for the Advanced Monitoring Systems Pilot,"
Environmental Technology Verification Program, prepared by Battelle, Columbus, Ohio,
October 1998.
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Appendix A
SOPs and EPA Test Methods
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METHOD 180.1
DETERMINATION OF TURBIDITY BY NEPHELOMETRY
Edited by James W. O'Dell
Inorganic Chemistry Branch
Chemistry Research Division
Revision 2.0
August 1993
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
180.1-1
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METHOD 180.1
DETERMINATION OF TURBIDITY BY NEPHELQMETRY
1.0 SCOPE AND APPLICATION
1.1 This method covers the determination of turbidity in drinking, ground, surface,
and saline waters, domestic and industrial wastes.
1.2 The applicable range is 0-40 nephelometric turbidity units (NTU). Higher
values may be obtained with dilution of the sample.
2,0 SUMMARY OF METHOD
2.1 The method is based upon a comparison of the intensity of light scattered by
the sample under defined conditions with the intensity of light scattered by a
standard reference suspension. The higher the intensity of scattered light, the
higher the turbidity. Readings, in NTU's, are made in a nephelometer
designed according to specifications given in Sections 6.1 and 6.2. A primary
standard suspension is used to calibrate the instrument. A secondary standard
suspension is used as a daily calibration check and is monitored periodically
for deterioration using one of the primary standards.
2,1.1 Forrnazin polymer is used as a primary turbidity suspension for water
because it is more reproducible than other types of standards
previously used for turbidity analysis.
2,1.2 A commercially available polymer primary standard is also approved
for use for the National Interim Primary Drinking Waier Regulations.
This standard is identified as AMCO-AEPA-1, available from Advanced
Polymer Systems.
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 surrogates analytes.
3.2 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.
3,3 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 wifh other samples. The LRB is used to determine if method
180.1-2
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analytes or other interferences are present in the laboratory environment, the
reagents, or the apparatus,
3,4 Linear Calibration Range (LCR) -- The concentration range over which the
instrument response 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 Primary Calibration Standard (PCAL) -- A suspension prepared from the
primary dilution stock standard suspension. The PCAL suspensions are used
to calibrate the instrument response with respect to analyte concentration.
3.7 Quality Control Sample (QCS) -- A solution of the method analyte of known
concentrations that is used to fortify an aliquot of LRB matrix. The QCS is
obtained from a source external to the laboratory, and is used to check
laboratory performance.
3.8 Secondary Calibration Standards (SCAL) - Commercially prepared, stabilized
sealed liquid or gel turbidity standards calibrated against properly prepared
and diluted formazin or styrenc divinylbenzene polymers.
3.9 Stock Standard Suspension (SSS) ~ A concentrated suspension containing the
analyte prepared in the laboratory using assayed reference materials or
purchased from a reputable commercial source. Stock standard suspension is
used to prepare calibration suspensions and other needed suspensions.
INTERFERENCES
4.1 The presence of floating debris and coarse sediments which settle out rapidly
will give low readings. Finely divided air bubbles can cause high readings.
4,2 The presence of true color, that is the color of water which is due to dissolved
substances that absorb light, will cause turbidities to be low. although this
effect is generally not significant with drinking waters.
4.3 Light absorbing materials such as activated carbon in significant concentrations
can cause low readings.
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 should be as low as reasonably achievable.
5,2 Each laboratory is responsible for maintaining a current awareness file of
OSHA regulations regarding the safe handling of the chemicals specified in
180.1-3
-------
this method. A reference file of Material Safety Data Sheets (MSDS) should be
made available to all personnel involved in the chemical analysts. The
preparation of a formal safety plan is also advisable.
5.3 Hydrazine Sulfate (Section 7.2.1) is a carcinogen. It is highly toxic and may be
fatal -if inhaled, swallowed, or absorbed through the skin. Formazin can
contain residual hydrazine sulfate. Proper protection should be employed.
6,0 EQUIPMENT AND SUPPLIES
6.1 The turbid imeter shall consist of a nephelometer, with light source for
illuminating the sample, and one or more photo-electric detectors with a
readout device to indicate the intensity of light scattered at right angles to the
path of the incident light. The turbidimeter should be designed so that little
stray light reaches the detector in the absence of turbidity and should be free
from significant drift after a short warm-up period.
6.2 Differences in physical design of turbidimeters will cause differences in
measured values for turbidity, even though the same suspension is used for
calibration. To minimize such differences, the following design criteria should
be observed;
6.2.1 Light source: Tungsten lamp operated at a color temperature between
220Q-30QO°K.
6.2.2 Distance traversed by incident light and scattered light within the
sample tube: Total not to exceed 10 cm.
6.2.3 Detector: Centered at 90° to the incident light path and not to exceed
±30° from 90°. The detector, and filter system if used, shall have a
spectra] peak response between 400 nm and 600 nm.
C.3 The sensitivity of the instrument should permit detection of a turbidity
difference of 0.02 NTU or less in waters having turbidities less than 1 unit.
The instrument should measure from 0-40 units turbidity. Several ranges may
be necessary to obtain both adequate coverage and sufficient sensitivity for low
turbidities.
6.4 The sample tubes to be used with the available instrument must be of clear,
colorless glass or plastic. They should be kept scrupulously clean, both inside
and out, and discarded when they become scratched or etched. A light
coating of silicon oil may be used to mask minor imperfections in glass tubes.
They must not be handled at all where the light strikes them, but should be
provided with sufficient extra length, or with a protective case, so that they
may be handled. Tubes should be checked, indexed and read at the
orientation that produces the lowest background blank value.
6.5 Balance -- Analytical, capable of accurately weighing to the nearest 0.0001 g.
180.1-4
-------
6,6 Glassware - Class A volumetric flasks and pipets as required.
7.0 REAGENTS AND STANDARDS
7.1 Reagent water, turbidity-free: Pass deionized distilled water through a Q.45u
pore size membrane filter, if such filtered water shows a lower turbidity than
unfiltered distilled water.
7.2 Stock standard suspension (Formazin):
7.2.1 Dissolve LOO g hydrazine sulfate, (NH^H^O^ (CASRN 10034-93-2) in
reagent water and dilute to 100 mL in a volumetric flask. CAUTION--
carcinogen.
7.2.2 Dissolve 10.00 g hexamethylenetetramine (CASRN 100-97-0) in reagent
water and dilute to 100 mL in a volumetric flask. In a 100 mL
volumetric flask, mix 5.0 mL of each solution (Sections 7.2.1 and 7.2.2).
Allow to stand 24 hours at 25 ±3°C, then dilute to the mark with
reagent water.
7.3 Primary calibration standards: Mix and dilute 10.00 mL of stock standard
suspension (Section 7.2) to 100 mL with reagent water. The turbidity of this
suspension is defined as 40 MTU. For other values, mix and dilute portions of
this suspension as required.
7.3.1 A new stock standard suspension (Section 7.2} should be prepared each
month. Primary calibration standards (Section 7.3) should be prepared
daily by dilution of the stock standard suspension.
7.4 Formazin in commercially prepared primary concentrated stock standard
suspension (SSS) may be diluted and used as required. Dilute turbidity
standards should be prepared daily.
7.5 AMCO-AEPA-1 Styrene Divinylbenzene polymer primary standards are
available for specific instruments and require no preparation or dilution prior
to use.
7.6 Secondary standards may be acceptable as a daily calibration check, but must
be monitored on a routine basis for deterioration and replaced as required.
8-° SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All bottles must be
thoroughly cleaned and rinsed with turbidity free water. Volume collected
should be sufficient to insure a representative sample, allow for replicate
analysis (if required), and minimize waste disposal.
8.2 Nf> chemical preservation is required. Cool sample to 4°C.
180.1-5
-------
8.3 Samples should be analyzed as soon as possible after collection. If storage is
required, samples maintained at 4°C may be held for up to 48 hours.
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 analysis of laboratory
reagent blanks and other solutions as a continuing check on performance. The
laboratory is required to maintain performance records that define the quality
of data generated,
9.2 INITIAL DEMONSTRATION OF PERFORMANCE,
9.2.1 The initial demonstration of performance is used to characterize
instrument performance (determination of LCRs and analysis of QCS).
9.2.2 Linear Calibration Range (LCR) -- The LCR must be determined
initially and verified every six months or whenever a significant change
in instrument response is observed or expected. The initial
demonstration of linearity must use sufficient standards to insure that
the resulting curve is linear. The verification of linearity must use a
minimum of a blank and three standards. If any verification data
exceeds the initial values by ±10%, linearity must be reestablished. If
any portion of the range is shown to be nonlinear, sufficient standards
must be used to clearly define the nonlinear portion.
