' O '
¦2. / Washington, DC
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United States Environmental Protection Agency
Office of Water
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NOTICE
The objective of the National Wetland Condition Assessment 2016 (NWCA 2016) project is to describe
the ecological condition of the nation's wetlands and stressors commonly associated with poor
condition. The complete documentation of overall project management, design, methods, quality
assurance, and standards is contained in four companion documents:
National Wetland Condition Assessment 2016: Field Operations Manual - 843-R-15-007
National Wetland Condition Assessment 2016: Quality Assurance Project Plan - 843-R-15-008
National Wetland Condition Assessment 2016: Laboratory Operations Manual - 843-R-15-009
National Wetland Condition Assessment 2016: Site Evaluation Guidelines - 843-R-15-010
This document (Laboratory Operations Manual) contains information on the methods for analyses of the
samples to be collected during the project, quality assurance objectives, sample handling, and data
reporting. Methods described in this document are to be used specifically in work relating to the NWCA
2016. All Project Cooperator laboratories should follow these guidelines. Mention of trade names or
commercial products in this document does not constitute endorsement or recommendation for use.
More details on specific methods for site evaluation, sampling, and sample processing in the field can be
found in the appropriate companion document.
The suggested citation for this document is:
USEPA. 2015. National Wetland Condition Assessment 2016: Laboratory Operations Manual. EPA-843-R-
15-009. U.S. Environmental Protection Agency, Office of Water, Washington, DC.
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VERSION HISTORY
Version
Date
Changes Made
1.0
2/12
n/a
1.1
4/18
Updated table 6.4 by adding column of acceptable reporting limits for
NWCA water chemistry and chlorophyll-a parameters; updated figures
for revised tracking forms; corrected minor grammatical errors and
typos
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TABLE OFCONTEMTS
NOTICE iii
TABLE OF CONTENTS v
LIST OF TABLES viii
LIST OF FIGURES viii
LIST OF ACRONYMS AND ABBREVIATIONS ix
1.0 INTRODUCTION 1
2.0 GENERAL LABORATORY GUIDELINES 3
2.1 Responsibility and Personnel Qualifications 3
2.2 Roles and Contact Information 3
2.3 Sample Tracking 3
2.4 Reporting 4
3.0 LABORATORY QUALITY CONTROL 5
3.1 Remote Evaluation/Technical Assessment 6
4.0 VEGETATION 9
4.1 Introduction 9
4.2 Receiving Voucher Samples 9
4.2.1 Definitions 9
4.2.2 Tracking information 10
4.3 Supplies and Equipment for Sample Handling 13
4.4 Handling Vegetation Samples 13
4.4.1 Plant Sample Label Form 13
4.4.2 Drying Samples 14
4.4.3 Treat samples for detritivores, molds, and pests 15
4.5 Identification of Vegetation Samples 15
4.5.1 Taxonomic standard. 15
4.5.2 Recording Identifications 15
4.6 Mounting and Storing Herbarium Sheets 15
4.7 Quality Assurance 16
4.7.1 Percent taxonomic disagreement (PTD) 17
4.8 References 17
5.0 SOILS 19
5.1 Introduction 19 £
5.2 Summary of Method 19 ^
5.3 Health and Safety Warnings 20 z
5.4 Sample Handling and Processing 20 8
5.4.1 Receiving Regulated Soils 20 o
5.4.2 Laboratory Sample Preparation 20 ^
5.5 Summary of Analytical Methods 21 <
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5.6 Quality Assurance / Quality Control (QA/QC) Procedures 23
5.6.1 Laboratory Performance Requirements 23
5.6.2 Laboratory Quality Control Samples 24
5.6.3 Data Reporting, Review, and Management 25
5.7 References 26
6.0 WATER CHEMISTRY AND CHLOROPHYLL A 29
6.1 Summary of Method 29
6.2 Health and Safety Warnings 29
6.3 Definitions and Required Resources (Personnel, Laboratories, and Equipment) 30
6.3.1 Definitions 30
6.3.2 General Requirements for Laboratories 31
6.3.3 Personnel 31
6.3.4 Equipment/Materials 32
6.4 Sample Receipt 32
6.5 Preparation of Water Chemistry Aliquots 33
6.6 Water Chemistry and Chlorophyll a Analysis: Requirements 35
6.7 Data Entry 38
6.8 Quality Measures 39
6.9 Sample and Record Retention 43
6.10 Literature Cited 43
7.0 ALGAL TOXIN (MICROCYSTIN) IMMUNOASSAY PROCEDURE 45
7.1 Summary of Method 45
7.2 Health and Safety Warnings 46
7.3 Definitions and Required Resources (Personnel, Laboratories, and Equipment) 46
7.3.1 Definitions 46
7.4 General Requirements for Laboratories 47
7.4.1 Expertise 47
7.4.2 Quality assurance and quality control requirements 48
7.4.3 Personnel 48
7.4.4 Equipment/Materials 48
7.5 Sample Receipt 49
7.6 Procedure 50
7.6.1 Sample Preparation 50
7.6.2 Additional Sample Preparation for Samples with Salinity>3.5 parts per thousand 51
7.6.3 Kit Prepara tion 52
7.6.4 Insertion of Con ten ts in to Wells 53
7.6.5 Dilutions (if needed) 58
1J Quality Measures 58
7.7.1 Assistance Visits 58
£ 7.7.2 QC Samples 58
m 7.7.3 Summary of QA/QC Requirements 59
z 7.8 Sample and Record Retention 60
u 7.9 References 61
U_
2 8.0 RESEARCH INDICATOR: SOIL ISOTOPES 62
I
< APPENDIX A: CONTACT INFORMATION 63
H
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APPENDIX B: LABORATORY REMOTE EVALUATION FORMS 65
APPENDIX C: DATA REPORTING TEMPLATES 79
APPENDIX D: SUPPLEMENTARY MATERIAL FOR VEGETATION - LISTS OF FLORISTIC RESOURCES 81
APPENDIX E: SUPPLEMENTARY MATERIAL FOR VEGETATION - PLANT PRESSING AND MOUNTING 91
APPENDIX F: USGS PROCEDURE FOR ANALYSIS OF ALGAL TOXINS (OGRL-SOP-5400) 95
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LIST
Table 2-1 Contact information 3
Table 4-1. Laboratory quality control activities for vegetation indicator 16
Table 5-1. Summary of NWCA 2016 soil analytical methods 19
Table 5-2. NWCA 2016 soil analytical methods. Analyses follow the standard operating procedures of the Kellogg
Soil Survey Lab (KSSL), National Soil Survey Center, Natural Resources Conservation Service 21
Table 5-3. Soil laboratory method performance requirements 23
Table 5-4. Soil data reporting criteria 25
Table 6-1. Water chemistry parameters measured by NWCA 2016 29
Table 6-2 Water Chemistry Login: Required Data Elements 32
Table 6-3 Water chemistry: acid preservatives added for various indicators 35
Table 6-4 Water Chemistry and Chlorophyll-a: Laboratory Method Performance Requirements 36
Table 6-5 Water Chemistry and Chlorophyll-a: Analytical Methods Used in Past NARS Surveys (EPA ORD-Corvallis)
37
Table 6-6 Water Chemistry and Chlorophyll-a: Data Elements for Each Sample 38
Table 6-7. Water chemistry reporting units and significant figures 39
Table 6-8 Water Chemistry and Chlorophyll-a: Quality control activities for water quality samples 39
Table 7-1 Microcystin: required data elements - login 50
Table 7-2 Microcystin: required data elements - data submission 56
Table 7-3 Microcystin: quality control - sample analysis 59
LIST
Figure 4-1. Unknown Plant Sample Tracking Form 11
Figure 4-2. QA Plant Sample Tracking Form 12
Figure 4-3. Plant specimen label 14
Figure 6-1. Water chemistry sample processing procedures 34
Figure 7-1 Microcystin: Abraxis microcystin test kit (from James, page 3, 2010) 45
Figure 7-2 Microcystin: sample template 54
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LIST : '/ 'AS ' ' ' ::
ASTM American Society for Testing and Materials NELAC
Ca calcium
CO2 carbon dioxide NELAP
CV coefficent of variation
Dl de-ionized NH4
DL detection limit NIST
DOC dissolved organic carbon NO2
ELISA enzyme-linked Immunosorbent assay NO3
EPA U.S. Environmental Protection Agency NTU
FOM Field Operations Manual NVC
GPS global positioning system NWCA
HCI hydrogen chloride PQL
HGM hydrogeomorphic PT
HDPE high density polyethylene PTD
HNO3 nitric acid QA
HQ headquarters QAPP
H2SO4 sulphuric acid QA/QC
IM information management QC
ISO International Organization for QCCS
Standardization QMP
ITIS Integrated Taxonomic Information System QRG
(ITIS) RL
K potassium RO
KC kit control RSD
LIMS Laboratory Information Management S
System SEG
LOM Lab Operations Manual SO2
LRL lower reporting limit SO4
Mg magnesium SOPs
MDL method detection limit SRM
MQO TMB
MSDS Materials Safety Data Sheet TN
N nitrogen TP
Na sodium UNK
NARS National Aquatic Resource Surveys USGS
NC negative control UV
ND non-detect
National Environmental Laboratory
Accreditation Conference
National Environmental Laboratory
Accreditation Program
ammonium
National Institute of Standards
nitrite
nitrate
Nephelometric Turbidity Units
National Vegetation Classification
National Wetland Condition Assessment
proficiency test
percent taxonomic disagreement
quality assurance
Quality Assurance Project Plan
quality assurance/quality control
quality control
quality control check solution
Quality Management Plan
Quick Reference Guide
reporting limit
reverse-osmosis
relative standard deviation
standard deviation
Site Evaluation Guidelines
sulphur dioxide
sulphate
Standard Operating Procedures
standard reference material
tetramethylbenzidine
total nitrogen
total phosphorus
unknown
United States Geological Survey
Ultraviolet
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1.0 INTRODUCTION
The U.S. Environmental Protection Agency (EPA), in partnership with state and tribal organizations, has
designed the National Wetland Condition Assessment (NWCA) 2016 to assess the condition of the
nation's wetlands. The NWCA is one in a series of National Aquatic Resource Surveys (NARS) conducted
to provide the public with a comprehensive assessment of the condition of the nation's waters. In
addition to wetlands, NARS assesses coastal waters, lakes, rivers, and streams in a revolving sequence.
This manual contains procedures for laboratory analysis of samples collected from wetlands throughout
the conterminous 48 states of the United States. The purposes of this manual are to:
1) document the standardized sample processing and analysis procedures used in the various
laboratories for the NWCA 2016
2) provide guidance for data quality and a performance-based method approach to obtain
comparable results across all participating laboratories.
Detailed laboratory procedures are described for the following indicators: algal toxins (microcystins),
soils, water chemistry and chlorophyll a, and vegetation. It should be noted that specific laboratory
analysis procedures for water chemistry samples are not presented here. A list of parameters to be
analyzed as well as the performance based methods and pertinent quality assurance/quality control
(QA/QC) procedures are outlined as requirements for laboratories to follow. Alternative analytical
methods for water chemistry are acceptable if they meet all specified performance requirements
described in this document. Acceptability is determined by the NWCA project management team (EPA
Office of Water).
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2.0 GENERAL LABORATORY GUIDELINES
2.1 Responsibility and Personnel Qualifications
All laboratory personnel shall be trained in advance in the use of equipment and procedures used for
the standard operating procedure (SOP) in which they are responsible. All personnel shall be responsible
for complying with all of the QA/QC requirements that pertain to the samples to be analyzed. Each lab
should follow its institutional or organizational requirements for instrument maintenance. Specific lab
qualification documentation required for analysis is contained in the Quality Assurance Project Plan
(QAPP).
2.2 Roles and Contact Information
The EPA Headquarters (HQ) Project Management Team consists of the Project Manager, Alternate
Project Manager, NARS QA Lead, Logistics Lead, and Laboratory Review Coordinator. The Team is
responsible for overseeing all aspects of the project and ensuring technical and quality assurance
requirements are properly carried out. The Team is the final authority on all decisions regarding
laboratory analysis.
The NARS Information Management (IM) Coordinator tracks the location of each NWCA 2016 sample
that involves post-processing. The coordinator will be the labs main point of contact in regards to
sample tracking and data submission.
Table 2-1 Contact information
Title Name Contact Information
EPA HQ NWCA Project
Manager
Gregg Serenbetz, OW
serenbetz.gregg(® eoa.gov
202-566-1253
EPA HQ NWCA Alternate
Project Manager
Chris Faulkner, OW
Faulkner.chris(® eoa.gov
202-566-1185
EPA HQ NARS QA Lead
Sarah Lehmann, OW
lehmann.sarah(® eoa.gov
202-566-1379
EPA HQ Logistics Lead
Colleen Mason, OW
Mason.colleen(® eoa.gov
202-343-9641
EPA HQ NWCA Laboratory
Review Coordinator
Kendra Forde, OW
kendra.forde@eoa.gov
202-564-0417
NARS IM Coordinator
Marlys Cappaert, SRA
International Inc.
caooaert.marlvs(® eoa.gov
541-754-4467
2.3 Sample Tracking
Samples are collected by a large number of different field crews during the index period (April through
September). The actual number of wetlands sampled on a given day will vary widely during this time.
Field crews will submit electronic forms when they have shipped samples and the NARS IM Center will
input each sample into the NARS IM database. Laboratories can track sample shipment from field crews
by accessing the NARS IM database. Participating laboratories will be given access to the NARS IM
system, where they can acquire tracking numbers and information on samples that have been shipped
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to them by field crews (either by overnight shipment for perishable samples or batch shipments for
preserved samples). Upon sample receipt, the laboratory must immediately log in to the database and
confirm that samples have arrived. Overnight samples may not be loaded into the database prior to
sample arrival, but should be tracked by the laboratory and receipt information inputted into the
database when sample information is loaded. Each lab will make arrangements with the NARS IM
Coordinator, listed above, to ensure access is granted.
When the samples arrive from the field crews, laboratories should also receive tracking forms in the
shipment (refer to the NWCA 2016 FOM). These forms will list the samples that should be included in
the shipment. Laboratory personnel should cross check the forms with the samples received to verify
that there are not any inconsistencies. If any sample is missing or damaged, contact the NARS IM
Coordinator immediately.