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 analysis of a QCS. 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 continuing with
on-going analyses.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) - The laboratory must analyze at least
one LRB with each batch of samples. Data produced are used to assess
contamination from the laboratory environment.
9.3,2 Instrument Performance Check Solution (IPC) -- For all determinations,
the laboratory must analyze the IPC (a mid-range check standard) and
a calibration blank immediately following daily calibration, after every
tenth sample (or more frequently, if required) and at the end of the
sample run. Analysis of the IPC solution and calibration blank
immediately following calibration must verify that the instrument is
180,1-6
-------
within ±10% of calibration. Subsequent analyses of the IPC solution
must verify the calibration is still within ±10%. 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, NOTE: Secondary calibration
standards (SS) may also be used as the IPC.
9.3.3 Where additional reference materials such as Performance Evaluation
samples 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 Turbidimeter calibration; The manufacturer's operating instructions should be
followed. Measure standards on the turbidimeter covering the range of
interest. If the instrument is already calibrated in standard turbidity units, this
procedure will check the accuracy of the calibration scales. At least one
standard should be run in each instrument range to be used. Some
instruments permit adjustments of sensitivity so that scale values will
correspond to turbidities. Solid standards, such as those made of lucite blocks,
should never be used due to potential calibration changes caused by surface
scratches. If a pre-calibrated scale is not supplied, calibration curves should be
prepared for each range of the instrument.
11.0 PROCEDURE
11.1 Turbidities less than 40 units: if possible, allow samples to come to room
temperature before analysis. Mix the sample to thoroughly disperse the solids.
Wait until air bubbles disappear then pour the sample into the turbidimeter
tube. Read the turbidity directly from the instrument scale or from the
appropriate calibration curve.
11.2 Turbidities exceeding 40 units: Dilute the sample with one or more volumes
of turbidity-free water until the turbidity falls below 40 units. The turbidity of
the original sample is then computed from the turbidity of the diluted sample
and the dilution factor. For example, if 5 volumes of turbidity-free water were
added to 1 volume of sample, and the diluted sample showed a turbidity of 30
units, then the turbidity of the original sample was 180 units.
11.2.1 Some turbidimeters are equipped with several separate scales. The
higher scales are to be used only as indicators of required dilution
volumes to reduce readings to less than 40 NTU.
180.1-7
-------
Note; Comparative work performed in the Environmental Monitoring
Systems Laboratory - Cincinnati (EMSL-Cincinnati) indicates a
progressive error on sample turbidities in excess of 40 units.
12-° DATA ANALYSIS AND CALCULATIONS
12.1 Multiply sample readings by appropriate dilution to obtain final reading.
12.2 Report results as follows;
MTU Record to Neargst:
0,0- 1.0 0.05
1 - 10 0.1
10-40 ]
40 - 100 5
100 - 400 10
400 - 1000 50
>1000 100
13,0 METHOD PERFORMANCE
13.1 In a single laboratory (EMSL-Cincinnati). using surface water samples at levels
of 26, 41, 75, and 180 NTU, the standard deviations were ±0.60, ±0,94, ±1.2.
and ±4.7 units, respectively.
13,2 The interlaboratory precision and accuracy data in Table 1 were developed
using a reagent water matrix. Values are in NTU,
H.O 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 The quantity of chemicals purchased should be based on expected usage
during its shelf life and disposal cost of unused material. Actual reagent
preparation volumes should reflect anticipated usage and reagent stability.
14.3 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
180.1-8
-------
Chemical Society's Department of Government Regulations and Science Policy,
1155 16th Street N.W., Washington D.C. 20036, (202)872-4477.
15*° 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. Excess reagents, samples and method process wastes should be
characterized and disposed of in an acceptable manner. 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 waste discharge permit 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 14.3.
16.0 REFERENCES
1. Annual Book of ASTM Standards, Volume 11.01 Water (1), Standard D1889-
88A, p. 359, (1993).
2. Standard Methods for the Examination of Water and Wastewater, 18th Edition,
pp. 2-9, Method 2130B. (1992).
180.1-9
-------
17.0 TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA
TABLE 1. INTERLABORATORY PRECISION AND ACCURACY DATA
Number of
Values
Reported
373
374
289
482
484
489
640
487
288
714
641
True
Value
CD
0,450
0,600
0.65
0,910
0.910
1.00
1.36
3.40
4.8
5.60
5.95
Mean
(X)
0.4864
0.6026
0.6931
0,9244
0.9919
0,9405
1.3456
3.2616
4.5684
5.6984
5.6026
Residual
forX
0.0027
-0.0244
0.0183
0.0013
0.0688
-0,0686
-0.0074
-0,0401
-0.0706
0,2952
-0.1350
Standard
Deviation
(S)
0,1071
0.1048
0.1301
0.2512
0.1486
0.1318
0.1894
0.3219
0.3776
0.4411
0.4122
Residual
forS
-0.0078
-0.0211
0.0005
0,1024
-0.0002
-0.0236
0.0075
-0.0103
-0.0577
-0.0531
-0.1078
REGRESSIONS: X = 0.955T + 0.54, S - 0.074T + 0.082
180.1-10
-------
Ortho Phosphate
Method
-------
QuikChem® Method 31-115-01-lS
DETERMINATION OF ORTHOPHOSPHATE BY FLOW
INJECTION ANALYSIS
Written by Krista Knepel and Karin Bogren
Applications Group
Revision Date:
4 January 2001
LACHAT INSTRUMENTS
6645 WEST MILL ROAD
MILWAUKEE, WI53218-1239 USA
-------
^fat INSTRUMENTS
QuikChem® Method 31-115-01-1-H
Orthophosphorous in Sea waters
5to400ngP/L
0.16 to 12.91 pM P/L
- Principle -
Ammonium molybdate and antimony potassium tartrate reacts in an acid medium with 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 in the sample. Though there is a density difference between seawater and reagent
water the bias is less than 2%.
Though the method is written for seawater and brackish water it is also applicable to non-saline
sample matrixes. The method is calibrated using standards prepared in deionized .water. Once
calibrated, samples of varying salinities (0 to 35 ppt) may be analyzed. The determination of
background absorbance is necessary only for samples, which have color absorbing at 880 nm.
- Interferences -
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 and coastal waters.
2. High iron can cause precipitation of and subsequent loss of phosphate from the dissolved
phase.
3. Using ascorbic acid as the reductant, the color intensity is not influenced by variations in
salinity. Stannous chloride reductant does show a significant salt effect.
4. Turbidity is removed by filtration.
5. Hydrogen sulfide effects, such as occur in samples "from deep anoxic basins, can be
treated by simple dilution since high sulfide concentrations are most often associated with
high phosphate values.
- Special Apparatus -
Please see Parts and Price list for Ordering Information
1. Heating Unit
2. Glass calibration vials must be used for this method (Lachat Part No. 21304)
Written and copyrighted © by K. Knepel and K. Bogren on 4 January 2001 by Lachat Instruments, 6645 West
Mill Road Milwaukee, WI 53218-1239 USA. Phone:414-358-4200 FAX: 414-358-4206. This document is the
rvrr»rn»f*-ij r\f T o^.U«* T_-J ,._ TT « .
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CONTENTS
1. SCOPE AND APPLICATION l
2. INTERFERENCES . . :
3. SAFETY..:..........;.;;......... ....;;..... ... !
4. EQUIPMENT AND SUPPLIES 2
5. REAGENTS AND STANDARDS 2
5.1. PREPARATION OF REAGENTS ... 2
5.2. PREPARATION OF STANDARDS 4
6. SAMPLE COLLECTION, PRESERVATION AND STORAGE 4
7. PROCEDURE 5
7.1. CALIBRATION PROCEDURE 5
7.2. SYSTEMNOTES 5
8. DATA ANALYSIS AND CALCULATIONS... g
9. METHOD PERFORMANCE 6
10. REFERENCES 6
11. TABLE, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 8
11.1. DATA SYSTEM PARAMETERS FOR QUKCHEM 8000..... 8
11.2. SUPPORT DATA FOR QUIKCHEM 8000 9
11.3. ORTHOPHOSPHATE MANIFOLD DIAGRAM 14
-------
QuikChem® Method 31-115-01-1-H
DETERMINATION OF ORTHOPHOSPHORUS BY FLOW
INJECTION ANALYSIS
1. SCOPE AND APPLICATION
1.1. Though the method is written for seawater and brackish water, it is also applicable to
non-saline sample matrixes. The method is calibrated using standards prepared in
deionized water. Once calibrated, samples of varying salinities (0 to 35 ppt) may be
analyzed. The determination of background is necessary only for samples, which have
color absorbing at 880 nm.
1.2. The method is based on reactions that are specific for the orthophosphate ion.
1.3. The applicable range is 5 to 400 ng/L. The claimed method detection limit is 1.0 u.g P/L.
The method throughput is 48 injections per hour.