2.4 Reporting
All laboratories must provide data analysis information to the HQ Project Management Team and the
NARS IM Center by March 30, 2017 or as stipulated in contractual agreements. These reports must
include the following information:
• Sample Type (indicator)
• Site ID (ex: NWCA16-1001)
• Sample ID (ex: 999000)
• Pertinent information to the indicator
• Metadata for all fields
See Appendix C for a list of reporting templates that laboratories will submit electronically. Electronic
reporting templates will be provided on EPA's NARS Sharepoint site.
The submitted file name must state the following:
• Indicator name (e.g., water chemistry)
• Date of files submission to NARS IM Center by year, month, and day (e.g., 2016_11_01)
• Lab name (e.g., MyLab)
Combined, the file name would look as follows: WaterChemistry_2016_ll_01_MyLab.xlsx
As specified in the QAPP, remaining sample material and specimens must be maintained by the EPA's
designated laboratory or facilities as directed by the NWCA 2016 Project Lead. All samples and raw data
files (including logbooks, bench sheets, and instrument tracings) are to be retained by the laboratory for
3 years or until authorized for disposal, in writing, by the EPA Project Lead. Deliverables from
contractors and cooperators, including raw data, are permanent as per EPA Record Schedule 258. EPA's
project records are scheduled 501 and are also permanent.
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3.0 LABORATORY QUALITY CONTROL
As part of the NWCA 2016, field samples will be collected at each assessment site. These samples will be
sent to laboratories cooperating in the assessment. To ensure quality, each Project Cooperator
laboratory analyzing samples from the NWCA 2016 will receive an evaluation from an NWCA Lab
Evaluator. All Project Cooperator laboratories will follow these guidelines.
No national program of accreditation for lab processing for most NWCA indicators currently exists. For
this reason, a rigorous program of laboratory evaluation has been developed to support the NWCA
2016.
Given the large number of labs participating in the NWCA 2016, it is not feasible to perform an
assistance visit1 (AV) on each of these laboratories. An AV would include an on-site visit to the lab lasting
at least a day. As a result, the EPA Headquarters Project Management Team will conduct remote review
of lab certifications and accreditations of all labs. This process is called laboratory verification. If issues
arise from the remote review that cannot be resolved remotely then an on-site visit to the lab will be
performed. The NWCA 2016 Project Management Team believes this approach meets the needs of this
assessment and can ensure quality control on data generated by the participating labs. General
information is provided here and more specifics are provided in Section 3.1.
Competency. To demonstrate its competency, the laboratory shall provide analyte and matrix specific
information to EPA; or information specific to the relevant biological indicator. EPA will accept one or
more of the following as a demonstration of competency:
• Memorandum that identifies the relevant services that the laboratory provided for the National
Aquatic Resource Surveys in the past five years.
• Documentation detailing the competency of the organization, including professional
certifications for water-related analyses, membership in professional societies, and experience
with analyses that are the same or similar to the requirements of this method.
• Demonstration of competency with sediment samples in achieving the method detection limits,
accuracy, and precision targets.
O
Quality assurance and quality control requirements.
To demonstrate its competency in quality assurance and quality control procedures, the organization
shall provide EPA with copies of the quality-related documents relevant to the procedure. Examples
include Quality Management Plans (QMP), QAPPs, and applicable Standard Operating Procedures
(SOPs). |
u
To demonstrate its ongoing commitment, the person in charge of quality issues for the organization t
shall sign the NWCA QAPP Certification Page. <
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QC
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3.1 Remote Evaluation/Technical Assessment
Procedural review and assistance personnel are trained to the specific implementation and data
collection methods detailed in this NWCA 2016 LOM. Laboratory evaluation reinforces the specific
techniques and procedures for both field and laboratory applications. A remote evaluation procedure
has been developed for performing assessment of all labs.
Laboratory evaluation will be conducted prior to data analysis to ensure that specific laboratories are
qualified and that techniques are implemented consistently across the multiple laboratories generating
data for the program. Laboratory evaluation plans have been developed to ensure uniform
interpretation and guidance in the procedural reviews.
The procedure being utilized involves requesting the laboratory to provide documentation of its policies
and procedures. For the NWCA 2016 project, we have requested that each participating laboratory
provide the following documentation:
• The laboratory's Quality Manual, Quality Management Plan or similar document
• Standard Operating Procedures (SOPs) for each analysis to be performed
• Long term Method Detection Limits (MDLs) for each instrument used and Demonstration of
Capability for each analysis to be performed
• A list of the laboratory's accreditations and certifications, if any
• Results from Proficiency Tests for each analyte to be analyzed under the NWCA project
If a laboratory has clearly documented procedures for sample receiving, storage, preservation,
preparation, analysis, and data reporting; has successfully analyzed Proficiency Test (PT) samples (if
required by EPA, EPA will provide the PT samples); has a Quality Manual that thoroughly addresses
laboratory quality including standard and sample preparation, record keeping and QA non-conformance;
participates in a nationally recognized or state certification program; and has demonstrated ability to
perform the testing for which program/project the audit is intended, then the length of an on-site visit
will be minimum, if not waived entirely. A final decision on the need for an actual on-site visit should be
made after the review and evaluation of the documentation requested.
If a laboratory meets or exceeds all of the major requirements and is deficient in an area that can be
corrected remotely, suggestions will be offered and the laboratory will be given an opportunity to
correct the issue. A correction of the deficiency will then be verified remotely. The on-site visit should
O only be necessary if the laboratory fails to meet the major requirements and is in need of help or fails to
h produce the requested documentation.
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QC
All labs must sign the NWCA 2016 QAPP signature page. In addition, all labs must sign a Lab Signature
Form (Appendix B) indicating that they will abide by the following:
1. Utilize procedures identified in the NWCA 2016 LOM (or equivalent). If using equivalent
procedures, please provide procedures manual to demonstrate ability to meet the required
< minimum quality objectives (MQO).
QC
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2. Read and abide by the NWCA 2016 Quality Assurance Project Plan (QAPP) and related Standard
Operating Procedures (SOP).
3. Have an organized IT system in place for recording sample tracking and analysis data.
4. Provide data using the template referenced in the LOM.
5. Provide data results in a timely manner. This will vary with the type of analysis and the number
of samples to be processed. Sample data must be received no later than May 1, 2017 or as
otherwise negotiated with EPA.
6. Participate in a lab technical assessment or audit if requested by EPA NWCA staff (this may be a
conference call or on-site audit).
If a lab is participating in biology analyses, they must, in addition, abide by the following:
1.
Use taxonomic standards outlined in the NWCA 2016 LOM.
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4.0 ¥EC
4.1 Introduction
Wetland plant species 1) represent diverse adaptations, ecological tolerances, and life history strategies,
and 2) effectively integrate environmental conditions, species interactions, and human-caused
disturbance. Data describing plant species composition and abundance and vegetation structure are
powerful, robust, and relatively easy to gather. They can be used to derive myriad metrics or indicators
that are useful descriptors of ecological integrity or stress (e.g., Lopez and Fennessy 2002, USEPA 2002,
Pino et al. 2005, Bourdaghs et al. 2006, Quetier et al. 2007, Magee et al. 2008, Magee et al. 2010, Mack
and Kentula 2010). NWCA collects data on plant species composition and abundance, on vegetation
structural attributes, and on ground surface attributes within in vegetation plots at each sample site.
The vegetation data are later used during analysis to calculate numerous metrics in a variety of
categories that inform the development of Vegetation Multimetric Indices that serve as indicators of
wetland vegetation condition. Thus, the vegetation data collected in the field by the Vegetation Team
are central to the key descriptors of ecological condition for the NWCA. The field data and metrics can
also be used to characterize wetland vegetation across the NWCA target population or subpopulations.
For NWCA, crews will collect unknown plant specimens ("unknown species vouchers") and known plant
species for quality assurance purposes ("QA vouchers") from each site and send to a designated
laboratory/herbarium for identification.
4.2 Receiving Voucher Samples
Plant samples will arrive at the laboratory/herbarium pressed in shipping boxes. Each plant sample
should arrive with a Plant Specimen Label (see Section 4.4.1).
4.2.1 Definitions
For the NWCA, a voucher sample is a pressed and dried plant sample, ideally comprised of leaves,
stems, flowers, fruits and roots. An integral component of each voucher sample is written data
describing the location, date of collection, habitat, plant habit, characteristic features, and other
information. Vouchers provide physical evidence that confirms the presence of plant species at specific
locations.
For all NWCA field work, whenever the identity of a species cannot be confirmed in the field, a sample is
collected (see Vegetation Chapter of Field Operations Manual (FOM)) for later identification in the office
or lab. All unknown species located in one of five Vegetation Plots arrayed across a site's Assessment
Area that are mature and have key structures needed for identification are collected (unknown species
voucher). Unknown species that are immature or senescent comprising more than 5% cover are also
collected. If an unknown species specimen is collected at a previous site, it is collected at subsequent
sites, until the field Botanist/Ecologist learns the identity of the species and can reliably sight-recognize
it in the field. This is particularly important for species in difficult-to-identify wetland genera and
families, such as those that include sedges, rushes, grasses, and submerged aquatic vegetation. The
Botanist/Ecologist will ship unknown samples to the identifying botanist at the laboratory/herbarium
for initial identification (Vegetation Chapter of FOM).
For the purposes of this manual, the identifying botanist represents the person identifying and
processing unknown samples. This could be a field botanist/ecologist; university, state, national or
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regional herbarium botanist; or an EPA contractor that has qualifying credentials in plant taxonomy. The
identifying botanist is responsible for ensuring all plant identification and processing tasks outlined in
this manual are completed. In some cases this may require lab partners to assist with the work.
In addition to all unknown specimens, field crews will be submitting five known plant voucher samples
(randomly selected from species identified by the Vegetation Team) for quality assurance (NWCA 2016
QAPP). These QA voucher specimens will be sent to a QA verifying botanist for re-
identification/verification (Vegetation Chapter of FOM). Collecting voucher samples of known species
both provides a quality assurance check on species identity data, and a permanent record of the
occurrence of a particular species at a given location.
The QA verifying botanist is responsible for re-identification/verification of the QA vouchers as well as a
random selection of 10% of the unknown specimens that were initially determined by the identifying
botanist in the lab.
If the unknown species specimens and QA voucher samples are planned to be sent to the same
institution, it is important that all quality assurance activities be completed by a taxonomist that did not
participate in the identification of unknown specimens. .
4.2.2 Tracking information
In the field, each voucher sample collected is assigned a set of tracking information, which is recorded
on the Plant Sample Tracking Forms (Figures 4-1 and 4-2). At the end of the sampling week, the
Vegetation Team will remove the samples and newspaper sleeves from the press, ensuring they retain
the Plant Specimen Label (Figure 4-3), and ship them in a sturdy box to either the identifying botanist for
unknown samples or the QA verifying botanist for the five known specimens (Vegetation Chapter of
FOM). If a sample listed on the tracking form is not part of the shipment, or a sample arrives at the lab
without the proper label, contact the EPA Project Management Team immediately.
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Y FORM T-4: NWCA 2016 UNKNOWN PLANT SAMPLE TRACKING
State of Site Location:
Crew:
Date Sent:
/ / 2 0 1 6
Sender:
Sender Phone: -
-
Shipped by: O FedEx O UPS O Hand Delivery Airbill/Tracking Number:
OR Retained for Identification By:
Phone:
-
Date:
/ / 2 0 16
Site ID: NWCA16-
Visit: O 1 O 2
Date Collected:
/ / 2 0 1 6
* ' ' ' ' ' ' '
ln st ructions:
1. Complete all shipping and site information above.
2. Fill in bubbles of all plant samples being shipped. Use the All bubble if all 10 species in thatcolumn ar.
5 unknown samples but later identified U1 and U2 and corrected Form V-2 for these, you would ml/ b.
only fill in bubbles for U3, U4, and U5.
D-iri^ shipped. If you collected
dipping U3-U5. In this case,
Shipped:
Shipped: Shipped: Rh.'pptd:
Shipped:
O All (U1-U10)
O All (U11-U20) O All (U21-U30) O All (J31-U40)
O All (U41-U50)
OU1
OU11 O U21
•*> J31
O U41
OU2
O U12 O U22
OU32
OU42
OU3
O U13 O U23
OU33
OU43
O U4
O U14 O U24
OU34
O U44
OU5
O U15 O U2f
OU35
OU45
OU6
O U16 O U2S
OU36
O U46
OU7
OU17 S pljE7
OU37
OU47
OU8
O U18 O U28
OU38
OU48
OU9
O U19 O U29
OU39
OU49
OU10
O U20 O U30
OU40
OU50
Comments:
Samples Shipped to
Save Completed Form as:
Tracking Related Inquiries:
O EnviroScience:
O
SitelD V# T4
Martys Cappaert
Phone: 541-754-4467
Michelle Gover
Phone: 541-754-4793
Michael Liptak
EnwiroScience
Email to:
5070 Stow Road
Stow, OH 44224
sampletracklrg@epa.gov
Or fax to: 541-754-4637
Chris T urner
Phone: 715-829-3737
^ 9441238685
03/07/2016 T-4 NWCA 2016 Tracking - UNK Plant Sample
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Figure 4-1. Unknown Plant Sample Tracking Form tu
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r
State of Site Location:
Sender:
FORM T-5: NWCA 2016 QA PLANT SAMPLE TRACKING ^
Crew: Date Sent: I J 2 0 16
Sender Phone: -
I I I I I 1 i I
Shipped by: O FedEx O UPS O Hand Delivery Airbill/Tracking Number:
OR Retained for identification By:
Phone:
I I 2 0 16
Site ID: NWCA16-
VIsit: O 1 O 2 Date Collected:
I I 2 0 16
Instructions:
1. Complete all shipping and site information above.
2. Fill in bubbles of all plant sample collection numbers. If all 5 samples were collected, use the All bubble. r, z'I were not collected, fill
individually. For example, if you collected 2 samples, fill bubbles Ql, and Q2.
v
Collected #:
O All (Q1-Q5) OQ1 OQ2 O T3 OQ4 OQ5
Samples Shipped to
Save Completed Form as:
Tracking Related Inquiries:
O EnviroScience:
Michael Liptak
EnviroScience
5070 Stow Road
Stow, OH 44224
o
STATE LAB NAME
STATE LAB ADDRESS
STATE
SitelD V#T5
Email to:
sampletracking@epa.gov
Or fax to: 641-754-4637
Marlys Cappaert
Phone: 541-754-4467
Michelle Gover
Phone: 541-754-4793
Chris Turner
Phone: 715-829-3737
^ 8622408549
03/07/2016 T-5 NWCA 2016 Tracking - QA Plant Sample
LU
>
Figure 4-2. QA Plant Sample Tracking Form
12
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4.3 Supplies and Equipment for Sample Handling
¦ Plant dryer
¦ Dissecting microscope
¦ Dissecting tools (e.g., single edge razor blades, forceps, dissecting needles)
¦ Regional floras and plant lists
¦ USDA PLANTS taxonomic standard http://plants.usda.gov/java/
¦ Plant nomenclatural forms
¦ Plant sample tracking forms
¦ Plant sample folders
¦ Storage cabinet or sealable plastic boxes for storing dried plant samples prior to identification
¦ OPTIONAL: Freezer or laboratory approved treatment supplies for killing pests on dried plant
material
¦ OPTIONAL: Mounting materials (herbarium sheets, mounting glue, forceps, weights for holding
samples with wet glue to the herbarium sheets, etc.)