2. INTERFERENCES
2.1. Interferences caused by copper, arsenate and silicate are minimal relative to the
orthophosphate determination because of the extremely low concentrations normally
found in estuarine and coastal waters.
2.2. High iron can cause precipitation of and subsequent loss of phosphate from the dissolved
_phase.
2.3. Using ascorbic acid as the reductant, the color intensity is not influenced by variations in
salinity. Stannous chloride reductant does show a significant salt effect.
2.4. Turbidity is removed by filtration.
2.5. Hydrogen sulfide effects, such as occur in samples from deep anoxic basins, can be
treated by simple dilution since high sulfide concentrations are most often associated with
high phosphate values.
• • n
3. SAFETY
3.1. 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
should be as low as reasonably achievable. Cautions are included for known extremely
hazardous materials.
3.2. Each laboratory is responsible for maintaining a current awareness file of the
Occupational Health and Safety Act (OSHA) regulations regarding the safe handling of
the chemicals specified in this method. A reference file of Material Safety Data sheets
(MSDS) should be made available to all personnel involved in the chemical analysis.
The preparation of a formal safety plan is also advisable.
3.3. The following chemicals have the potential to be highly toxic or hazardous, for detailed
explanation consult the MSDS.
3.3.1. Sulfuricacid
-------
4. EQUIPMENT AND ST7PPTJRSS
4. 1 . Balance -- analytical, capable of accurately weighing to the nearest 0.0001 g.
4.2. Glassware - Class A volumetric flasks and pipettes or plastic containers as required.
Samples may be stored in plastic or glass.
4.3. How injection analysis equipment designed to deliver and react sample and reagents in
the required order and ratios.
4.3.1. Sampler
4.3 .2. Multichannel proportioning pump
4.3.3. Reaction unit or manifold
4.3.4. Colorimetric detector
4.3.5. Data system
4.4. Special Apparatus
.4.1. Heating unit
4.4.2. Glass calibration vials must be used in this method (Lachat Part No. 21304)
4.5. Phosphate-Free Glassware and Polyethylene Bottles
4.5.1. All labware used in the determination must be low in residual phosphate to avoid
sample or reagent contamination. Washing with 10% (v/v) HC1 and thoroughly
rinsing with distilled, deionized water was found to be effective.
4.5.2. Use membrane or glass fiber filters, 0.45 uM nominal pore size and check for
contamination by analyzing blanks.
4.5.3. When degassing reagents, DO NOT DEGAS USING AN INVASIVE
PROCEDURE SUCH AS A WAND TO AVOID CONTAMINATION. Degas
by vacuum or sonication.
5. REAGENTS AND STANDARDS
5.1* PREPARATION OF REAGENTS
Use deionized water (10 megohm) for all solutions.
Degassing:
To prevent bubble formation, DO NOT DEGAS USING AN INVASIVE PROCEDURE
SUCH AS A WAND TO AVOID CONTAMINATION. Degas by vacuum or sonication.
AH reagents need to be thoroughly degassed.
Reagent 1. Stock Ammonium Molybdate Solution
The molybdate solid may take up to four hours on a stir plate to dissolve. Store in plastic
and refrigerate. Discard after six months.
By Volume: Li a 1 L volumetric flask, dissolve 40.0 g ammonium molybdate
tetrahydrate [(NH4)6Mo7O24-4H2O)] in approximately 800 mL water. Dilute to the
mark and mix with a magnetic stirrer for at least four hours. Store in plastic and
refrigerate.
-------
By Weight: To a tared 1 L container add 40.0 g ammonium molybdate tetrahydrate
[(NH4)6Mo7024-4H2O)] and 983 g water. Mix with a magnetic stirrer for a least four
hours. Store in plastic and refrigerate.
i/Reagent 2. Stock Antimony Potassium Tartrate Solution
By Volume: In a 1L volumetric flask, dissolve_3.0j^ antimony "potassium tartrate-..
(potassium antimonyl tartrate hemihydrate K(SbO)C2H4O6-1/2 H2O) or dissolve 3.22 g "
antimony potassium tartrate (potassium antimonyl tartrate trihydrate C8H4Oi2K2Sb2 '
3H20) in approximately 800 mL water. Dilute to the mark and mix with a magnetic
stirrer until dissolved. Store in a dark bottle and refrigerate.
By Weight: To a 1 L dark, tared container add 3.0 g antimony potassium tartrate
(potassium antimonyl tartrate hemihydrate K(SbO)C2H4O6-l/2 H2O) or 3.22 g antimony
potassium tartrate (potassium antimonyl tartrate trihydrate C8H4Oi2K2Sb2 ' 3H20) and
995 g water. Mix with a magnetic stirrer until dissolved. Store in a dark bottle and
refrigerate.
Reagent 3. Molybdate Color Reagent
This reagent may be stored at room temperature. Discard when the solution turns blue.
By Volume: To a 1 L volumetric flask add 35.0 mL concentrated sulfuric acid to
about 500 mL water, (CAUTION: The solution will get hot!) Swirl to mix. Add 213
mL Ammonium Molybdate Solution (Reagent 1) and 72 mL Antimony Potassium
Tartrate Solution (Reagent 2). Dilute to the mark and invert to mix. Degas by vacuum
or sonicatioh.
By Weight: To a tared 1 L container add 680 g DI water and 64.4 g concentrated
sulfuric acid (CAUTION: The solution will get hot!) Swirl to mix. Add 213 mL
Ammonium Molybdate Solution (Reagent 1) and 72 mL Antimony Potassium
Tartrate Solution (Reagent 2). Dilute to the mark and invert to mix. Degas by vacuum
or sonication.
/ Reagent 4. Ascorbic Acid Reducing Solution
By Volume: To a 1 L volumetric flask, dissolve 60.0 g granular ascorbic acid
(Spectrum Chemicals, Catalogue # AS-102) in about 700 mL DI water. Dilute to ffie "
jnarkjndinvert to mix^Degas by vacuum or sonication for at least 5 minutes. Add 1.0 g
C_sodium dodecylsuffi^SDS CH3(CH2)nOSO3Na). Degas prior to the addition of
SDS; "Prepare fresh" weekly.
By Weight: To a tared 1 L container, add 60.0 g granular ascorbic acid (Spectrum
Chemicals, Catalogue # AS-102) and 975 g DI water. Invert to mix. Degas by vacuum
or sonication for at least 5 minutes. Add 1.0 g sodium dodecyl sulfate (SDS
/ CH3(CH2)nOSO3Na). Degas prior to the addition of SDS. Prepare fresh w^ldy -*C
^Reagerit5. Sodium Hydroxide-EDTA Rinse
Dissolve 65 g sodium hydroxide (NaQH) and 6 g tetrasodium ethylenediamine
tetraacetic acid (N^EDTA) hrl.0 L or 1.0 kg DI water. Use daily at the end of the day
(-10 minutes, followed by a DI rinse) or if the baseline begins to drift upwards.
-------
5.2. PREPARATION OF STANDARDS
To prepare the stock and working standards, the following containers will be requires:.
By Volume: Three 1 L and five 250 mL volumetric flasks.
By Weight: Three 1L and five 250 mL containers.
Standard 1. Stock Standard 1000 mg P/L
By Volume: In a 1 L volumetric flask dissolve 4.39 g potassium
dihydrogenphosphate (KH2PC>4) in approximately 500 mL DI water. Dilute to the mark"
with DI water and invert to mix. This solution may be refrigerated and stored in glass for
up to one month,
Standard 2. Working Standard 1 • 1000 ing P/L
By Volume: In a 1L volumetric flask add 1 mL of Standard 1 (1000 mg P/L).
Dilute to the mark with DI water and invert to mix. Prepare fresh daily.
Standard 3. Working Standard 2 -100 ug P/L
By Volume: In a 1 L volumetric flask, add 100 mL of Working Standard 1 (1000 pig
P/L). Dilute to the mark with DI water and invert to mix. Prepare fresh daily.
Working Standards (Prepare Daily)
Concentration ug P/L
A
400
B
200
C
100
D
25
•^•^^•••^^^W^^HI
E
5
. F
0.0
By Volume
Volume (mL) of Working standard 1
diluted to 250 mL with DI water
Volume (mL) of Working standard 2
diluted to 250 mL with DI water
100
—
50
—
—
250
—
62.5
—
12.5
—
—
By Weight
Weight (g) of Working standard 1
diluted to final weight (-250 g)
divided by factor below with DI water
Weight (g) Working standard 2
diluted to final weight (-250 g)
divided by factor below with DI water
Division Factor
Divide exact weight of the standard by
this factor to give the final weight
100
—
0.4
50
— .