¦ OPTIONAL: Herbarium sample labels
4.4 Handling Vegetation Samples
Plant samples may arrive at the laboratory/herbarium in several conditions: 1) as dried, pressed
samples, or 2) pressed but still wet plant material enclosed in a plant press.
1. If samples are pressed and dried, proceed to Section 4.4.3 (Treat samples for detritivores,
molds, and pests).
2. If samples arrive in a press, but are still wet they should be placed on a plant dryer to complete
drying, and then be treated for pests.
4.4.1 Plant Sample Label Form
Every sample will arrive with a Plant Specimen Label. This label includes the original identification and
diagnostic information for known and unknown species collected including location, date of collection,
habitat, plant habit, and abundance information. Voucher samples are considered incomplete without
this information. An example of the Plant Specimen Label is provided in Figure 4-3. If a sample does not
have any of the following information, contact the EPA Project Management Team immediately:
Plant Specimen Label Information
¦ Specimen Type: Samples collected for QA purposes will have QA Voucher filled in, while
unknown samples will have Unknown Species filled in.
¦ Plant Sample ID Number: NWCA Site Number-Plant collection number. Plant collection
numbers for samples are assigned consecutive numbers depending on the specimen type
(unknown specimens are prefaced with the letter U and OA specimens are prefaced with the
letter Q) for each site beginning with one. For example, the sample number for the 14th
unknown specimen collected at NWCA16-9999 would be NWCA16-9999-U14.
¦ Visit Number: Indicates whether it was the first visit (1) or a repeat visit (2). Most sites are only
visited once.
Collection Date: Date is numerical: month, day, year, e.g. 06/14/2016.
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¦ County and State: Information on county and State where specimen was collected.
¦ Species Name or Pseudonym: Species name from data form if known or descriptive name used
on data forms (e.g., Carex sp. 1) if unknown.
¦ Collector(s) name: Lists the first name, middle initial and surname of the person or persons who
collected the sample.
¦ Abundance of Plant: Indicates whether the species is dominant, common, sparse or uncommon
at the site.
¦ Habitat: The type of plant community or setting where the plant is growing, (e.g., such as
wetland type (Cowardin, HGM, NVC), wetland community type (forested wetland, emergent
marsh, wet prairie, mountain bog, etc.), anthropogenic disturbances (urban setting type), and,
other plants growing in association (associated species information would be available from the
plot).
¦ Growth habit: Describes key features of the plant such as growth form (tree, shrub, vine, herb),
approximate height, longevity (annual, biennial, perennial), clonal, rhizomatous, tussock-
forming, etc. Lists any characteristics of the plant which may be lost upon drying, such as
flower/fruit color, fragrance, and leaf orientation.
PLANT SPECIMEN LABEL
O - OA Voucher O * Unknown Spedes
Plant Sample ID Number {SitelD+V8+Collectfang):
NWCA16- Visit#: Ql Q2
(Sits#) (Collection#)
Date: / /2Q16
County: State:
Species Name or Pseudonym:
Collectors) Mame(s):
Abundance of Plant fflll appropriate circle):
O Dominant O Common O Sparse O Uncommon
Habitat:
Growth Habst:
Figure 4-3. Plant specimen label
4.4.2 Drying Samples
-z.
O Plant samples may arrive wet and in the plant press. The pressed plants must be thoroughly dried
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Low ambient humidity and good airflow around and through the presses is important for rapid and
thorough drying of plant material. Rapid drying over low heat promotes preservation of color and
morphology resulting in high quality samples. Dry air circulating through the press also may kill many
insects and insect eggs, which may protect the samples from some insect damage. These conditions are
most easily obtained by placing full presses on an electric plant dryer that provides steady bottom heat
(95°F to 113°F), where plants usually dry in 12 to 48 hours. However, presses placed in a warm dry place
will be sufficient if a plant dryer is not available.
4.4.3 Treat samples for detritivores, molds, and pests
Dried plant material is highly susceptible to contamination by detritivores, molds, and pests that can
destroy herbaria collections. Therefore it is important to treat all incoming samples to kill potential
contaminates.
Standard pest procedures of the herbaria should be implemented. A common method for sample
treatment is to freeze them (-20°C or below) for at least three days for loosely stacked samples and
seven days for tightly packed samples.
To protect the collection from infestation, plant samples should be stored in herbarium cabinets or
sealable plastic containers when not in use. Under no circumstances should samples be left out
overnight. If samples are found that have been left out overnight or if a cabinet/plastic container has
been left open, all samples may need to be decontaminated again.
4.5 Identification of Vegetation Samples
4.5.1 Taxonomic standard
The recognition and identification of particular classes of plants such as families, genera, and species is a
critical and difficult element of collecting accurate vegetation plot data. To complicate matters, not all
botanical authorities agree about which name to apply to a particular plant species. The NWCA uses the
taxonomic nomenclature of the USDA Plants Database as its taxonomic standard. To effectively key
plants and identify them in the field, however, field crews may use local floras appropriate to each
region or state (Appendix D). This means numerous taxonomies will likely be applied across the 48
conterminous states comprising the study area. The identifying botanist will reconcile all species names
to the standard found in USDA-NRCS PLANTS at http://plants.usda.gov/.
4.5.2 Recording Identifications
All identifications are recorded in an Excel database (2016 NWCA Plant ID Lab Spreadsheets). The Excel
database includes user information tabs that provide quick reference lists and instructions for recording
data. For example, a list of growth habit codes as well as floras of field guides are included for quick
reference while other tabs provide examples and specific instructions on how to fill out the various data
fields of the Excel spreadsheets for the QA voucher and Unknown specimen spreadsheets. Once the
spreadsheets in the database have been completed, copies are then sent to the project facilitator (QAPP
Section 5.1.6).
4.6 Mounting and Storing Herbarium Sheets
Once the samples are dried, pressed, and identified, they are to be stored at the herbarium for at least
five years. Vouchers should be kept in sealable plastic containers in a cool dry climate and must be
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accessible to the EPA. However, the herbarium is encouraged to incorporate the NWCA vouchers into
their permanent collections as desired. Vouchers from the national survey mounted on herbarium
sheets should be labeled to indicate that they were collected as part of the NWCA. For an example of
commonly used mounting and labeling methods see Appendix E.
4.7 Quality Assurance
A subset of plant samples collected as unknown specimens and later identified by a State or National
Plant Laboratory botanist ("identifying botanist") will be verified by a QAtaxonomist ("verifying
botanist") for additional quality assurance. The lab will randomly select 10% of the identified unknown
samples for re-identification by another experienced taxonomist who did not participate in the original
identifications. The NWCA QA Team will evaluate differences in the taxonomic identification of plant
specimens between the identifying and verifying botanists. Substantial disagreements between the two
will be investigated and logged for indication of error patterns or trends, but all values will generally be
considered acceptable for further analysis, unless the investigation reveals significant problems.
Quality control procedures associated with sample handling and processing at laboratories handling
NWCA QA and unknown plant vouchers are summarized in Table 4-1.
Table 4-1. Laboratory quality control activities for vegetation indicator.
O
16
Quality Control
Activity
Frequency
Acceptance Criteria
Corrective Action
Demonstrate
competency for
identifying samples
to meet the
performance
measu res
Once
Demonstration of past
experience relevant to
identifying plants collected
from wetlands
EPA will not approve any
laboratory for NWCA voucher
identifications if the laboratory
cannot demonstrate
competency. In other words,
EPA will select another
laboratory that can
demonstrate competency.
Verify that plant
voucher has arrived
in acceptable
condition
All vouchers
The condition must allow for
positive identification
Lab will consult immediately
with EPATOCOR if voucher does
not arrive in acceptable
condition.
Sample Log-in
All vouchers
Plant vouchers logged into
NARS IM system within 24
clock hours of receipt.
Discrepancies, damaged or
missing samples are reported to
EPA Project Manager and
Laboratory Review Coordinator.
Store sample
appropriately
All vouchers
Vouchers must be treated to
kill potential contaminants
and properly stored dry in a
condition that prevents
contamination by detritivores,
molds, and pests (typically in
herbarium cabinets or
sealable plastic containers).
EPA expects that the laboratory
will exercise every effort to
maintain vouchers in proper
storage conditions.
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Quality Control
Activity Frequency Acceptance Criteria Corrective Action
Use widely/
commonly accepted
taxonomic
references and
reconcile to USDA-
NRCS PLANTS
taxonomic
nomenclature
All identifications
Full citations for floras and
field guides used in plant
identification must be
provided and;
identifications must be
reconciled to the taxonomic
nomenclature of the USDA-
NRCS PLANTS database
Lab will provide explanation and
discuss deviances with EPA
TOCOR.
Identification by
laboratory
When field plant ID
specialist cannot
identify specimen
Identification by lab plant ID
specialist (who must be a
different individual than the
field plant ID specialist)
Replace field crew's "unknown"
identification with
determination by lab
Unknowns QC
Approximately 10% of
all unknown vouchers
independently
identified in the lab
PTD < 15%
If PTD > 15%, review data for
possible explanations;
otherwise, insert data qualifier
for laboratory identifications
Conduct assistance
visit
EPA may choose to
visit any laboratory
Visit conducted using checklist
Performance and any
recommended improvements
described in debrief with
laboratory staff
4.7.1 Percent taxonomic disagreement (PTD)
PTD is a measure of taxonomic precision comparing the number of agreements (positive comparisons,
comppos) of the first plant ID specialist ("identifying botanist") and the second plant ID specialist
("verifying botanist") for unknown vouchers. In the following equation, N is the total number of
specimens in the larger of the two counts. PTD should be <15%.
PTD =
1-
compi
N~
: 100
The NWCA QA Team will monitor differences in the taxonomic identification of plant specimens
between the identifying botanists providing the initial identification and the verifying botanists providing
the independent re-identifications. Substantial disagreements between the two will be investigated and
reasons for the discrepancies examined and corrected.
4.8 References
Kansas State University. 2006. Integrated Pest Management Plan, http://www.k-
state.edu/herbarium/pests.html. Accessed November 17, 2009.
Lee, M. T., R. K. Peet, S. D. Roberts, and T. R. Wentworth. 2008. CVS-EEP protocol for recording
vegetation: All levels of plot sampling. Version 2008. The Carolina Vegetation Survey (CVS,
http://cvs.bio.unc.edu) and the North Carolina Ecosystem Enhancement Program (EEP,
http://www.nceep.net).
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Mack, J. J. 2007. Integrated Wetland Assessment Program. Part 9: Field Manual for the Vegetation
Index of Biotic Integrity for Wetlands v. 1.4. Ohio EPA Technical Report WET/2004-9. Ohio
Environmental Protection Agency, Wetland Ecology Group, Division of Surface Water, Columbus, Ohio.
Magee, T. K., S. E. Gwin, R. G. Gibson, C. C. Holland, J. E. Honea, P. W. Shaffer, J. C. Sifneos, and M. E.
Kentula. 1993. Research Plan and Methods manual for the Oregon Wetlands Study. EPA/600/R-
93/072. Environmental Protection Agency, Environmental Research Laboratory, Corvallis, Oregon.
Stribling, J.B., S.R. Moulton, and G.T. Lester. 2003. Determining the quality of taxonomic data. Journal
of the North American Benthological Society 22:621-631.
University of Florida Herbarium. 2009. Preparation of Plant Samples for Deposit as Herbarium Vouchers.
http://www.flmnh.ufl.edu/herbarium/voucher.htm, Accessed June 12, 2009.
USEPA. 2002. Methods for Evaluating Wetland Condition: #10 Using Vegetation to Assess
Environmental Conditions in Wetlands. EPA-822-R-02-020, Office of Water, U.S. Environmental
Protection Agency, Washington, DC.
USEPA. 2006. Survey of the Nation's Lakes. Laboratory Methods Manual. EPA841-B-06-005. U.S.
Environmental Protection Agency, Washington, DC.
USEPA. 2006. Survey of the Nation's Lakes. Quality Assurance Project Plan. EPA841-B-07-003. U.S.
Environmental Protection Agency, Washington, DC.
USEPA. 2011. National Wetland Condition Assessment. Laboratory Operations Manual. EPA843-R10-
002. U.S. Environmental Protection Agency, Washington, DC.
USEPA. 2011. National Wetland Condition Assessment. Quality Assurance Project Plan. EPA843-R10-003.
U.S. Environmental Protection Agency, Washington, DC.
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5.0 SOILS
5.1 Introduction
Soils play an important role in wetland ecosystems, cycling nutrients, regulating water movement and
storage, and serving as a growth medium or habitat for plants, microbes, and macroinvertebrates.
Wetland soils develop distinct characteristics as a result of the hydrology and biota (e.g., microbes,
vegetation) associated with wetlands, as well as other factors that influence soil development across all
environments (e.g., climate, geology). These characteristics impact the functions and processes
occurring in the soil and reflect ecological condition.
This chapter describes the methods for analyzing chemical and physical properties of soil samples
collected in the NWCA.
5.2 Summary of Method
Three types of soil samples will be collected from each site:
• Standardized Depth Soil Core - collected from the Soil Plot, represents a layer from the soil
surface to the 10 cm depth. Analysis will include chemical parameters and particle size
distribution (soil texture).
• Horizon Bulk Density Sample - collected from the Soil Pit, three samples are collected from all
horizons greater than or equal to 8 cm thick to a depth of 1.0 m. Samples will be analyzed for
bulk density.
• Horizon Chemistry Sample - collected from the Soil Pit, a sample is collected from every horizon
to a depth of 1.0 m. Analysis will include chemical parameters and particle size distribution (soil
texture).