0.2
~,
250
—
—
62-5
0.25
—
12.5
0.05
—
—
—
6. SAMPLE COLLECTION, PRESERVATION AND STORAGE
6.1. Analysis should be commenced within two hours of sample collection. Glass bottles
should be used, unless acceptable storage stability studies have been completed with
plastic bottles. If long term storage is necessary, samples should be filtered on-site with
-------
0.45 uM pore size membrane filters (washed with greater than 200 mL of sample) and
frozen at -10°C. Samples should be frozen in plastic bottles leaving about 30%
headspace for expansion. Frozen samples may be. stored for up to three months.
6.2. Thaw samples by immersion in warm water with occasional mixing to ensure uniform
sample temperature.. Do not warm samples-above ambient temperature. Since the
analyte is in the liquid portion of the thawing,, care should be taken to ensure complete
thawing.
6.3. Samples should be collected in plastic or glass bottles. All bottles must be thoroughly
cleaned and rinsed with reagent water. The volume collected should be sufficient to
insure a representative sample, allow for replicate analysis (if required), and minimize
waste disposal.
6.4. If samples must be chemically preserved, add 2 mL of 8 N H2SO4 per liter of sample.
Store in glass or polyethylene at 4°C. Analyze within two months.
6.5. Some researchers have found serious errors when investigating the effects of filtration.
Others have found phosphate to absorb on the walls of polyethylene bottles. It is
imperative that the analyst examines preservation techniques before routine testing.
7. PROCEDURE
7.1. CALIBRATION PROCEDURE
7.1.1. Prepare reagent and standards as described in section 5.
7.1.2. Set up manifold as shown in section 11.
7.1.3. Input data system parameters as shown in section 11.
7.1.4. Pump DI water through all reagent lines and check for leaks and smooth flow.
Switch to reagents and allow the system to equilibrate until a stable baseline is
achieved.
7.1.5. Place samples and/or standards in the sampler. Input the information required by
the data system, such as concentration, replicates and QuikChem scheme (See
section 11).
7.1.6. Calibrate the instrument by injecting the standards. The data system will then
associate the concentrations with the instrument responses for each standard.
7.2. SYSTEM NOTES
7.2.1. For information on system maintenance and troubleshooting refer to the
Troubleshooting Guide in the System Operation Manual. This guide is also
available on request from Lachat.
7.2.2. Allow 15 minutes for the heating unit to warm up to 45°C.
7.2.3. Glassware contamination is a problem in low level phosphorus determination.
Glassware should be washed with 1:1 HC1 solution and rinsed with deionized
water. Commercial detergents should rarely be needed but, if they are used, use
special phosphate-free preparations for lab glassware.
7.2.4. Reagent recipes from other automated wet chemistry analyzers should not be
substituted.
-------
7.2.5. It is very important to use granular ascorbic acid as opposed to powder. If the
ascorbic acid solution turns yellow upon preparation or storage it should be
discarded.
7.2.6. Over time a blue film may accumulate on the walls of the flowcell and in the
manifold tubing. This may be removed by pumping the manifold rinse solution
(Reagent 5) for 5 minutes followed by rinsing DI water for 5 minutes.
7.2.7. The blank in this method should not give a peak. If the blank peak is negative,
the carrier is contaminated. If the blank peak is positive, the blank is
contaminated.
7.2.8. If samples are colored, this background can be subtracted. First calibrate in the
normal manner. Next, replace the molybdate reagent with a solution "containing
35 mL H2SC>4/L. finally, reanalyze the samples. The color interference
concentration is then subtracted from the original determined concentration.
8. DATA ANALYSIS AND CALCULATIONS
8.1. Calibration is done by injecting standards, The data system will then prepare a
calibration curve by plotting response versus standard concentration. Sample
concentration is calculated from the regression equation.
8.2. Report only those values that fall between the lowest and highest calibration standards.
Samples exceeding the highest standard should be diluted and reanalyzed.
8.3. Report results in jig P/L.
9. METHOD PERFORMANCE
9.1. The method support data are presented in section 11. This data was generated according
to a Lachat Work Instruction during development of the method.
9.2. Although Lachat Instrument publishes method performance data, including MDL,
precision, accuracy and carryover studies, we cannot guarantee that each laboratory will
be capable of meeting such performance. Individual laboratory and instrument
conditions, as well as laboratory technique play a major role in determining method
performance. The support data serves as a guide of the potential method performance.
Some labs may not be able to reach this level of performance for various reasons, while
other labs may exceed it.
10. REFERENCES
10.1. 40 CFR, 136 Appendix B. Definition and Procedure for Determination of the Method
Detection Limit. Revision 1.1.
10.2. Grasshoff.K., Methods of Seawater Analysis, Verlag Chemie, Federal Republic of
Germany, Second Edition, 1976,419 pages.
10.3. Murphy J. and Riley, J.P., A Modified Single Solution Method for the Determination of
Phosphate in Natural Waters, Anal. Chim. Acta, Vol. 27,1962, p. 31-36.
10.4. Murphy J. and Riley, J.P., the Storage of Seawater Samples for the Determination of
Dissolved Inorganic Phosphate, Anal. Chim. Acta, Vol. 14,1956, p. 318-319.
-------
10.5. MacDonald, R.W. and F.A. McLaughlin. The Effect of Storage by Freezing on
Dissolved Inorganic Phosphate, Nitrate and Reactive Silicate for Samples from Coastal
and Estuarine Waters, Water Research, Vol. 16; 1982 p. 95-104.
10.6. Johnson, K., and Petty, R., Determination of Phosphate in Seawater by Flow Injection
Analysis with Injection of Reagent, Analytical Chemistry, Vol. 54,1982, p. 1185-1187.
10.7. Lachat Instruments Inc., QuikChem Method 31-115-01-1-F written by Amy Huberty and
David Diamond on 29 December 1998.
10.8. Lachat Instruments Inc., QuikChem Method 31-115-01-1-G written by David Diamond
on 30 December 1998.
-------
11. TABLE. DIAGRAMS. FLOWCHARTS, AND VALIDATION DATA
11.1. DATA SYSTEM PARAMETERS FOR QUIKCHEM 8000
The timing values listed below are approximate and will need to be optimized using
graphical events programming.
Sample throughput:
Pump Speed:
Cycle Period:
Analyte Data:
Concentration Units:
Chemistry:
Inject to BW Baseline Start
Inject to BW Basehne End
Inject to BW Integ Start
Inject to BW Integ End
Calibration Data:
48 samples/h, 75 s/sample
35
75
Brackish
3.7s
62.0s
20.9s
27.3
Level
Concentration fig P/L
1
400
2
200
3
100
4
25
5
5
6
0
Calibration Rep Handling:
Calibration Fit Type:
Weighting Method:
Force through zero:
Sampler Timing:
Mm. Probe in Wash Period:
Probe in Sample Period:
Valve Timing:
Load Time:
Load Period:
Inject Period:
Average
2nd Order Polynomial
None
No
19s
45s
Os
30s
-------
11.2. SUPPORT DATA FOR QUIKCHEM 8000
Calibration Data for Orthophosphorus
File Name: 201200c4.fdt
Acq. Date: 20 December 2000
Calibration Graph and Statistics
Level
1
2
3
4
5
6
Area
4231523
2104093
1028960
244912
43507
-158
HgP/L
400
200
100
25
5
0
Determined
395.2
199.82
98.5
24.55
4.99
0
Replicate
%RSD
1.2
0.9
1.5
1.8
0.3
-1124.7
% residual
0.0
-0.4
0.9
2.7
-1.1
—
Scaling: None
Weighting: None
2nd Older Poly
Cone - -3.493a-013 Arerf + 9.576e-OOS Area + 3.930*001
t- 1.0000
-------
«A*p 0.952305 von*
4=*44C^
Mrs
.irvur
JrsJJrs
H H M M M
i i i i
i i i i i i i i i
i i i
j
I
3
h N
n a
n N
N N
Method Detection Limit for Orthophosphorus using 2.5 fxg P/L standard
MDL= 033 fig P/L * Claimed MDL is 1.0 jig P/L due to Y-intercept and carryover.
Standard Deviation (s) = 0.13 |ig P/L, Mean (x) = 2.4 |^g P/L, Known value = 2.5 ug/L
Acq. Date: 20 December 2000
File Name: 201200c3.fdt
Precision data for Orthophosphorus using 100 jig P/L standard
% RSD = 0.86 %
Standard Deviation (s) = 0.85 jig P/L, Mean (x) = 98.6 jig P/L, Known value = 100 (ig P/L
Acq. Date: 20 December 2000
File Name: 201200c4.fdt
-------
Carryover Study: 400 pig P/L standard followed by 4 blanks
Carryover Passed
Acq. Date: 20 December 2000
File Name: 201200c4.fdt
-------
Various seawater samples showing different salinities and their spikes.
1M<.t!S.<»a Amf: (Ugm Volu
V 'V 'V
i i i
2 3 3
i i
i i i
i i i
8 8
Acq. Date: 20 December 2000
File Name: 201200c6.fdt
Sample Type
Artificial Seawater
_
Artificial Seawater plus
50 ppb spike
*Nutrient Depleted
Seawater
*Nutrient Depleted
Seawater plus
50 ppb spike
*NCSU sample water
Results ^g P/L
2.9688
2.3585
2.6638
45.4061
45.0773
46.0375
27.8034
27.7725
28.3994
73.2411
74.4015
74.3447
42.6480
42.2650
42.1551
Averages |ig P/L
2.6637
45.5069
27.992
73.995
42.356
Spike Recoveries
85.69%
-
92.01%
...
*These samples were overrange. They were diluted after the spike with a 1:2 dilution to bring them within range.
-------
Interference Study using Silicate Standard
0.94
V
:
! i -i
"35 3T
The selectivity of the method against silicate is 1112.4. For 1000 mg of silicate, the response
would be 0.853 mg P/L. The amount of silicate in the above samples was 1700 (xg Si/L.
Conclusion: Silicate is not a significant interferent in this method.
-------
11.3. ORTHOPHOSPHATE MANIFOLD DIAGRAM
pump flow
white
white
yellow
green
>.
•
probe rinse t
9-
Molybdate Color Reagent
Ascorbic Acid Y
carrier 2, . 3
sample l(-fflX.>4
\ /- 1 to port 6
flow cell
Carrier: DI water
Manifold Tubing: 0.8 mm (0.032 in) i.d. This is .5.2 uL/cm.
AE Sample Loop: 150 cm x 0.042 in i.d.
QC8000 Sample Loop: 150 cm x 0.042 in i.d.
Interference Filter: 880 nm
Apparatus: An injection valve, a 10 mm path length flow cell, and a colorimetric detector
module is required. The ~T^M~ shows 175 cm of tubing wrapped around the
heater block at the specified temperature.
8: 168 cm of tubing on a 8 cm coil support
Note 1: The sample loop should be cut on a 30°-45° angle for the best fit. One o-ring can
also be used instead of 2.
Note 2: Backpressure coil of 200 cm x 0.5 mm (0.022 in) i.d.
Note 3: For the Cetac sampler: From the probe to the sample pump tube is 190 cm x 0.032
in i.d. From the sample pump tube to port 6 is 20 cm x 6.032 in i.d.
For the AJ/Gilson samplers: From probe to sample pump tube is 130 cm x 0.032
in i.d. From the sample pump tube to port 6 is 20 cm x 0.032 in i.d.
Note 4: Glass calibration vials must be used with this method (Lachat Part No. 21304).
-------
Nitrate and Nitrite
Method
-------
QuikChem® Method 31-107-04-1-D
DETERMINATION OF NITRATE AND/OR NITRITE IN BRACKISH
WATERS BY FLOW INJECTION ANALYSIS
Written by Lynn Egan
Applications Group
Revision Date:
20 November 2000
LACHAT INSTRUMENTS
6645 WEST MILL ROAD
MILWAUKEE, WI53218-1239 USA
-------
LrtCHrfT
^sfc, INSTRUMENTS
QuikChem® Method 31-107-04-1-D
DETERMINATION OF NITRATE AND/OR NITRITE IN BRACKISH
WATER BY FLOW INJECTION ANALYSIS
0.5 to 14 jig N/L as NO2 and/or NO3 __-?
(0.036-1 MM N as NO2 and/or NO3)
- Principle -
Nitrate is quantitatively reduced to nitrite by passage of the sample through a copperized
cadmium column. The nitrite (reduced nitrate plus original nitrite) is then determined by
diazotization with sulfanilamide under acidic conditions to form a diazonium ion. The
diazonium ion is then coupled with N-(l-naphthyl)ethylenediamine dihydrochloride. The
resulting pink dye absorbs at 540 nm. Nitrate concentrations are obtained by subtracting nitrite
values, which have been previously analyzed, from the nitrite + nitrate values. Though the
method is written for seawater and brackish water, it is also applicable to non-saline sample
matrixes.
The method is calibrated using standards prepared in deionized water. Heat is used to improve
linearity. Once calibrated, samples of varying salinites (0 to 35 ppt) may be~analyzed. The
determination of background absorbance is necessary only for samples mat have color absorbing
at540nm.
\
- Interferences -
1. Sample turbidity interferes. Remove turbidity by filtration with a 0.45 |jm. pore diameter
- membrane filter prior to analysis.
2. A positive error could be obtained for samples that contain high concentrations of iron,
copper, or other metals. Na2EDTA in the buffer helps to prevent this interference.
3. Samples that contain oil and grease will coat the surface of the cadmium, causing it to
lose reduction efficiency. This interference can be eliminated by pre-extracting the
sample with an organic solvent.
4. Sample color may be subtracted by analyzing the samples with a substitute color reagent,
which does not contain the diazotizing agent. This is done by replacing the
sulfanilamide-NED-phosphoric acid reagent with a solution containing 100 mL of
phosphoric acid per liter.
- Special Apparatus -
Please see Parts and Price list fbrOrdering Information
1. Heating Unit
2. Cadmium Reduction Column (Lachat Part No. 50327 or 50327R)
3. 60 position racks for samples are required to allow replicate sample analyses from a single
tube. XYZ with 60 Position rack (Lachat Part No. A81122 [110VJ/A81222 [220V]); RAS
A81136 [110VJ/A81236 [220V])
4. Sample tubes are needed for 60 Position Samplers (Lachat Part No. 21042)
5. PVC pump tubes and RP-100'pump must be used with this method.
Written by Lynn Egan and copyrighted © on 20 November 2000 by Lachat Instruments, 6645 West Mill Road,
Milwaukee, WI 53218-1239 USA. Phone: 414-358-4200 FAX: 414-358-4206. This document is the property of
T .ai-Viat TnctriiTT»»nfc TTnn'.,tt,^«n<.J ~~~,,;~rr ^Pfl,4« ,-].,„,,„.„„* :_ ujuj* l
-------
CONTENTS
1. SCOPE AND APPLICATION 2
2. INTERFERENCES 2
3. SAFETY............ ........; ....... .........;..... 2
4. EQUIPMENT AND SUPPLIES 3
5. REAGENTS AND STANDARDS 3
5.1. PREPARATION OF REAGENTS 3
5.2. PREPARATION OF STANDARDS '. 5
6. SAMPLE COLLECTION, PRESERVATION AND STORAGE 6
7. PROCEDURE 7
7.1. CALIBRATION PROCEDURE 7
. 7.2. SYSTEMNOTES 7
8. DATA ANALYSIS AND CALCULATIONS ;.... 8
9. METHOD PERFORMANCE 9
10. REFERENCES 9
11. TABLE, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 10
11.1. DATA SYSTEM PARAMETERS FOR QUKCHEM'8000 10
11.2. SUPPORT DATA FOR QUIKCHEM 8000 11
11.3. NrrRATE/NrrRTTE MANIFOLD DIAGRAM 15
-------
QuikChem® Method 31-107-04-1-D
DETERMINATION OF NITRATE AND/OR NITRITE IN BRACKISH
WATERS BY FLOW INJECTION ANALYSIS
1. SCOPE AND APPLICATION
1.1. This method covers the determination of nitrate/nitrite in brackish, seawater, or non-saline
sample matrices.
1-2. The method is based on reactions that are specific for the nitrite (NO2~) ion.
1.3. The applicable range is 0.5 to 14.0 \ig N/L (0.036-lpM). The method detection limit is
0.014 f4M N (0.20 jig N/L). The method throughput is 19 injections per hour.
2. INTERFERENCES
2.1. Sample turbidity will interfere by causing artificially high results. Remove turbidity by
filtration with a 0.45 um pore membrane filter prior to analysis.
2.2. A positive error could be obtained for samples that contain high concentrations of iron,
copper, or other metals. Na2EDTA in the buffer helps to prevent this interference.
2.3. Samples that contain oil and grease will coat the surface of the cadmium, causing it to
lose reduction efficiency. This interference can be eliminated by pre-extracting the
- sample with an organic solvent.
2.4. Sample color may be subtracted by analyzing the samples with a substitute color reagent
which does not contain the diazotizing agent. This is done by replacing the
sulfanilamide-NED-phosphoric acid reagent with a solution containing 100 mL of
phosphoric acid per liter.
3. SAFETY
3.1. 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
should be as low as reasonably achievable. Cautions are included for known extremely
hazardous materials.
3.2. Each laboratory is responsible for maintaining a current awareness file of the
Occupational Health and Safety Act (OSHA) regulations regarding the safe handling of
the chemicals specified in this method. A reference file of Material Safety Data sheets
(MSDS) should be made available to all personnel involved in the chemical analysis.