The Standardized Depth Soil Core, Horizon Bulk Density Samples, and Horizon Chemistry Samples will be
analyzed by the Kellogg Soil Survey Lab (KSSL), National Soil Survey Center (NSSL). A total of 13 analytical
methods will be performed to characterize the soil chemical and physical properties (Table 5-1). Soil
bulk density measurements will be made on Horizon Bulk Density Samples, all other parameters will be
measured on the Standardized Soil Depth Core and the Horizon Chemistry Samples.
Table 5-1. Summary of NWCA 2016 soil analytical methods.
Analysis Method
Analyte(s) Measured
Particle Size Distribution Analysis (PSDA), < 2mm, air dry
Clay, Silt, Sand
Calcium Carbonate Equivalent, < 2mm
CaC03
Calcium Carbonate Equivalent, < 20 mm
CaC03
Total Carbon, Nitrogen, and Sulfur
C, N, S
PH
1:1 HzO, 1:2 0.01 M CaCh
Cation Exchange Capacity and Base Cations
CEC, Ca2+, K+, Mg2+, Na+
Ammonium Oxalate Extraction
Al, Fe, Mn, P, Si
Electrical Conductivity
EC
Dithionite-Citrate Extraction
Al, Fe, Mn
Olsen Phosphorus
P
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Analysis Method
Analyte(s) Measured
Mehlich Phosphorus
P
Trace Elements
Ag, As, Ba, Be, Cd, Co, Cr, Cu, Hg, Mn, Mo, Ni, P,
Pb, Sb, Se, Sn, Sr, V, W, Zn
Bulk Density
Dbf
5.3 Health and Safety Warnings
The laboratory must require its staff to abide by appropriate health and safety precautions. Specific
safety warnings and guidelines for each of the analytical methods can be found in the Kellogg Soil Survey
Laboratory Methods Manual (Soil Survey Staff, 2014).
5.4 Sample Handling and Processing
5.4.1 Receiving Regulated Soils
Soils that may contain pests (i.e., bacteria, plant viruses, fungi, nematodes, and life stages of destructive
mollusks, acari, and insects) are regulated by U.S Department of Agriculture's Animal and Plant Health
Inspection Service (APHIS). Areas within states that are under Federal quarantine must follow the
conditions and safeguards prescribed by APHIS before shipping to another part of the country. To
ensure that the NWCA is in compliance with APHIS recommendations, all soils collected for the survey
will be shipped as regulated soils. Participating labs are responsible for obtaining and maintaining a valid
permit for receiving regulated soils (see example, USDA APHIS PPQ 525-A, Figure 5-1 below).
Upon arrival at the lab, soil samples will be separated into regulated and non-regulated based on their
county and state of origin (as recorded on the water proof label affixed to the outside of the sample
bag). The lab is responsible for following all APHIS protocols when handling or disposing regulated soils
as found in 7 CFR 330.300.
[Placeholder for copy of APHIS permit (Figure 5-1) when received from NRCS]
5.4.2 Laboratory Sample Preparation
Each sample type collected is preassigned a six-digit site specific sample ID number. Samples are also
labeled with Site ID, visit number, and horizon number. Laboratory identification numbers and
preparation codes are assigned to each soil sample by the NRCS laboratory. These unique identification
numbers carry important information about the soil sample (e.g., site, year sampled, soil horizon,
replicate). Laboratory preparation codes depend on the properties of the sample and the requested
analyses. Identification numbers and preparation codes are reported on the KSSL Primary
Characterization Data Sheets. Refer to the Soil Survey Investigations Report No. 45, Soil Survey
Laboratory Information Manual (Soil Survey Staff, 2011), for a detailed explanation of sample
identification numbers. Detailed information on the current preparation codes as they appear on the
Primary Characterization Data Sheets may be obtained from the KSSL upon request.
For most standard chemical, physical, and mineralogical analysis, the field sample is air-dried, crushed,
and sieved to <2 mm. The protocol for preparing soil samples and descriptions of preparation methods
for specific analyses are given in Kellogg Soil Survey Laboratory Methods Manual, Soil Survey
Investigations Report No. 42, Version 5.0 (Soil Survey Staff, 2014).
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5.5 Summary of Analytical Methods
Analytical methods used at the Kellogg Soil Survey Laboratory are summarized in Table 5-2. Method
procedures are described in detail in the Kellogg Soil Survey Laboratory Methods Manual, Soil Survey
Investigations Report No. 42, Version 5.0 (Soil Survey Staff, 2014). These are the standard operating
procedures of the Lab, and are standard methods, peer-recognized methods, KSSL-developed methods,
and/or methods specified in Keys to Soil Taxonomy (2014).
Table 5-2. NWCA 2016 soil analytical methods. Analyses follow the standard operating procedures of the Kellogg
Soil Survey Lab (KSSL), National Soil Survey Center, Natural Resources Conservation Service.
Analyte
Method
Summary of Method
KSSL Method
Clay
Silt
Sand
PSDA, <2 mm, air
dry
Organic matter removed; sand fraction removed by wet
sieving; clay and fine silt fractions determined by
pipetting following sedimentation; coarse silt is the
difference between 100% and the sum of sand, clay, and
fine silt.
3Alala
CaC03
Calcium Carbonate
Equivalent, <2mm
Samples are treated with HCI; evolved CO2 is measured
manometrically; carbonate in the soil is calculated as
4Elalalal
Calcium Carbonate
Equivalent, >20
mm
percent CaC03.
4Elalala2
C
N
S
Total Carbon,
Nitrogen, and
Sulfur
Total Carbon, Nitrogen, and Sulfur are measured by dry
combustion; released measuring components (N2, CO2,
and SO2) are measured using an elemental analyzer.
4H2al-3
PH
1:1 H20
The pH is measured in soil-water (1:1) and soil-salt (1:2
4Cla2ala-bl
1:2 0.01 M CaCh
CaCh) solutions using a combination pH-reference
electrode.
4Cla2a2a-bl
CEC
Ca2+
K+
Mg2+
Na+
Cation Exchange
Capacity by
NH4OAc, pH 7
The CEC and base cations are determined by a
displacement procedure. Sample is leached using 1 N
NH4OAC. Exchange sites are saturated by an index cation
(NH4+) adsorbed by the soil, and soil is washed free of
excess saturated salt. The index cation is displaced by
rinsing with KCI and the leachate is analyzed by steam
distillation and titration to determine the NH4+ adsorbed
on the soil exchange complex. The NH4OAC extract is
diluted with an ionization suppressant (La20s) and
analytes (Ca2+, K+, Mg2+, and Na+) are measured by an
atomic absorption spectrophotometer (AAS).
4Blalalal
4Blalbl-4
Al
Fe
Mn
P
Si
Ammonium
Oxalate Extraction
Soil sample is extracted with a mechanical vacuum
extractor in a 0.2 M ammonium oxalate solution
buffered at pH 3.0 under darkness. The ammonium
oxalate extract is weighed, diluted, and analytes are
measured by an inductively coupled plasma atomic
emission spectrophotometer (ICP-AES).
4G2alal-5
EC
Electrical
Conductivity
Soil sample is mixed with water and allowed to stand
overnight; electrical conductivity (EC) of the mixture is
measured using an electronic bridge. The EC by this
method is used to indicate the presence of soluble salts.
4Flalalal
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Analyte
Method
Summary of Method
KSSL Method
Al
Fe
Mn
Dithionite-Citrate
Extraction
Soil sample is mixed with sodium dithionite, sodium
citrate, RODI water, and shaken overnight; solution is
centrifuged and extract is diluted; analytes are measured
by an atomic absorption spectrophotometer (AAS).
4Glal-3a-bl
P (Olsen)
Olsen Extraction
This extractant is most applicable to neutral to
calcareous soils (Buurman et al., 1996).
Soil sample is shaken with Olsen sodium-bicarbonate
extracting solution at pH 8.5, centrifuged, and filtered;
clear extract is diluted with a color reagent; absorbance
of the solution is read using a spectrophotometer at 882
nm.
4D5ala-bl
P (Mehlich
No. 3)
Mehlich No. 3
Extraction
Mehlich No. 3 is used as an index of available P in the
soil. Extraction of P by Mehlich No. 3 is designed to be
applicable across a wide range of soil properties with
reaction ranging from acid to basic (Mehlich, 1984), and
correlates with Olsen extractant on calcareous soils
(R2=0.918), even though the quantity of Mehlich No. 3
extractable P is considerably higher (Soil and Plant
Analysis Council, 1999).
Soil sample is shaken with Mehlich No. 3 extracting
solution, centrifuged, and filtered; clear extract is diluted
with a working solution; absorbance of the solution is
read using a spectrophotometer at 882 nm.
4D6ala-bl
Ag
As
Ba
Be
Cd
Co
Trace Elements
Microwave digestion methodology utilizing HNO3 and
HCI. Analyte concentrations are determined using an
inductively coupled plasma mass spectrometer (ICP-MS).
This method follows EPA Method 3051A.
4H la la la 1-20
Cr
Cu
Hg
Mn
Mo
Ni
P
Pb
Sb
Se
Sn
Sr
V
W
Zn
Bulk
Density
Bulk Density Core
Method
Bulk density was determined for field-moist soil cores of
known volume. The field-state bulk density (Dbf) value is
the bulk density of a soil sample including the water
content of the soil in the field at the time of sampling.
3B6a
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Analyte
Method
Summary of Method
KSSL Method
A metal cylinder is pressed or driven into the soil; the
cylinder is removed, extracting a sample of known
volume. The moist sample weight is recorded, sample is
dried in an oven and weighed. Dbf is the oven dry weight
of the soil divided by the core volume and corrected for
rock fragments (if present).
5.6 Quality Assurance / Quality Control (QA/QC) Procedures
Standardized lab protocols, consistent training of all lab technicians, lab assistance visits to all labs, and
availability of experienced technical personnel to respond to site-specific questions as they arise are
important to ensuring the quality of lab data. Additionally, control measures to minimize measurement
error among lab technicians and laboratories include the use of laboratory quality control samples and a
data review and validation process (QAPP Section 5.2.5).
5.6.1 Laboratory Performance Requirements
Table 4.3 summarizes the pertinent laboratory performance requirements for the soil indicators.
Table 5-3. Soil laboratory method performance requirements.
Analyte
Method
Units
MDL
PQL
Potential
Accuracy
Sample Range1
Objective
Clay
PSDA, <2 mm, air dry
%
na
na
Oto 93.1
Silt
0.1 to 100
Sand
0 to 94.5
CaC03
Calcium Carbonate
%
0.5
2.5
nd to 105
Equivalent, <2mm
Calcium Carbonate
%
0.5
2.5
nd to 96
Equivalent, <20mm
C
Total Carbon,
%
0.04
0.2
nd to 62.43
0.01%
N
Nitrogen, and Sulfur
nd to 11.193
0.001%
S
nd to 21.86
0.01%
PH
1:1 H20
PH
na
na
2.4 to 10.5
0.1 pH unit
1:2 0.01 M CaCh
PH
na
na
2.4 to 10.5
0.1 pH unit
CEC
Cation Exchange
cmol(+) kg1
0.1
0.6
nd to 252
0.1 cmol(+) kg1
Ca2+
Capacity by NH4OAC,
0.07
0.4
nd to 507.3
0.1 cmol(+) kg1
K+
pH 7
0.06
0.3
nd to 17.4
0.1 cmol(+) kg1
Mg2+
0.01
0.07
nd to 147.1
0.1 cmol(+) kg1
Na+
0.2
1.0
nd to 650.3
0.1 cmol(+) kg1
Al
Ammonium Oxalate
%
0.002
0.009
nd to 15.62
0.01%
Fe
Extraction
%
0.0001
0.0006
nd to 20.15
0.01%
Mn
mg kg1
0.1
0.6
nd to 15730.7
1.0 mg kg1
1 nd = non-detect, tr = trace
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Analyte
Method
Units
MDL
PQL
Potential
Accuracy
Sample Range1
Objective
P
mg kg1
26
129
nd to 16926.4
1.0 mg kg1
Si
%
0.0002
0.001
nd to 6.13
0.01%
EC
Electrical Conductivity
mmhos cm 1
0.001
0.005
nd to 167.4
0.01 mmhos cm 1
Al
Dithionite-Citrate
%
0.001
0.006
nd to 8.6
0.1%
Fe
Extraction
0.01
0.07
nd to 36.6
0.1%
Mn
0.0006
0.003
nd to 3.6
0.1%
P (Olsen)
Olsen Extraction
mg kg1
0.1
0.7
nd to 399.7
0.1 mg kg1
P (Mehlich
Mehlich No. 3
mg kg1
0.1
0.6
nd to 1232.3
0.1 mg kg1
No. 3)
Extraction
Ag
Trace Elements
mg kg1
0.001
0.01
nd to 175.62
As
mg kg1
0.002
0.01
nd to 1808.06
Ba
mg kg1
"0"
"0"
0.02 to 4415.8
Be
mg kg1
0.001
0.01
nd to 29.98
Cd
mg kg1
0.001
0.01
nd to 85.68
Co
mg kg1
"0"
"0"
nd to 1125.58
Cr
mg kg1
0.006
0.03
nd to 2020.31
Cu
mg kg1
0.002
0.01
nd to 1036.28
Hg
Mg kg1
1.3
6.50
nd to 26060
Mn
mg kg1
0.002
0.01
nd to 692942
Mo
mg kg1
0.001
0.01
nd to 235.17
Ni
mg kg1
0.009
0.05
tr to 3347.36
P
mg kg1
0.4
2.07
nd to 70708.6
Pb
mg kg1
0.001
0.01
nd to 12287.4
Sb
mg kg1
0.002
0.01
nd to 42.01
Se
Mg kg1
1.8
9.00
nd to 16523.1
Sn
mg kg1
0.005
0.03
nd to 1117.66
Sr
mg kg1
0.001
0.01
nd to 10895
V
mg kg1
"0"
"0"
nd to 1064.65
W
mg kg1
"0"
"0"
nd to 137.39
Zn
mg kg1
0.006
0.028
0.06 to 10379.1
Bulk
Bulk Density Core
g cm 3
na
na
0.15 to 2.6
0.01 g cm 3
Density
Method
5.6.2 Laboratory Quality Control Samples
Laboratory quality control samples for the soil indicators include control samples and blank samples.
A control sample represents a sample of known concentration for a particular attribute. A control
sample is collected in bulk for an attribute and repetitively analyzed to determine statistical control
limits (i.e., range of expected values) for the particular method. A control sample is analyzed in
conjunction with every batch of samples to ensure the method was run correctly. If the value of the
control sample falls outside the expected range of values then the process has failed and the batch is
flagged for reanalysis.