The preparation of a formal safety plan is also advisable.
3.3. The following chemicals have the potential to be highly toxic or hazardous. For detailed
explanations consult the MSDS.
-------
3.3.L Sodium hydroxide
3.3.2. Ammonium chloride
3.3.3. Phosphoric acid
3.3.4. Sulfanilamide
3.3.5. N-(i-naphthyl)-ethylenediamine(NED)
3.3.6. Cadmium
3.3.7. Ammonium hydroxide
3.3.8. Hydrochloric acid
4. EQUIPMENT AND SUPPLIES
4.1. Balance ~ analytical, capable of accurately weighing to the nearest 0.0001 g.
4.2. Glassware - Class A volumetric flasks and pipettes or plastic containers as required.
Samples should be stored in plastic.
4.3. Flow injection analysis equipment designed to deliver and react sample and reagents in
the required order and ratios. '
4.3.1. Sampler
4.3.2. Multichannel proportioning pump
4.3.3. Reaction unit or manifold
4.3.4. Colorimetric detector
.~ 4.3.5. Data system
4.4. Special Apparatus
1^4.4.1. Heating unit
4.4.2. Cadmium Column (Lachat Part No. 50327 or 50327R)
4.4.3. 60 position racks for samples are required to allow replicate sample analyses from a
single tube. XYZ with 60 Position rack (Lachat Part No. A81122 [110V]/A81222
[220V]); RAS A81136 [110V]/A81236 [220V])
4.4.4. Sample tubes are needed for 60 Position Samplers (Lachat Part No. 21042)
4.4.5. PVC pump tubes and RP-100 pump must be used with this method.
5. REAGENTS AND STANDARDS
5.1. PREPARATION OF REAGENTS
Use deionized water (18 megohm-cm or greater is strongly recommended for successful
trace-level analysis) for reagents, standards, carrier and washbath solution.
Degassing with helium:
To prevent bubble formation, degas all solutions except the standards with helium. Use
He at 140kPa (20 lb/in2) through a helium degassing tube (Lachat Part No. 50100.)
Bubble He through the solution for one minute. (Dedicated degassing tubes will prevent
cross contamination).
-------
Reagent 1. 15 N Sodium Hydroxide
By Volume: Add 150 g NaOH very slowly to about 125 ml. of DI water in a 250 mL
volumetric flask . CAUTION: This solution will get very hot! Stir until dissolved. Dilute
to volume when cool. Store in a plastic bottle.
Reagent 2. Ammonium Chloride Buffer, pH 8.5
By Volume: In a 1 L volumetric flask, dissolve 85.0 g ammonium chloride (NH4C1) and
4.0 g disodium ethylenediamine tetra-acetic acid dihydrate (Na2EDTA'2H2O) in about
800 mL DI water. Dilute to the mark and invert to mix. Adjust the pH to 8.5 with 13 N
sodium hydroxide solution.
By Weight: To a tared 1 L container, add 85.0 g ammonium chloride (NHUCl), 4.0 g
disodium ethylenediamine tetraacetic acid dihydrate (Na2EDTA'2H20) and 938 g DI
water. Shake or stir until dissolved. Then adjust the pH to 8.5 with 13 N sodium
hydroxide solution.
ACS grade ammonium chloride has been found occasionally to contain significant nitrate
contamination. (The main symptom of this type of contamination would be a larger than
normal increase in baseline when the cadmium column is placed in line). An alternative
recipe for the ammonium chloride buffer is:
By Volume: CAUTION: Fumes!!! In a hood, to a 1 L volumetric flask, add 500 mL DI
water, 105 mL concentrated hydrochloric acid (HCI), 95 mL ammonium hydroxide
(NEUOH), and 4.0 g disodium EDTA dihydrate. Dissolve and dilute to the mark. Invert to
mix. Adjust the pH to 8.5 with HCI or 13 N NaOH solution.
By Weight: CAUTION: Fumes!!! In a hood, to a tared 1 L container, add 800 g DI
water, 126 g concentrated hydrochloric acid (HCI), 85 g ammonium hydroxide (NHtOH)
and 4.0 g disodium EDTA dihydrate. Stir until dissolved. Adjust the pH to 8.5 with HCI
or 13 N NaOH.
(Note: The ammonium chloride solution, pH 8.5, is used as a buffer prior to the reduction of
NO3" to NO2". At pH 8.5, ammonia gas is evolved and may cause contamination of reagents,
standards, and samples in nearby ammonia, total nitrogen, or TKN determinations. For this
reason, it is important to seal the buffer and waste containers with laboratory film, and to
keep this container tightly closed when not in use).
Reagent 3. Sulfanilamide Color Reagent
By Volume: To a 1L volumetric flask add about 600 mL DI water. Then add 100 mL 85%
p^£p™°^.laHd^3PO4), 40.0 g sutfanaajmidejind 5.0 g N-(l-naphthyl)-ethylenedianiine
3ffiiyrdrocHroride (NED). Shake to wet, and stifto dissolve for 30 min. Dilute to m"e" mark,
and invert to mix. Filter through a 0.45 um filter prior to use. Store in a dark bottle and
discard when the solution turns pink.
By Weight: To a tared, dark 1 L container add 875 g DI water, 170 g 85% phosphoric
acid (H3PO4), 40.0 g sulfanilamide, and 5.0 g N-(l-naphthyl)ethylene- diamine
dihydrochloride (NED). Shake until wetted and stir with a stir bar for 30 min. until
dissolved. Filter through a 0.45 ym filter prior to use. Store in a dark bottle and discard
when the solution turns pink.
-------
Reagent 4: Artificial Seawater (ASW).
This solution contains the major constituents of seawater. (Alternatively, ammonia-free, low
nutrient natural seawater can be used if available). This is used to determine brackish timing
parameters. An ASW "blank" is injected, along with low-level spikes at one or two levels.
(This tray must include a calibration for accurate results). While this reagent will contain
some level of nitrate, low level spikes aid in determination of the proper area of the peak to
integrate.
By Volume: In a 2 L volumetric flask, dissolve 58.6 g sodium chloride (NaCI), 18.8 g
magnesium sulfate heptahydrate (MgSO4 ' 7H2O), and 0.44 g sodium bicarbonate
(NaHCO3) in about 1500 mL of DI water. Stir to mix and dilute to the mark.
By Weight: To a tared 2 L container, add 58.6 g sodium chloride (NaCI),. 18.8 g
magnesium sulfate heptahydrate (MgSO4 ' 7H2O), and 0.44 g sodium bicarbonate
(NaHCO3) to 1962 g of DI water. Stir to mix.
5.2. PREPARATION OF STANDARDS
To prepare the stock and working standards, the following containers will be required:
By Volume: Two 1 L, four 500 mL three 250 mL, and one 200 mL volumetric flasks
By Weight: Two 1 L, four 500 mL, three 250 mL and one 200 mL containers.
Standard 1. Stock Standard 100.0 mg N/L
^
Nitrate: In a 1 L volumetric flask dissolve 0.607 g sodium nitrate (NaNO3) in about
800 mL water. Dilute to the mark and invert to mix. Refrigerate. Prepare Monthly.
Nitrite: In a 1 L volumetric flask dissolve 0.493 g sodium nitrite (NaNO2) in about 800
mL water. Dilute to the mark and invert to mix. Refrigerate. Prepare weekly.
Standard 2. Stock Standard 10 mg N/L.
By Volume: To a 500 mL volumetric flask add 50 mL of Standard 1 (100 mg N/L as
NO3 or NO2). Dilute to the mark with DI water and invert to mix. Prepare fresh daily.
Standard 3. Stock Standard 100 jig N/L as NO3 or NO2
By Volume: To a 500 mL volumetric flask add 5 mL of Standard 2 (10 mg N/L as
NO3 or NO2). Dilute to the mark with DI water and invert to mix. Prepare fresh daily.
(Note: The 14 U£ N/L as N02 is to be used for assessment of column efficiency. It,
along with the 14 ng N/L as NO3 standard should be analyzed when the column is
exchanged or reduction efficiency is suspect. If nitrite alone is being analyzed, then only
nitrite standards need to be prepared, and the reduction column is kept off-line during the
analysis). If nitrate + nitrite are being measured, then nitrate standards and the 14 fig
nitrite standard should be prepared.
-------
Working Standards (Prepare Daily)
Concentration u,g N/L as NO, or NO2
A
14
B
7
C
3.5
D
1.75
E
0.5
••^MVMI^MMVH
F
0.00
By Volume
Volume (mL) of stock standard 3
diluted to 250 mL with DI water
Volume (mL) of working standard C
Diluted to 250 mL with DI water
Volume (mL) of stock standard 3
diluted to 200 mL with DI water
35
—
...