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A blank sample is used to ensure equipment is thoroughly cleaned before each use. A blank sample is
especially important when measuring soil chemistry (i.e., trace metals) because concentrations may be
quite small. A blank sample is analyzed in conjunction with every batch of samples to ensure that proper
equipment cleaning protocols are followed. If the value of the blank sample does not equal zero or falls
below the method detection limit, then the equipment is not clean and the batch is flagged for
reanalysis.
5.6.3 Data Reporting, Review, and Management
The data validation process involves four data reviews, first by the Bench Analysts, second by the Lead
Analyst, third by the Project Coordinator Soil Scientist, and fourth by a Soil Scientist Liaison with
expertise in soils from the region where the samples are from. The Bench Analysts verifies that blank
and control samples return results that fall within established control limits. The Lead Analyst examines
the data for inconsistencies and apparent anomalies; inconsistencies usually take the form of
unexpected high or low values for a particular analyte or values that do not fit with the expected trend
of a soil profile. The Project Coordinator will use professional judgment to determine whether the
project data are self-consistent and congruent with the site data collected in the field; incongruities
within the data that can be explained either by site data or the results of other analytes are recorded.
Data reviews include range checks, summary statistics, and/or exploratory data analysis. Identified
reporting errors are corrected or data is qualified as suspect or invalid as appropriate. A final review is
given by a Soil Scientist Liaison to the area of sample origin, before the data are released. Data reporting
units and significant figures are given in Table 5-4. Indicator QC coordinator determines impact and
possible limitations on overall usability of data based on the specific issue. The NWCA 2016 Project QA
Officer is ultimately responsible for ensuring the validity of the data, although performance of the
specific checks may be delegated to other staff members.
Table 5-4. Soil data reporting criteria.
Analyte
Method
Units
Number of
Significant Figures
Maximum Number of
Decimal Places
Clay
PSDA, <2 mm, air dry
%
3
1
Silt
3
1
Sand
3
1
CaC03
Calcium Carbonate
Equivalent, <2mm
%
3
0
Calcium Carbonate
%
3
0
Equivalent, <20mm
C
Total Carbon, Nitrogen,
%
4
2
N
and Sulfur
4
2
S
4
2
PH
1:1 H20
PH
2
1
1:2 0.01 M CaCh
PH
2
1
CEC
Cation Exchange Capacity
cmol(+) kg 1
3
1
Ca2+
by NH4OAC, pH 7
4
1
K+
3
1
Mg2+
4
1
Na+
4
1
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Analyte
Method
Units
Number of
Significant Figures
Maximum Number of
Decimal Places
Al
Ammonium Oxalate
%
4
2
Fe
Extraction
%
4
2
Mn
mg kg1
6
1
P
mg kg1
6
1
Si
%
3
2
EC
Electrical Conductivity
mmhos cm 1
4
2
Al
Dithionite-Citrate
%
2
1
Fe
Extraction
3
1
Mn
2
1
P (Olsen)
Olsen Extraction
mg kg1
4
1
P (Mehlich No. 3)
Mehlich No. 3 Extraction
mg kg1
5
1
Ag
Trace Elements
mg kg1
5
2
As
mg kg1
6
2
Ba
mg kg1
5
2
Be
mg kg1
4
2
Cd
mg kg1
4
2
Co
mg kg1
6
2
Cr
mg kg1
6
2
Cu
mg kg1
6
2
Hg
Mg kg1
4
0
Mn
mg kg1
6
2
Mo
mg kg1
5
2
Ni
mg kg1
6
2
P
mg kg1
7
2
Pb
mg kg1
7
2
Sb
mg kg1
4
2
Se
Mg kg1
7
2
Sn
mg kg1
6
2
Sr
mg kg1
7
2
V
mg kg1
6
2
W
mg kg1
5
2
Zn
mg kg1
7
2
Bulk Density
Bulk Density Core Method
g cm"3
3
2
5.7 References
Buurman, P., B. van Lagen, and E.J. Velthorst. 1996. Manual for soil and water analysis. Backhuys Publ.,
Leiden, the Netherlands.
Mehlich, A. 1984. Mehlich 3 soil test extractant: A modification of Mehlich 2 extractant.
Communications in Soil Science and Plant Analysis 15:1409-1416.
Soil and Plant Analysis Council, Inc. 1999. Handbook on Reference Methods for Soil Analysis. Council on
Soil Testing and Plant Analysis. CRC Press, Boca Raton, FL.
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Soil Survey Staff. 2011. Soil Survey Laboratory Information Manual. Soil Survey Investigations Report No.
45, Version 2.0. R. Burt (ed.). U.S. Department of Agriculture, Natural Resources Conservation
Service.
Soil Survey Staff. 2014. Kellogg Soil Survey Laboratory Methods Manual. Soil Survey Investigations
Report No. 42, Version 5.0. R. Burt and Soil Survey Staff (ed.). U.S. Department of Agriculture,
Natural Resources Conservation Service.
USEPA. 2011. National Wetland Condition Assessment. Laboratory Operations Manual. EPA843-R10-
002. U.S. Environmental Protection Agency, Washington, DC.
USEPA. 2011. National Wetland Condition Assessment. Quality Assurance Project Plan. EPA843-R10-003.
U.S. Environmental Protection Agency, Washington, DC.
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6.0 WATER CHEMISTRY AND CHLOROPHYLL A
This chapter describes the analysis requirements for water quality samples. The purpose is to determine
concentrations of water quality parameters in water quality samples collected in the NWCA 2016. The
laboratory shall perform analysis to determine levels of conductivity, pH, ammonia (NH3), nitrate-nitrite
(NO3-NO2), total nitrogen (TN), total phosphorous (TP), turbidity, dissolved organic carbon (DOC) and
chlorophyll a found in freshwater and saline wetlands, and sulfate (S04) and chloride (CI) for freshwater
samples only.
Table 6-1. Water chemistry parameters measured by NWCA 2016.
Analyte
Units
Comments
Conductivity
|j.S/cm at 25°C
All samples
pH (laboratory)
Standard (Std) Units
All samples
Turbidity
Nephelometric Turbidity Units (NTU)
All samples
Dissolved Organic Carbon (DOC)
mg C/L
All samples
Ammonia (NHs)
mg N/L
All samples
Nitrate-Nitrite (NO3-NO2)
mg N/L
All samples
Total Nitrogen (TN)
mg/L
All samples
Total Phosphorus (TP)
Hg P/L
All samples
Sulfate (SO4)
mg SO4/L
Freshwater samples only
Chloride (CI)
mg Cl/L
Freshwater samples only
Chlorophyll a
|ag/L (in extract)
All samples
6.1 Summary of Method
As an alternative to specifying laboratory methods for sample analysis, NWCA 2016 uses a performance-
based approach that defines a set of laboratory method performance requirements for data quality.
Method performance requirements for this project identify detection limit, precision, and accuracy
objectives for each parameter. As described in Section 6.6, unless otherwise contractually bound by
other requirements, the laboratory may choose to use any method that meets EPA's specifications for
water chemistry measurements.
6.2 Health and Safety Warnings
The laboratory must require its staff to abide by appropriate health and safety precautions. In addition
to the laboratory's usual requirements such as a Chemical Hygiene Plan, the laboratory must adhere to
the following health and safety procedures:
1. Laboratory facilities must properly store and dispose of solutions of weak acid.
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g.
lab coat, protective eyewear, gloves).
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3. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel
must avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must
avoid contacting skin and mucous membranes with acid. If skin contact occurs, remove
clothing immediately. Wash and rinse the affected skin areas thoroughly with large amounts
of water.
6.3 Definitions and Required Resources (Personnel, Laboratories, and
Equipment)
This section provides definitions and required resources for using the procedure.
6.3.1 Definitions
The procedure uses the following terms:
CI: Chloride
Detection Limit is the minimum concentration at which the analyte can be detected with
confidence. In other words, the outcome can be reported with confidence that it is greater than
zero (i.e., present in the sample) Also see "Sample-Specific Detection Limit."
DOC: Dissolved Organic Carbon
Duplicates are defined as two aliquots of the same sample which are analyzed separately using
identical procedures. The results are used to evaluate the precision of the laboratory analyses.
FIA: Flow Injected Analysis
IC: Ion Chromatography
NH3: Ammonia
NO3-NO2: Nitrate-nitrite
Percent Recovery: Recovery is measured by comparing the concentrations of a sample split into two
parts; and one part is spiked with a known concentration value. Cs is the concentration measured in
the spiked part; C is the concentration measured in the unspiked part; and s is the known
concentration amount for the spike. The following equation is used to calculate the percent
recovery:
%Rs =
cs- c
x 100
s
Relative Standard Deviation (RSD): The precision at each concentration is reported in terms of the
RSD. To calculate the RSD, first calculate the standard deviation, 5, as follows:
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S =
n
ht,(c°~cy'
k=1
1/2
where n is the number of replicate samples, C, is the concentration measure for the kth sample, and
C is the average concentration of the replicate samples. Then, RSD is calculated as:
RSD =
X 100
Reporting Limit: A reporting limit is the point at which the measured value of the analyte can be
reported with confidence.
Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal to the
method's detection limit. For diluted samples, the sample-specific detection limit will be the product
of the method's detection limit and the dilution factor. Typical values for the dilution factors will be
10 or 100.
Spiked Sample: See Percent Recovery definition for purpose of spiked samples.
SO4: Sulfate
TN: Total nitrogen
TP: Total phosphorous
6.3.2 General Requirements for Laboratories
Expertise. To demonstrate its competency/expertise to address each of the applicable parameters, the
laboratory shall provide EPA with performance data demonstrating their proficiencies in analyzing water
quality samples. See Appendix B for more information.
Quality assurance and quality control requirements. To demonstrate its expertise in quality assurance
and quality control procedures, the organization shall provide EPA with copies of the quality-related
documents relevant to the procedure. Examples include Quality Management Plans (QMP), Laboratory
Quality Assurance Manuals, QAPPs, and applicable Standard Operating Procedures (SOPs). See
Appendix B for more information.
To demonstrate its ongoing commitment, the person in charge of quality issues for the organization
shall sign the NWCA QAPP Certification Page.
6.3.3 Personnel
The procedure refers to the following personnel:
Laboratory Technician: This procedure may be used by any laboratory technician who is familiar
with the NWCA Quality Assurance Project Plan, and this procedure in the NWCA Laboratory
Operations Manual.
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6.3.4 Equipment/Materials
The analytical method, selected by the laboratory, identifies the necessary equipment.
6.4 Sample Receipt
Because EPA initiates tracking procedures designed to recover any missing shipment, the laboratory
personnel responsible for tracking samples must start the following login steps within 24 clock hours of
receiving a delivery. When samples are received, if they are not logged in and processed immediately,
they must be stored at 4 °C and processed within 48 hours. For each sampled site, the lab will receive
the following samples on wet ice:
• One 1 liter bulk water sample labeled 'CHEM' for water chemistry analysis
• A filter in a 50 ml tube for chlorophyll a labeled 'CHLA'
The laboratory technician must inspect the samples promptly on receipt and:
1. Log the samples into the National Aquatic Resource Survey Information Management
system (NARS-IM) within 24 clock hours. Alternatively, for shipments with a large number of
samples, the laboratory may email a spreadsheet with the sample login and sample
condition information to NARS-IM.
2. Check that each shipping container has arrived undamaged. Check the temperature of one
of the samples in the cooler using a thermometer that reads to at least -20 ^C (i.e., the
expected temperature of frozen samples), or an infra-red (IR) temperature "gun" and record
the reading. Temperature of the wet ice shipments should be 4 ^C or at less. Record the
condition and temperature of the sample in the database using the codes in Table 6-2.
3. Verify that all required data elements, per Table 6-2, have been recorded in the NARS IM
database. If any data elements are missing, then enter them into the database.
4. Transfer the samples for storage as follows:
a. Water chemistry aliquots are prepared following the requirements in Section 6.5 and
then are stored in a refrigerator at 4° C in darkness.
b. Chlorophyll-a filters to the freezer for no more than 30 days before analysis. Except
during processing and analysis stages, the filter must be stored frozen to less than or
equal -20 °C ± 2°.
5. Notify the EPA immediately about any problems involving sample integrity, conformity, or
inconsistencies as soon as possible following sample receipt and inspection.
Table 6-2 Water Chemistry Login: Required Data Elements
Variable
Type
Description
SITE ID
Character
Site identification code
SAMPLE ID
Character
Sample number
DATE_COLLECT
Date
Date that the field crew collected the sample
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Variable
Type
Description
ANALYSIS_TYPE
Character
Water Chemistry (CHEM) or Chlorophyll a (CHLA)
ARRIVAL_TEMP
Numeric
Temperature of sample upon arrival at the laboratory (will
be on wet ice);
CONDITION_CODE
Character
Condition codes describing the condition of the sample upon
arrival at the laboratory; leave blank for control
Flag
Definition
OK
Sample is in good condition
C
Sample container is cracked
L
Sample or container is leaking
ML
Sample label is missing
NF
Sample is not at proper
temperature
Q
Other quality concerns, not
identified above
CONDITION_COMMENT
Character
Explanation for Q FLAG (if needed)
6.5 Preparation of Water Chemistry Aliquots
Figure 6-1 presents the sample preparation processing steps for the water chemistry indicators,
including filtering and acidifying.
For nitrate-nitrite, DOC, ammonia, sulfate, and chloride, the laboratory technician will filter the sample
before processing. The laboratory technician will conduct the following steps:
1. Use 0.4nm pore size polycarbonate filters for all filtration.
2. Rinse vacuum filter funnel units thoroughly with reverse-osmosis (RO) or de-ionized (Dl)
water (ASTM Type II reagent water) five times before each use and in between samples.
After placing a filter in the funnel unit, run approximately 100 mL of RO or Dl water through
the filter, with vacuum pressure, to rinse the filter. Discard the rinse water.
3. Place the appropriate sample bottle under the funnel unit and filter sample directly into the
bottle. If a new filter is needed, remove the sample bottle, and rinse the new filter with 100
mL of RO or Dl water before continuing.
4. Split the sample into two aliquots as shown in Figure 6-1.
5. Add ultra-pure acid (H2S04, depending on the analytes, see Table 6-3) to one of the two
aliquots. Cap the bottle tightly and inverts the bottle several times to mix.