17.5
—
—
8.75
—
—
125
—
— -
— .
1.0
—
._
—
By Weight
Weight (g) of stock standard 3
diluted to final weight (-250 g)
divided by factor below with DI water
Weight (g) of working standard C
diluted to final weight (-250 g)
divided by factor below with DI water
Weight (g) of stock standard 3
diluted to 200 mL with DI water
Division Factor
Divide exact weight of the standard by
this factor to give the final weight
35.0
—
—
01.14
17.5 "
—
—
0.07
8.75
—
—
0.035
—
125
—
0.5
._.
—
1.0
0.005
...
—
—
—
6.2.
6. SAMPLE COLLECTION, PRESERVATION AND STORAGE
6.1. Nitrite will be oxidized by air (O2) to nitrate in a few days. If analysis cannot be made
within 24 hours, the sample should be preserved by refrigeration at 4°C.
When samples must be stored for more than-24 hours,, they should be preserved either
with sulfuric acid (addition of a maximum of 2 mL of concentrated H2SO4 per liter) and
stored refrigerated (at 4°C) or frozen. CAUTION: Samples must not be preserved with
mercuric chloride or thiosulfate because this will degrade the cadmium column. Frozen
samples must be completely thawed and be well mixed prior to analysis to obtain a
representative sample.
Samples should be collected in plastic or glass bottles. All bottles must be thoroughly
cleaned and rinsed with reagent water. The volume collected should be sufficient to
ensure a representative sample, allow for replicate analysis (if required), and minimize
waste disposal. (NOTE: If samples are to be analyzed for silicate as well, plastic
containers must be used).
6.3.
-------
7. PROCEDURE
7.1. CALIBRATION PROCEDURE
7.1.1. Prepare reagents and standards as described in section 5.
7.1.2. Set up manifold as shown in section 11.
7.1.3. Input data system parameters as shown in section 11.
7.1.4. With the cadmium column off-line, pump DI water through all reagent lines and
check for leaks and smooth flow. Switch to reagents and allow the system to
equilibrate until bubbles are no longer visible in the manifold. Place the cadmium
column in line, and allow reagents to pump until a stable baseline is achieved.
7.1.5. Place samples and/or standards in the sampler., Input the information required by
the data system, such as concentration, replicates and QuikChem scheme (See
section 11).
7.1.6. Calibrate the instrument by injecting the standards. The data system will then
associate the concentrations with the instrument responses for each standard.
7.1.7. Because this is a trace level seawater method, which requires the use of brackish
timing, it is strongly recommended that a calibration be run with every tray.
7.1.8. Standards may be chosen to bracket sample concentrations as long as the range of
the method is not exceeded.
7.2. SYSTEM NOTES
7.2.1 For information on system maintenance and troubleshooting refer to the
Troubleshooting Guide in the System Operation Manual. This guide is also
available on request from Lachat.
7.2.2 When running this chemistry as part of a multi-channel method, the sequential
filling of large-volume sample loops may limit the cycle time and consume extra
sample. To prevent these effects, the sample stream is split prior to the pump by
adding a tee at the pump inlet and using two red/red sample pump tubes in place
of a single green/green one. This allows each sample line to feed 2-4 channels.
7.2.3. Reagent recipes from other automated wet chemistry analyzers should not be
substituted. It is important to check column efficiency each time the column is
replaced or when efficiency is suspect. Once the efficiency is known, a nitrite
standard can be inserted in the sample tray to verify that the column remains
efficient. To do this, a nitrate and nitrite standard of equal concentrations should
be run side by side. If the value for nitrate does not fall within 90-110% the value
obtained for nitrite, the column should be replaced.
7.2.4. Poor correlation coefficients are sometimes the result of sub-standard column
performance. If the standards are freshly prepared and the calibration fails
consistently, replace the column.
7.2.5. Because this method is for the measurement of trace levels of nitrate and/or
nitrite, poor correlation coefficients may also be caused by improperly or
carelessly prepared standards, contaminated standard or sample cups, or the use of
water of less than optimum quality.
-------
7.2.6. If sample tube or standard container materials other than polystyrene are used,
standards and samples in these containers should be analyzed to investigate
absorption or contamination.
7.2.7. The blank in this method should result in a very minute peak or no peak at all. If
the blank peak is negative, the carrier is contaminated. If the blank peak is
positive, the blank is contaminated. It is crucial that high-quality water be used
for preparation of both reagents and standards (18.O M£2-cm or better is strongly
suggested). Dilutions must also be very carefully carried out.
7.2.8. If samples are colored, this interference can be determined and subtracted. First,
calibrate in the standard fashion. Next, replace the color reagent with a solution
containing 100 mL H3PC>4/L. Finally, reanalyze the samples. The determined
concentration due to color interference can then be subtracted from the original
determined concentration.
7.2.9. It is critical that the peak be detected on the "flat portion" of the seawater peaks.
This is done by injecting a seawater blank. If the integration window is not on the
"flat portion", the peak start time should be adjusted. Correct positioning can be
tested by the use of ASW (or low nutrient seawater) as a blank and spiked at one
or two low levels. Spike recoveries of 80-120% should be achieved.
7.2.10. To ascertain that the standard peak is properly positioned in the window, observe
the peaks on screen. The baseline trace should be as flat as possible where the
baseline start and stop times are set. If the peak is not properly positioned, adjust
the timing.
,7.2.11. For low level analysis it is recommended that samples be analyzed in duplicate
from each sample cup. This is done by entering Replicates = 2 when entering
sample information.
7.2.12. Due to low analyte levels, a noisy baseline will adversely affect the results. Pump
tube wear will cause increased noise as well as changes in timing. Noise will also
be increased by the use of unfiltered color reagent.
7.2.13. If spike recoveries in the seawater matrix are high, additional Na2EDTA may be
required in the ammonium chloride buffer reagent. (High spike recoveries are
likely to be caused by precipitate formed at higher pH by cations. This precipitate
appears to be signal from the analyte).
8. DATA ANALYSIS AND CALCULATIONS
8.1. Calibration is done by injecting standards. The data system will then prepare a
calibration curve by plotting response versus standard concentration. Sample
concentration is calculated from the regression equation.
8.2. Report only those values that fall between the lowest and highest calibration standards.
Samples exceeding the highest standard should be diluted and reanalyzed.
8.3. Report results in jig N/L or (jM N.
-------
9. METHOD PERFORMANCE
9.1. The method support data are presented in section 11. This data was generated according
to a Lachat Work Instruction during development of the method.
9.2. Although Lachat Instruments publishes method performance data, including MDL,
precision, accuracy and carryover studies, we cannot guarantee that each laboratory will
be capable of meeting such performance. Individual laboratory and instrument
conditions, as well as laboratory technique play a major role in determining method
performance. The support data serves as a guide of the potential method performance.
Some labs may not be able to reach this level of performance for various reasons, while
other labs may exceed it.
10. REFERENCES
10.1 Grasshoff, K. Methods of Seawater Analysis, Verlag Chemie, Second Edition, 1976
10.2 Zimmerman, Carl. F. and Keefe, Carolyn W., EPA Method 353.4, Determination of
Nitrate + Nitrite in Estuarine and Coastal Waters by Automated Colorimetric Analysis in
An Interim Manual of Methods for the Determination of Nutrients, in Estuarine and
Coastal Waters., Revision 1.1, June 1991.
10.3 Johnson, K.S. and Petty, R.L., Determination of Nitrate and nitrite in Seawater by Flow
Injection Analysis, Limnol. Oceanogr., 28(6) p. 1260-1266
10.4 Anderson, Leif, Simultaneous Spectrophotometric Determination of Nitrite and Nitrate
~by Flow Injection Analysis, Analytica Chimica Acta, Vol. 110,1979 p. 123 -128
10.5 Yamane, T. and Saito, M., Simple Approach for Elimination of Blank Peak Effects in
Flow Injection Analysis of Samples Containing Trace Analyte and Excess of another
Solute., Talanta, Vol. 39, No. 3,1992 p. 215-219.
10.6 MacDonald, R.W. and F.A. McLaughlin. The Effect of Storage by Freezing on Dissolved
Inorganic Phosphate, Nitrate, and Reactive Silicate for Samples from Coastal and
Estuarine Waters, Water Research, Vol. 16, 1982 p. 95 -104
10.7 Lachat Instruments Inc., QuikChem Method 31-107-04-1-C written by D. Diamond on 18
January 1994. Revised by L. Egan 27 June 2000.
-------
11. TABLE. DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA
11.1. DATA SYSTEM PARAMETERS FOR QUIKCHEM 8000
The timing values listed below are approximate and will need to be optimized using
graphical events programming.