6. Store all aliquots in a refrigerator at 4°C in darkness.
For the other water chemistry analytes (TP, TN, turbidity, conductivity, and pH), the laboratory
technician will complete the following steps:
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1. Add ultra-pure acid (H2S04,) to one unfiltered sample as show in Figure 6-1 for TP, TN and
DOC; and prepare one bottle without acid. Cap the bottle tightly and invert the bottle
several times to mix.
2. Store all aliquots in a refrigerator at 4°C in darkness.
Sample Receipt
1 L Bulk Sample
Inspect samples and complete
tracking form
Store at 4°C in darkness
Process Sample within 24
hours
Filtration (0.4|jm)
HDPE bottle
Not acid washed
Store at 4 °C in
darkness
HDPE bottle
Acid washed
Preserve with
H SO.
J
Analyses
Nitrate-Nitrite
(freshwater) (IC)
(7 days)
Chloride (28 days)
Sulfate (28 days)
±
HDPE bottle
Acid washed
Preserve with
H2S04
Analyses
Ammonia (28 days)
Dissolved Organic Carbon (28
days)
Nitrate-Nitrite (brackish water)
(FIA) (28 days)
Not Filtered
_L
HDPE bottle
Not acid washed
Store at 4 °C in
darkness
Analyses
Total Phosphorus
(28 days)
Total Nitrogen (28 days)
Analyses
pH (3 days)
Turbidity (3 days)
Conductivity (28
days)
Figure 6-1. Water chemistry sample processing procedures
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Table 6-3 Water chemistry: acid preservatives added for various indicators
H2S04used for the following indicators
DOC
NHs
In
1p
no2-nc>3 when FIA method used)
6.6 Water Chemistry and Chlorophyll a Analysis: Requirements
The laboratory shall perform analysis of the samples to determine the ammonia (NH3), nitrate-nitrite
(NO3-NO2), total nitrogen (TN), total phosphorous (TP), dissolved organic carbon (DOC), conductivity,
turbidity, pH, and chlorophyll a. We are considering the addition of sulfate and chlorides for freshwater
samples. As an alternative to specifying laboratory methods for sample analysis (unless otherwise
required by contract), NWCA uses a performance-based approach that defines a set of laboratory
method performance requirements for data quality as shown in Table 6-4. Method performance
requirements for this project identify the reporting limit, precision, and accuracy objectives for each
parameter. NWCA is designating the reporting limit as the lowest value that the laboratory needs to
quantify (as opposed to just detecting the parameter in the sample), and is the value of the lowest non-
zero calibration standard that the laboratory must use. EPA has set the value to double the long-term
method detection limit (LT-MDL), following guidance presented in USGS (1999)1.
NWCA expresses precision and accuracy objectives in both absolute and relative terms following Hunt
and Wilson (1986). The transition value is the value at which performance objectives for precision and
accuracy switch from absolute (< transition value) to relative (> transition value). For pH, the objectives
are established for samples with higher and lower pH levels.
For standard samples (of known concentration), precision is estimated as the standard deviation of
repeated measurements across batches at the lower concentration range, and as percent relative
standard deviation of repeated measurements across batches at the higher concentration range.
Accuracy is estimated as the difference between the mean measured value and the target value of a
performance evaluation and/or internal reference samples at the lower concentration range measured
across sample batches, and as the percent difference at the higher concentration range.
Table 6-5 summarizes analytical methods used for past NARS surveys for the selected parameters (EPA
ORD-Corvallis). Participating laboratories may use alternative analytical methods for each target analyte
as long as they can satisfactorily demonstrate the alternative method is able to achieve the performance
requirements as listed in Table 6-4. Appendix B identifies the information that the laboratory should
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1 If a laboratory has questions related to meeting the -LT-MDL, they may contact the NWCA Laboratory Review
Coordinator to discuss concerns.
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provide to the NWCA Laboratory Review Coordinator to use in determining whether the laboratories
meet the necessary requirements.
Table 6-4 Water Chemistry and Chlorophyll-a: Laboratory Method Performance Requirements
Parameter
Units
Potential
Range
of
Samples1
Method
Detection
Limit
Objective2
Target
Reporting
Limit
Acceptable
Reporting
Limit
Transition
Value3
Precision
Objective4
Accuracy
Objective5
Conductivity
|iS/cm at 25"C
1 to 75,000
1.0
2.0
2.0
20
± 2 or ±10%
± 2 or 5%
PH
Std units
3.3 to 10.2
N/A
NA
NA
5.75, 8.25
<5.75 or
>8.25 =±0.07;
5.75-8.25 = ±0.15
<5.75 or
> 8.25 =±0.15;
5.75-8.25 =±0.05
Ammonia (NHj)
mg N/L
Oto 17
0.01 marine
(0.7 l-ieq/L)
0.02 freshwater
0.02
(1.4 1-ieq/L)
Max of 0.1
marine*
0.02
freshwater
0.10
± 0.01 or
±10%
± 0.01 or
±10%
Nitrate-Nitrite
(no3-no2)
mg N/L
Oto 360
(as nitrate)
0.01 marine
0.02 freshwater
0.02
0.05 marine
Max of 0.05
freshwater*
0.10
± 0.01 or
±10%
± 0.01 or
±10%
Total Nitrogen
(TN)
mg/L
0.1 to 90
0.01
0.02
Calculated
0.10
± 0.01 or
±10%
± 0.01 or
±10%
Total
Phosphorous
(TP)
Mg P/L
Oto 22,000
(as TP)
2.0
4.0
10
20.0
± 2 or
±10%
± 2 or
±10%
Dissolved
Organic Carbon
(DOC)
mgC/L
0.1 to 109
0.1
0.20
0.5
<1
> 1
±0.10 or ±10%
±0.10 or ±10%
Turbidity
Nephelometric
Turbidity Units
(NTU)
0 to 44,000
1.0
2.0
2
20
± 2 or ±10%
± 2 or ±10%
Chloride (CI)
mg Cl/L
Oto 5,000
0.10 (3 neq/L)
0.20
(6 1-ieq/L)
Max of 1*
1
±0.10 or ±10%
±0.10 or ±10%
Sulfate (SO4)
mg SO4/L
Oto 5,000
0.25 (5.2
Heq/L)
0.50
(10.4 neq/L)
Max of 1*
2.5
±0.25 or ±10%
±0.25 or ±10%
Chlorophyll-a
IJg/L in extract
0.7 to
11,000
0.5
0.5
0.5**
15
± 1.5 or
±10%
± 1.5 or
±10%
CL
o
1 Estimated from samples analyzed for NWCA 2011 and at the EPA Western Ecological Division-Corvallis laboratory between 1999 and
2005
2 The method detection limit is determined as a one-sided 99% confidence interval from repeated measurements of a low-level standard
across several calibration curves.
^ 3 Value for which absolute (lower concentrations) vs. relative (higher concentrations) objectives for precision and accuracy are used.
^ 4 For duplicate samples, precision is estimated as the pooled standard deviation (calculated as the root-mean square) of all samples at the
U lower concentration range, and as the pooled percent relative standard deviation of all samples at the higher concentration range. For
^ standard samples, precision is estimated as the standard deviation of repeated measurements across batches at the lower concentration
< range, and as percent relative standard deviation of repeated measurements across batches at the higher concentration range.
^ 5 Accuracy is estimated as the difference between the measured (across batches) and target values of performance evaluation and/or
internal reference samples at the lower concentration range, and as the percent difference at the higher concentration range.
^ * The national lab contractor shall provide the results of additional development with ion chromatography in attempting to achieve EPA's
^ reporting limits. If EPA determines that the contractor has made a good faith effort, EPA will accept the reporting limits that the
U contractor has been able to achieve up to a maximum of the value shown in Table 6-4.
oc ** yhe reporting limit assumes that the field crew provide enough filtered sample so that the lab does not need to adjust the reporting
b limit.
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Table 6-5 Water Chemistry and Chlorophyll-a: Analytical Methods Used in Past NARS Surveys (EPA ORD-
Corvallis)
Analyte
Summary of Method1
References2
WRS SOP3
pH (lab)
Automated, using ManSci PC-Titrate w/ Titra-
Sip autotitrator and Ross combination pH
electrode. Initial pH determination for ANC
titration
EPA 150.6 (modified)
WRS 16A.0 (April
2011)
Conductivity
Electrolytic, Man-Tech TitraSip automated
analysis
OR manual analysis, electrolytic
EPA 120.6
WRS 16A.0 (April
2011)
WRS 11A.4 (April
2011)
N itrate+N itrite, as N
Ion Chromatography (freshwater samples)
OR
FIA automated colorimetric (cadmium
reduction for brackish or freshwater
samples)4
EPA 300.6; SW-846 9056A;
APHA 4110B
EPA 353.2
APHA 4500-N03-N-E
Lachat 10-107-04-1-C
WRS 36A.0 (April 2011
WRS 40A.5 (May
2011)
Chloride and Sulfate
(potential)
Ion Chromatography (freshwater samples)
EPA 300.6; SW-846 9056A;
APHA 4110B
WRS 40A.5 (May
2011)
Ammonia, as N
FIA automated colorimetric (salicylate,
dichloroisocyanurate)
PEA 350.1, or modification
Lachat 10-107-06-3-D
WRS 30A.4 (April
2011)
Total nitrogen (TN)
Persulfate Digestion; FIA Automated
Colorimetric Analysis (Cadmium Reduction,
sulfanilamide)
EPA353.2 (modified)
APHA 4500-N-C (modified)
ASTM WK31786
U.S. EPA (1987)
Lachat 10-107-04-1-C
(modified)
WRS 34A.5 (April
2011)
Total phosphorus
(TP)
Persulfate Digestion
EPA 365.1
WRS 34A.5 (April
2011)
Dissolved Organic
Carbon (DOC)5
UV promoted persulfate oxidation to C02with
infrared detection
APHA 5310-C
U.S. EPA (1987)
WRS 21A.4 (May
2011)
Turbidity
Nephelometric; Man-Tech TitraSip
automated analysis,
OR
Manual analysis using Hach turbidimeter
(high turbidity samples)
APHA 214 A, EPA 180.1
U.S. EPA (1987)
WRS 16A.0 (April
2011)
WRS 13A.3 (April
2011)
Chlorophyll-a
(CHLA)
Extraction 90% acetone analysis by
fluorometry
EPA445.0, EPA446.0
WRS 71A.3 (April
2011)
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1 FIA=Flow injection analysis. AAS=Atomic Absorption Spectrometry
2 U.S. EPA, 1987. Handbook of Methods for Acid Deposition Studies: Laboratory Analyses for Surface Water Chemistry.
EPA/600/4-87/026. U.S. Environmental Protection Agency, Office of Research and Development, Washington D.C. APHA=
American Public Health Association (Standard Methods). ASTM=American Society of Testing and Materials.
3 WRS= Willamette Research Station. References are to laboratory SOP being used at central laboratory. Available upon request
from the EPA HQ Laboratory Review Coordinator.
4 Brackish samples that require use of the FIA method are those above 9000 uS/cm while those below 9000 uS/cm are
considered freshwater and can be run using either the IC or FIA method.
5 For DOC, "dissolved" is defined as that portion passing through a 0.45 [am nominal pore size filter. For other analytes,
"dissolved" is defined as that portion passing through a 0.4 [am pore size filter (Nucleopore or equivalent).
37
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6.7 Data Entry
Table 6-6 identifies the required data elements that laboratories must provide to EPA, preferably in
EPA's data template, available separately from EPA. Table 6-7 identifies reporting units and significant
figures.
Table 6-6 Water Chemistry and Chlorophyll-a: Data Elements for Each Sample
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Variable
Type
Description
SITE ID
Character
Site identification code or type of QC sample (e.g., LAB BLANK)
SAMPLE ID
Character
Sample number, LCS, QCCS, Blank, Matrix Spike, or CRM
ANALYSIS TYPE
Character
Water Chemistry (CHEM) or Chlorophyll a (CHLA)
REPEAT
Numeric
Duplicate
DATE COLLECT
Date
Date that the field crew collected the sample
ARRIVAL TEMP
Numeric
Temperature of sample upon arrival at the laboratory
CONDITION_CODE
Character
Condition codes describing the condition of the sample upon arrival at the
laboratory; leave blank for control
Flag
Definition
OK
Sample is in good condition
C
Sample container is cracked
L
Sample or container is leaking
ML
Sample label is missing
NF
Sample is not at proper temperature
Q
Other quality concerns, not identified above
CONDITION COMMENT
Character
Explanation for Q FLAG (if needed)
PARAMETER
Character
Analyte name
CAS NO
Character
CAS Registry number
LABNAME
Character
Laboratory name (abbreviation)
METHOD
Character
Laboratory method used
ANALYST
Character
Last name or initials of person who performed the analysis
REVIEWER
Character
Last name or initials of the person who provided a separate independent
review of the data
INSTRUMENT
Character
Identification of instrument used for the analysis - provide enough
information to identify the particular instrument in the laboratory
DATE PROCESSED
Date
Date that the analysis started
QC_BATCH_LOT
Character
Unique laboratory quality control lot numbers must be assigned to each
batch of samples. The lot number must associate each batch of field
samples to the appropriate laboratory control sample, matrix spike,
laboratory duplicate, method blank, and CRM samples.
HOLDING TIME
Y/N
Analysis performed within holding time
MATRIX
Character
Water
MDL
Numeric
Lab method detection limit (based upon lab's historical data)
LRL
Numeric
Lab reporting limit (based upon lab's historical data)
DILUTION
Numeric
Dilution of sample (blank or 1 if no dilution)
RESULT
Numeric
Concentration value
RESULT_QUAL
Character
Data qualifier (usually blank)
RESULT REASON
Character
Reason for qualification in RESULT_QUAL (usually blank)
UNIT
Character
Unit of measurement for RESULT, MDL, and LRL
QC_CODE
Character
Apply laboratory defined QC codes and describe in the comments field.
Provide set of laboratory's code as part of the case narrative
QC_COMMENT
Character
Explain situation that created QC code, or any unusual aspects of the
analysis
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Table 6-7. Water chemistry reporting units and significant figures.
No. Significant Maximum No.
Measurement Units Figures Decimal Places
Temperature
°C
2 1
PH
pH units
3 2
Dissolved Organic Carbon
mg/L
3 1
Conductivity
|_iS/cm at 25 °C
3 1
Total phosphorus
M-g/L
3 0
Total nitrogen
mg/L
3 2
Nitrate-Nitrite
mg/L
3 2
Ammonia
mg/L
3 2
Turbidity
NTU
3 0
Chlorophyll a
ug/L
3 2
Chloride and sulfate (Potential)
mg/L
3 1
6.8 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that the data
will meet NWCA's requirements. QC protocols are an integral part of all analytical procedures to ensure
that the results are reliable and the analytical stage of the measurement system is maintained in a state
of statistical control. The laboratory must conduct QC analyses for each batch of samples. Each batch
shall consist of no more than 20 samples. Unique laboratory quality control lot numbers must be
assigned to each batch of samples. The lot number must associate each batch of field samples to the
appropriate measures such as laboratory control sample, matrix spike, laboratory duplicate, and method
blank samples. Also, each laboratory QC samples (i.e., preparation and instrument blanks, laboratory
control sample (LCS), spike/duplicate, etc.) must be give a unique sample identification. Table 6-8
provides a summary of the quality control requirements.
Table 6-8 Water Chemistry and Chlorophyll-a: Quality control activities for water quality samples
QC Sample
Type and
Description
Indicators
Description
Frequency
Acceptance
Criteria
Corrective Action
Demonstrate
competency for
analyzing water
samples to meet
the performance
measures
All
Demonstration of
past experience
with water
samples in
achieving the
method detection
limits
Once
See Appendix A
EPA will not approve
any laboratory for
NWCA sample
processing if the
laboratory cannot
demonstrate
competency. In other
words, EPA will select
another laboratory
that can demonstrate
competency for its
NWCA samples.
39
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QC Sample
Type and
Description
Indicators
Description
Frequency
Acceptance
Criteria
Corrective Action
Check condition
of sample when
it arrives.
All
Sample issues such
as cracked
container; missing
label;
temperature;
adherence to
holding time
requirements;
sufficient volume
for test.
Once
No sample issues
or determination
that sample can
still be analyzed
Lab determines if the
sample can be
analyzed or has been
too severely
compromised (e.g.,
contamination).
Assign appropriate
condition code
identified in Table 1.
Store sample
appropriately.
All
Check the
temperature of
the refrigerator
per laboratory's
standard operating
procedures.
Record temperature
of sample upon
arrival at the
laboratory. Check
temperature of the
refrigerator/freezer
where samples are
stored at least daily
if using a
continuous
temperature logger
and twice daily
(once at beginning
of the day and once
at the end) not
using a continuous
logger.
While stored at
the laboratory,
the sample must
be kept at a
maximum
temperature of
4° C (for aliquots
except
chlorophyll a)
and -20° C for
the chlorophyll a
sample.
If at any time samples
are warmer than
required, note
temperature and
duration (either from
the continuous
temperature log or
from the last manual
reading) in comment
field. Lab will still
perform test. EPA
expects that the
laboratory will
exercise every effort
to maintain samples
at the correct
temperature.
Analyze sample
within holding
time
All
The test must be
completed
within the
holding time
specified in the
analytical
method.
Perform test in all
cases, but note
reason for
performing test
outside holding time.
EPA expects that the
laboratory will
exercise every effort
to perform tests
before the holding
time expires.
Analyze
Laboratory/
Reagent Blank
All
Once per day prior
to sample analysis
Control limits <
MDL
Prepare and analyze
new blank.
Determine and
correct problem (e.g.,
reagent
contamination,
instrument
calibration, or
contamination
introduced during
filtration) before
proceeding with any
sample analyses.
Reestablish statistical
control by analyzing
three blank samples.
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QC Sample
Type and
Description
Indicators
Description
Frequency
Acceptance
Criteria
Corrective Action
Analyze
Filtration Blank
All dissolved
analytes
ASTM Type II
reagent water
processed through
filtration unit
Prepare once per
week and archive
Prepare filter blank
for each box of 100
filters, and examine
the results before
any other filters are
used from that box.
Measured
concentrations
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QC Sample
Type and
Description
Indicators
Description
Frequency
Acceptance
Criteria
Corrective Action
Analyze
Standard
Reference
Material
(SRM)
When
available for
a particular
indicator
One analysis in a
minimum of five
separate batches
Manufacturers
certified range
Analyze standard in
next batch to confirm
suspected inaccuracy.
Evaluate calibration
and QCCS solutions
and standards for
contamination and
preparation error.
Correct before any
further analyses of
routine samples are
conducted.
Reestablish control
by three successive
reference standard
measurements that
are acceptable.
Qualify all sample
batches analyzed
since the last
acceptable reference
standard
measurement for
possible reanalysis.
Analyze Matrix
Spike Samples
Only
prepared
when
samples with
potential for
matrix
interferences
are
encountered
One per batch
Control limits for
recovery cannot
exceed 100±20%
Select two additional
samples and prepare
fortified subsamples.
Reanalyze all
suspected samples in
batch by the method
of standard additions.
Prepare three
subsamples
(unfortified, fortified
with solution
approximately equal
to the endogenous
concentration, and
fortified with solution
approximately twice
the endogenous
concentration).
Use consistent
units for QC
samples and
field samples
All
Verify that all units
are provided
consistently within
each indicator.
Data reporting
For each
indicator, all field
and QC samples
are reported
with the same
measurement
units
If it is not possible to
provide the results in
consistent units, then
assign a QC code and
describe the reason
for different units in
the comments field of
the database.
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QC Sample
Type and
Description
Indicators
Description
Frequency
Acceptance
Criteria
Corrective Action
Maintain
completeness
All
Determine
completeness
Data reporting
Completeness
objective is 95%
for all indicators
(useable with or
without flags).
Contact EPA HQ
NWCA Laboratory
Review Coordinator*
immediately if issues
affect laboratory's
ability to meet
completeness
objective.
*Chapter 2 and Appendix A provides contact information for the EPA HQ NWCA Laboratory Review Coordinator. Laboratories
under contract to EPA must contact the Task Order's Contracting Officer's Representative (TOCOR) instead of the Laboratory
Review Coordinator.
6.9 Sample and Record Retention
The laboratory shall retain:
1. The sample materials for a minimum of 1 year after collection. During this time, the laboratory
shall store the materials cold (e.g., 4 0 C) and in darkness. The lab shall retain the sample
materials from the 1 year point until the EPA publishes the final report at ambient
temperatures.
2. Original records, including laboratory notebooks for a minimum of 10 years from the date that
EPA publishes the final report.
After the stated time periods, the laboratory shall follow its internal protocols for disposal.
6.10 Literature Cited
Hunt, D.T.E. and A.L. Wilson. 1986. The Chemical Analysis of Water: General Principles and Techniques.
2nd ed. Royal Society of Chemistry, London, England.
USEPA, 1987. Handbook of Methods for Acid Deposition Studies: Laboratory Analyses for Surface Water
Chemistry. EPA/600/4-87/026. U.S. Environmental Protection Agency, Office of Research and
Development, Washington D.C.
USEPA. 1997. Methods for the Determination of Chemical Substances in Marine and Estuarine
Environmental Matrices-2nd Edition. EPA No. 600-R-97-072. U.S. Environmental Protection Agency,
Office of Research and Development, Washington, DC, retrieved June 30, 1997 from
http://www.epa.gov/microbes/documents/marinmet.pdf.
USEPA. September 1997. Method 353.4 "Determination of Nitrate and Nitrite in Estuarine and Coastal
Waters by Gas Segmented Continuous Flow Colorimetric Analysis, Revision 2.0", retrieved June 30, 2014
from http://www.epa.gov/microbes/documents/m353_4.pdf.
USGS. 1999. "New reporting procedures based on long-term method detection levels and some
considerations for interpretations of water-quality data provided by the U.S. Geological Survey National
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Water Quality Laboratory." Open-File Report: 99-193 by Childress, Oblinger, et al., retrieved June 30,
2014 from http://pubs.usgs.gov/of/1999/0193/report.pdf.
Youden, W.J. 1969. Ranking laboratories by round-robin tests. In Precision Measurement and
Calibration. H.H. Ku, ed. NBS Special Publication 300, Vol. 1. U.S. GPO Washington, D.C.
USEPA. 2011. National Wetland Condition Assessment. Laboratory Operations Manual. EPA843-R10-
002. U.S. Environmental Protection Agency, Washington, DC.
USEPA. 2011. National Wetland Condition Assessment. Quality Assurance Project Plan. EPA843-R10-003.
U.S. Environmental Protection Agency, Washington, DC.
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7.0 ALGAL TOXIN (MICROCYSTIN) IMMUNOASSAY PROCEDURE1
This chapter describes an immunoassay procedure that measures concentrations of total microcystins in
water samples. In applying the procedure, the laboratory uses Abraxis' Microcystins-ADDATest Kits
("kits", Figure 7-1). Each kit is an enzyme-linked immunosorbent assay (ELISA) for the determination of
microcystins and nodularins in water samples. Microcystins refers to the entire group of toxins, all of the
different congeners, rather than just one congener. Algae can produce one or many different congeners
at any one time, including Microcystin-LR (used in the kit's calibration standards), Microcystin-LA, and
Microcystin-RR. The different letters on the end signify the chemical structure (each one is slightly
different) which makes each congener different.
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Figure 7-1 Microcystin: Abraxis microcystin test kit (from James, page 3, 2010) q
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7.1 Summary of Method ^
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The procedure is an adaption of the instructions provided by Abraxis for determining total microcystins o
concentrations using its ELISA-ADDA kits.2 For freshwater samples, the procedure's reporting range is §
0.15 ng/Lto 5.0 |ig/L, although, theoretically, the procedure can detect, not quantify, microcystins ^
concentrations as low as 0.10 |ig/L. For samples with higher concentrations of microcystins, the ^
procedure includes the necessary dilution steps. The procedure also provides additional sample §
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1 Algal toxin samples collected in NWCA will be processed and analyzed by the USGS Organic Geochemistry ~
Research Laboratory (OGRL) and by State operated laboratories. The SOP used by the USGS OGRL to analyze for x
the algal toxin microcystin is provided in Appendix F. P
I
2 Abraxis, "Microcystins-ADDA ELISA (Microliter Plate): User's Guide R021412." Retrieved on January 14, 2014 from ^
http://www.abraxiskits.com/uploads/products/docfiles/278 Microcvstin%20PL%20ADDA%20users%20R120214.pdf. —J
45
MICROCYSTINS NODULARINS
ELISA KIT
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preparation steps for samples with salinities>3.5 ppt. The results then are adjusted by a factor of 1.75
for a reporting range of 0.263 ng/L to 8.75 ng/L.
7.2 Health and Safety Warnings
The laboratory must require its staff to abide by appropriate health and safety precautions, because the
kit substrate solution contains tetramethylbenzidine (TMB) and the stop solution contains diluted
sulfuric acid. In addition to the laboratory's usual requirements such as a Chemical Hygiene Plan, the
laboratory must adhere to the following health and safety procedures:
1. Laboratory facilities must properly store and dispose of solutions of weak acid.
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g. lab
coat, protective eyeware, gloves).
3. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel must
avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must avoid
contacting skin and mucous membranes with the TMB and stopping solution. If skin contact
occurs, remove clothing immediately. Wash and rinse the affected skin areas thoroughly with
large amounts of water.
7.3 Definitions and Required Resources (Personnel, Laboratories, and
Equipment)
This section provides definitions and required resources for using the procedure.
7.3.1 Definitions
The following terms are used throughout the procedure:
LI-
CO
£ Absorbance (A) is a measure of the amount of light in a sample. A standard statistical curve is used to
q convert the absorbance value to the concentration value of microcystins.
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§ Brackish and Seawater Samples, for the purposes of the ABRAXIS microcystins test procedure, are
^ samples with salinity greater than or equal to 3.5 parts per thousand (ppt). (EPA is using different
5i definitions for the water chemistry samples.) EPA recognizes that brackish water is usually defined as 0.5
< ppt, and seawater as 35 ppt, but for this immunoassay procedure, it is important to use additional steps
z described in Section 7.6.2 for any sample with salinity greater than or equal to 3.5 ppt. The sample
^ labels provide the salinity levels.
5" Calibration Range is the assay range for which analysis results can be reported with confidence. For
^ undiluted samples, it ranges from the reporting limit of 0.15 ng/L to a maximum value of 5.0 ng/L.
b Values outside the range are handled as follows. If the value is:
QC
^ • < 0.10 ng/L, then the laboratory reports the result as being non-detected ("<0.10 ng/L").
~z.
g • Between 0.10 ng/L and the reporting limit of 0.15 ng/L (i.e., >0.10 ng/L and <0.15 ng/L), the
laboratory should record the value, but assign a QC code to the value (i.e., DATA_FLAG=J).
<
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• 5.0 ng/L, the laboratory must dilute and reanalyze the sample.
Coefficient of Variation (CV): The precision for a sample is reported in terms of the percent CV of its
absorbance values. To calculate the %CV, first calculate 5 (standard deviation) as follows:
where n is the number of replicate samples, Ais the absorbance measured for the /'th replicate. Samples
are evaluated in duplicate (i=l or 2); controls are either evaluated in duplicate or triplicate (i=l, 2, 3). A
is the average absorbance of the replicates. Then, calculate %CV as:
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable.
Detection Limit is the minimum concentration at which the analyte can be detected with confidence. In
other words, the outcome can be reported with confidence that it is greater than zero (i.e., present in
the sample). The detection limit is less than the reporting limit of 0.15 ng/L at which the measured value
of the analyte can be reported with confidence. Also see "Sample-Specific Detection Limit."
Duplicates are defined as two aliquots of the same sample which are analyzed separately using identical
procedures. The results are used to evaluate the precision of the laboratory analyses. Per Section 7.6.4,
controls are evaluated in duplicate or triplicate (i.e., three aliquots).
Relative Standard Deviation (RSD) is the same as the coefficient of variation (%CV). Because many of
the plate reader software programs provides the CV in their outputs, the procedure presents the quality
control requirement in terms of %CV instead of RSD.
Reporting Limit: For undiluted freshwater sample, the reporting limit is 0.15 ng/L. A reporting limit is
the point at which the measured value of the analyte can be reported with confidence.
Standard Deviation (S) shows variation from the average
Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal to the
method's detection limit of 0.1 ng/L. For diluted samples, the sample-specific detection limit will be the
product of the method's detection limit of 0.1 ng/L and the dilution factor. Typical values for the dilution
factor will be 10 or 100.
Seawater Sample: See definition for brackish and seawater samples.