Sample throughput: 19 samples/h, 190 s/sample
Pump Speed: 35
Cycle Period: 190
Analyte Data:
Concentration Units:
Chemistry
Inject to BW Baseline Start
Inject to BW Baseline End
Inject to BW Integ Start
Inject to BW Integ End
Calibration Data:
u,g N/L or fiM N
Brackish
20.4s
209.4s
42s
75s
Level
Concentration \ig N/L
Concentration juM N
1
14.0
1
2
7
0.5
3
3.5
0.25
4
1.75
0.125
5
0.5
0.036
6
0
0
Calibration Rep Handling:
Calibration Fit Type:
Weighting Method:
Force through zero:
Sampler Timing:
Min. Probe in Wash Period:
Probe in Sample Period:
Valve Timing:
Load Time:
Load Period:
Inject Period:
Average
2nd Order Polynomial
None
No
9s
65s
Os
53s " -:
137s '-
-------
11.2. SUPPORT DATA FOR QUIKCHEM 8000
Calibration Data for Nitrate/Nitrite
135-
File Name: Ino3pre3.fdt
Acq. Date: 16 November 2000
Calibration Graph and Statistics
Level
1
2
3
4
5
6
Area
2062314
1098590
588874
321618
96171
19127
M,gN/L
14.00
7.00
3.50
1.75
0.5
0
Determined
14.028
6.916
3.51
1.83
0.46
—
% residual
-0.2
1.2
-0.3
-4.4
8.0
—
Scaling: None
Weighting: None
2nd Order Polji
Cone = 3.487a-013 Area1 + 6.711 e-006 Area -1.724e-001
r- 0.3999
-------
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u M a
333
Method Detection Limit for Nitrate using 0.5 jig N/L standard
MDL= 0.20 Jig N/L (0.014 (AM N)*
Standard Deviation (s) =0.068 (ig N/L, Mean (x) = 0.382 [Xg N/L, Known value = 0.5 fxg N/L, %RSD = 16.14
File Name: Ino3pre3.fdt
Acq. Date: 16 November 2000
Results are noise limited, and may be improved by the use of a longer cycle period
TlM 11770.95 S*co«d» Amp; -(UM356 Veto
3 33 3 3 3
o » s N o n l-o-"
10:00 nocc
Precision data for Nitrate using 7 pig N/L standard
% RSD = 1.44
Standard Deviation (s) = 0.099 (ig N/L, Mean (x) = 6.89 jig N/L, Known value = 7 p,g N/L,
File Name: Ino3pre3.fdt
Acq. Date: 16 November 2000
-------
-or
-
-------
Spikes Into Artificial Seawater Matrix.
tu
3
I
0
File Name: spike3.fdt
Acq. Date: 20 November2000
0.5 and 2.1 jj,g N/L as NO3 was spiked into an artificial seawater matrix. Recoveries of 80.62%
and 101.66% were obtained, respectively. Conclusion: Recoveries of 80-120% can be
obtained in the artificial matrix.
-------
11.3. NITRATE/NITRITE MANIFOLD DIAGRAM
pump flow
orange-white
red
white
red
Note 1
probe rinse
Sulfanilamide NED Color Reagent
12
NoteS
flow cell
Ammonium Chloride Buffer
Carrier
Sample
Noted
to port 6 of next valve
or waste
Carrier: DI water
Manifold Tubing: 0.8 mm (0.032 in) i.d. This is 5.2 \jJJcm.
QC8000 Sample Loop: 125 cm x 0.042" i.d.
Interference Filter: 540 nm
Apparatus: An injection valve, a 10 mm path length flow cell, and a colorimetric detector
module is required. The
' shows 175 cm of tubing wrapped around the
heater block at the specified temperature.
12: 150 cm of tubing on a 12 cm alternating coil support
Note: PVC PUMP TUBES MUST BE USED FOR TfflS METHOD.
Note 1: Sample line is connected to a red-red pump tube
Note 2: Sample loop ends should be cut at a 30-45° angle for the best fit.
Note 3: 12-cm.<.alternating coil support wrapped'with 150 cm of 0.032" i.d. tubing.
Note 4: Two-state switching valve for the cadmium column-
Note 5: 175 cm of 0.032" tubing wrapped on the heater at 45°C. This section of tubing
must not be used for phosphate or silicate chemistries, as the phosphoric acid in
the nitrate color reagent can interfere in these chemistries. This section of tubing
should be dedicated to nitrate only.
Note 6: Flow is to waste. Backpressure loop (50 cm x 0.022" i.d.) should be used only if
outgassing is problematic, as it may cause increased noise.
-------
Chlorophyll A
Standard Operating Procedure
-------
ECOTOX/SOP 00-005
Page 1 of3
Effective Date; 06 December, 2000
Tide: FLUOROMETRIC DETERMINATION OF CHLOROPHYLL A
Author: Date:
Marie E, DeLorcnzo
Program Manager: Date:
Michael H. Fulton
Branch Chief: Date;.
Geoffrey I. Scott
1.0 OBJECTIVE
Chlorophyll a is used to estimate phototrophie biomass. The purpose of this method is to quantify
chlorophyll a concentration from water samples. This method was adapted from Glover and Morris
(1979).
2.0 HEALTH AND SAFETY
Personnel should wear lab coats and chemical resistant gloves.
3.0 PERSONrm/TRAINING/RESPONSmnjrnES
Personnel should not perform this method until training by experienced individuals is complete.
4.0 REQUIRED AND RECOMMENDED MATERIALS
20 mL plastic scintillation vials
acetone
magnesium carbonate (MgCQ?)
deionized water
glass fiber filters (Type GF/F, 25 mm diameter)
filter apparatus
filter forceps
fluorometer (e.g. Sequoia-Turner Model 450)
-------
ECOTOX/SOP 00-005
Page 2 of3
Effective Date: 06 December, 2000
disposable, borosiUcafe glass test tubes for ffuorometer
Vortex mixer
1 and 10 mL pipettes/bulbs
5.0 PROCEDURE
5.1 Chlorophyll extraction
« Sample volume required wffl vary, depending on file chlorophyll concentration in fee
water. For PFU samples, 10 mL is typically adequate.
• Filter water sample onto glass fiber filter (Type GF/F, 25 mm diameter),
* Just before all sample passes the filter, rinse the column with two separate aliquots (1.0
mL each) deionized water. Continue vacuum until all liquid is gone,
* Release vacuum, disassemble filter tower apparatus, and remove filter with forceps.
• Place filter face up on bottom of scintillation vial, add 1 mL MgQDj and freeze until
analysis.
» Samples should be kept in the dark for file-rest of the procedure. To extract the
samples, add 9 mL of acetone to each scintillation vial and shake weU.
* Refrigerate samples overnight in the dark at 4 QC .shake the samples the nextday.and
refrigerate overnight again.
* The next day, bring the samples to room temperature and read on a fluorometer.
5.2 Fluorometrk measurement
5.2.1 Chlorophyll a
» Before using the fiuorometer for unknowns, a standaM oirve should be created with
pure chlorophyll a extracts (available from Sigma).
» "Zero* each door opening of the fluorometer immediately prior to use, using a tube of
90% acetone.
-------
ECOTQX/SOP 00-005
Page 3 of3
Effective Date: 06 December, 2000
• Decant the chlorophyll extract from the scintillation vial into fee fluorometer test tube.
Care should be taken not to transfer particulates from the filter into the tube.
* Wipe off the sides of the test tube with a Kim Wipe and place the test tube in the
fluorometer,
* Record the fluorescence units, door setting and gain setting.
* Samples that are too concentrated may be diluted with 90% acetone.
* A new test tube should be used for each sample.
5.2.2 Phaeo-pigments
This extra step is performed to determine and correct for the concentration of phaeo-pigments
(chlorophyll degradation products) in the samples. It is usually not necessary with the GF/F
filtering technique, but is provided here for those using other collection methods.
* After the first reading is taken on the fluorometer, remove the tube and add 2 drops of
5% v/v hydrochloric acid.
• Mix contents of tube with a vortex mixer.
• Take a second reading 30-60 seconds later, after a stable value is reached.
53 Calculations
Chlorophyll a Oig/mL)= (door factor*(cMorophyll fluorescence reading-phaeo-pigment
reading)*gain eoirection*acetone vohimeyvolume filtered*gain.
6.0 QUALITY CONTROL/QUALI1Y ASSURANCE
A minimum of three replicates per site or treatment is recommended It is important that each sample is
well-mixed prior to filtration and that the samples are kept in the dark after collection onto the filters.
7.0 REFERENCES
Glover H.E. and Morris 1.1979. PhotosynthetiG carboxylating enzymes in marine phytoplankton.
Umnol Oceanogr 23:510-519
-------
Appendix B
Data Sheet/Chain of Custody
-------
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