7.4 General Requirements for Laboratories
n
1/2
i=1
s
%CV = -= x 100
A
7.4.1 Expertise
To demonstrate its expertise, the laboratory shall provide EPA with one or more of the following:
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• Memorandum that identifies the relevant services that the laboratory provided for the National
Aquatic Resource Surveys in the past five years.
• Documentation detailing the expertise of the organization, including professional certifications
for water-related analyses, membership in professional societies, and experience with analyses
that are the same or similar to the requirements of this method.
7.4.2 Quality assurance and quality control requirements
To demonstrate its expertise in quality assurance and quality control procedures, the organization shall
provide EPA with copies of the quality-related documents relevant to the procedure. Examples include
Quality Management Plans (QMP), QAPPs, and applicable Standard Operating Procedures (SOPs).
To demonstrate its ongoing commitment, the person in charge of quality issues for the organization
shall sign the NWCA 2016 QAPP Certification Page.
7.4.3 Personnel
Laboratory Technician: This procedure may be used by any laboratory technician who is familiar with
the NWCA 2016 QAPP, and this procedure in the NWCA 2016 LOM (which differs from the Abraxis
instructions). The laboratory technician also must be familiar with the use of a multichannel pipette and
plate readers.
External QC Coordinator is an EPA staff person who is responsible for selecting and managing the "QC
contractor." To eliminate the appearance of any inherent bias, the QC contractor must be dedicated to
QA/QC functions, and thus, must not be a primary laboratory or a field sampling contractor for NWCA.
The QC contractor is responsible for complying with instructions from the External QC Coordinator;
coordinating and paying for shipments of the performance samples to participating laboratories;
comparing immunoassay results from the laboratories; and preparing brief summary reports.
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QC
>1 • Abraxis ADDA Test Kit, Product #520011
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Q • Adhesive Sealing Film (Parafilm) for Micro Plates (such as Rainin, non-sterile, Cat. No. 96-SP-
O 100): Used to cover plates during incubation.
n
Data Template - See Appendix C.
Distilled or Deionized Water: For diluting samples when necessary.
ELISA evaluation software
Glass scintillation, LC, vials (two vials of 2 mL each)
Glass vials with Teflon-lined caps of size:
o 20 mL
o 4 mL (for dilutions)
l/l
b
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• Multichannel Pipette & Tips: A single-channel and an 8-channel pipette are used for this
method.
• Norm-ject syringes (or equivalent)
• Paper Towels: For blotting the microtiter plates dry after washing.
• Permanent Marker (Sharpie Fine Point): For labeling samples, bottles, plates and covers.
• Plate Reader (e.g., Metertech Model M965 AccuReader; ChroMate"; or equivalent readers with
software to read the microtiter plates and measure absorbances).
• Reagent Reservoirs (e.g., Costar Cat Number 4870): Plain plastic reservoir for reagents that
accommodate the use of a multi-channel pipette.
• Test tubes: For dilutions, if needed.
• Timer: For measuring incubation times.
• Vortex Genie: For mixing dilutions.
• Whatman Glass fiber syringe filter (25mm, GF 0.45 pim filter)
7.5 Sample Receipt
Field crews keep the microcystins samples cool while in the field and then pack the samples in ice for
delivery to a central facility ("batching laboratory") or the State's laboratory. The batching and State
laboratories freeze the samples upon receipt. Periodically, the batching laboratory ships samples to the
microcystins laboratory. The batching and microcystins laboratory may retain the frozen samples for
several months before analysis.
Because EPA initiates tracking procedures designed to recover any missing shipment, the laboratory
personnel responsible for tracking samples must start the following login steps within 24 clock hours of
receiving a delivery.
1. Report receipt of samples in the NARS IM sample tracking system (within 24 clock hours).
2. Inspect each sample THE SAME DAY THEY ARE RECEIVED:
a. Verify that the sample IDs in the shipment match those recorded on the:
i. Chain of custody forms when the batching laboratory sends the samples to the
microcystins laboratory; or
ii. Sample tracking form if the field crew sends the shipment directly to the State
laboratory.
b. Record the information in Table 7-1 into NARS IM, including the Condition Code for each
sample:
i. OK: Sample is in good condition
ii. C: Sample container was cracked
iii. L: Sample container is leaking
iv. ML: Sample label is missing
v. NF: Sample not frozen
c. If any sample is damaged or missing, contact the EPA HQ Laboratory Review Manager to
discuss whether the sample can be analyzed. (See contact information in Table 2-1).
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3. Store samples in the freezer until sample preparation begins.
4. Maintain the chain of custody or sample tracking forms with the samples.
Table 7-1 Microcystin: required data elements - login
FIELD
FORMAT
DESCRIPTION
LAB ID
text
Name or abbreviation for QC laboratory
DATE RECEIVED
MMDDYY
Date sample was received by lab
SITE ID
text
NWCA site id as used on sample label
VISIT NUMBER
numeric
Sequential visits to site (1 or 2)
SAMPLE ID
numeric
Sample id as used on field sheet (on sample label)
DATE COLLECTED
MMDDYY
Date sample was collected
CONDITION CODE
text
Condition codes describing the condition of the sample upon arrival at the
laboratory.
Flag
Definition
OK
Sample is in good condition
C
Sample container is cracked
L
Sample or container is leaking
ML
Sample label is missing
W
Sample is warm (>8 °C)
Q
Other quality concerns, not identified above
CONDITION
text
Comments about the condition of the sample. If the condition code='W' then
COMMENT
provide the temperature
7.6 Procedure
The following sections describe the sample and kit preparation and analysis.
7.6.1 Sample Preparation
oo
£ For each frozen sample (125 mL per sample), the laboratory technician runs it through a freeze-thaw
q cycle three times to lyse the cells as follows:
LU
U
§ 1. All cycles: Keep the samples in dark or dimly lit areas (i.e., away from sunlight, but under
incandescent lighting is acceptable).
CL
>
<
$ 2. First freeze-thaw cycle:
O
a. Start with a frozen 125 ml sample.
^ b. Thaw the sample to room temperature (approximately 25° C). Swirl the sample to check for
^ ice crystals. At this temperature, no ice crystals should be present in the sample.
5" c. Shake well to homogenize the sample, then transfer 10 mL to an appropriately labeled clean
^ 20 mL glass vial.
O 3. Second freeze-thaw cycle:
QC
^ a. Freeze the vial.
b. Keep the large sample bottle (from the 125 mL initial sample) frozen for future use.
g c. Thaw the sample vial contents to room temperature.
<
50
4. Third freeze-thaw cycle:
a. Freeze the vial.
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b. Thaw the vial contents to room temperature.
c. Filter the vial contents through a new, syringe filter (0.45 pirn) into a new, labeled 20 mL
glass scintillation vial. Norm-ject syringes and Whatman Glass fiber syringe filters (25mm, GF
0.45 pirn filter) or other similar alternative are acceptable. One new syringe and filter should
be used per sample.
7.6.2 Additional Sample Preparation for Samples with Salinity>3.5 parts per thousand
For any sample with salinity of 3.5 parts per thousand (ppt) or greater (the salinity will be marked on
sample vials), the laboratory technician needs to perform the following additional steps provided by
Abraxis. 1 For all other samples (i.e. with salinity less than 3.5 ppt), the technician skips this section (i.e.,
Section 7.6.2) and goes directly to kit preparation as described in Section 7.6.3. For samples with salinity
>3.5 ppt the technician:
1. Prepares the column as follows:
a. Place a small amount of glass wool into the top of a 5 %" glass Pasteur pipette. Using a 9"
glass Pasteur pipette, push the glass wool into to the bottom of the 5 %" pipette to form the
base of the column. The depth of the glass wool should be approximately 5 mm. Place the
column into a 12x75 mm test tube.
b. Each column will require approximately 1.5 g of Seawater Sample Clean-Up Resin. Calculate
and add the appropriate amount of Microcystins-ADDA Seawater Sample Clean-Up Resin to
a 20 mL glass vial.
c. Add distilled or deionized water at an approximately 2:1 ratio to the Microcystins- ADDA
Seawater Sample Clean-Up Resin (for example, 10 mL of deionized or distilled water per 5 g
of Resin). Shake or vortex.
d. Pipette the Resin in water solution into the column using the 9" Pasteur pipette. Avoid the u.
00
formation of air bubbles in the column bed by keeping the tip of the pipette at the surface ^
of the bed being created. Fill the column to the indentation approximately 2 cm from the ^
top of the pipette. This will create an approximately 8 cm column. u
O
e. Allow the deionized or distilled water to drain from the column.2. Lift the tip of the column ^
at least 1 cm above the surface of the water in the tube. Place the pipette bulb against the ^
top of the column (do not attach the bulb to the column) and push the remaining water out $
of the column. Avoid allowing the tip of the column to come into contact with the water in O
the tube to prevent aspiration of water back into the column. 3
f. Place the column into an appropriately labeled 4 mL glass vial. ^
2 Additional correspondence between EPA and Abraxis notes that this step leaves the resin in the column.
l/l
>
u
1 Reformatted from Abraxis, "Microcystins in Brackish Water or Seawater Sample Preparation" Retrieved on §
January 14, 2014 from http://abraxiskits.com/uploads/products/docfiles/385 MCT- ^
ADDA%20in%20Seawater%20Sample%20Prep%20%20Bulletin%20R041112.pdf. Reproduced with permission.
Except for Abraxis' solutions labeled as seawater, EPA has removed references to "brackish" and "seawater" which 5
typically are defined as having different cutpoints than 3.5 ppt for salinity. O
<
51
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2. Cleans up the sample as follows:
a. Add 1 mL of the sample to a clean, appropriately labeled 4 mL glass vial. Add 50 piL of
Microcystins-ADDA Seawater Sample Treatment Solution. Vortex.
b. Add 375 piL of the treated sample to the top of the column. Allow the sample to drain
through the column and collect in the vial.
c. Add a second 375 piL aliquot of the treated sample to the column. Allow to drain through
the column.
d. Lift the tip of the column at least 1 cm above the surface of the sample in the vial. Place the
pipette bulb against the top of the column (do not attach the bulb to the column) and push
the remaining sample out of the column. Avoid allowing the tip of the column to come into
contact with the sample in the vial to prevent aspiration of the sample back into the column
e. Lower the column back into the vial. Add 500 piL of distilled or deionized water to the top of
the column. Allow the rinse to drain through the column and collect with the sample.
f. Lift the tip of the column at least 1 cm above the surface of the sample/rinse in the vial.
Place the pipette bulb against the top of the column (do not attach the bulb to the column)
and push the remaining rinse out of the column. Avoid allowing the tip of the column to
come into contact with the sample in the vial to prevent aspiration of the sample back into
the column.
g. Remove the column and discard (columns are single use only). Cap vial and vortex. The
sample can then be analyzed using the Abraxis Microcystins-ADDA ELISA Kit beginning with
the next section (7.6.3).
7.6.3 Kit Preparation
oo The technician prepares the kits using the following instructions:
LU
CC
o 1. Check the expiration date on the kit box and verify that it has not expired. If the kit has expired,
LU
u discard and select a kit that is still within its marked shelf life. (Instead of discarding the kit,
^ consider keeping it for training activities.)
< 2. Verify that each kit contains all of the required contents:
i/i
O • Microtiter plate
¦z.
^ • Standards (6) referenced in this procedure as follows with the associated concentration:
— o SO: 0 ng/L
2" o SI: 0.15 ng/L
£ o S2: 0.40 ng/L,
g o S3: 1.0 ng/L
g o S4: 2.0 ng/L
^ o S5: 5.0 Hg/L
55 • Kit Control (KC): 0.75 ng/L
O
• Antibody solution
<
52
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• Wash Solution 5X Concentrate
• Color Solution
• Stop Solution
• Diluent
3. If any bottles are missing or damaged, discard the kit. This step is important because Abraxis has
calibrated the standards and reagents separately for each kit.
4. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.
5. Remove 12 microtiter plate strips (each for 8 wells) from the foil bag for each kit. The plates
contain 12 strips of 8 wells. If running less than a whole plate, remove unneeded strips from the
strip holder and store in the foil bag, ziplocked closed, and place in the refrigerator.
6. Store the remaining strips in the refrigerator (4-8° C).
7. Prepare a negative control (NC) using distilled water
8. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop solutions
are ready to use and do not require any further dilutions.
9. Dilute the wash solution with deionized water. (The wash solution is a 5X concentrated
solution.) In a 1L container, dilute the 5X solution 1:5 (i.e., 100 mL of the 5X wash solution plus
400 mL of deionized water). Mix thoroughly. Set aside the diluted solution to wash the
microtiter wells later.
10. Handle the stop solution containing diluted H2S04 with care.
Laboratory Operations Manual
Page 53 of 95
7.6.4 Insertion of Contents into Wells
This section describes the steps for placing the different solutions into the 96 wells. Because of the oo
LU
potential for cross contamination using a shaker table, the following steps specify manual shaking of the ^
kits instead mechanized shaking. Q
u
1. While preparing the samples and kit, turn the plate reader on so it can warm up. The plate §
reader needs a minimum of 30 minutes to warm up. ^
-
4. Using the lOO-piL pipette, add 50 piL, each, of the standards, controls, and samples to the q
appropriate wells in the plate. Place all six standards (0.00, 0.15, 0.40, 1.00, 2.0 and 5.0 ng/L), u
the kit control (0.75 piL), and negative control, in pairs, starting in the well in the upper left-hand ^
corner of the kit as shown in Figure 7-2. Verify that the software displays the same template or z
make any necessary corrections. O
i—
i
<
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1
2
3
4
5
6
7
8
9
10
11
12
A
SO
S4
NC
U4
U8
U12
U16
U20
U24
U28
U32
U36
B
SO
S4
NC
U4
U8
U12
U16
U20
U24
U28
U32
U36
C
SI
S5
Ul
U5
U9
U13
U17
U21
U25
U29
U33
U37
D
SI
S5
Ul
U5
U9
U13
U17
U21
U25
U29
U33
U37
E
S2
KC
U2
U6
U10
U14
U18
U22
U26
U30
U34
U38
F
S2
KC
U2
U6
U10
U14
U18
U22
U26
U30
U34
U38
G
S3
KC
U3
U7
Ull
U15
U19
U23
U27
U31
U35
U39
H
S3
NC
U3
U7
Ull
U15
U19
U23
U27
U31
U35
U39
Figure 7-2 Microcystin: sample template
Key:
S0-S5 = Standards;
KC = Control supplied with Kit (i.e., Kit Control);
NC = Negative Control;
U = Unknown (sample collected by the field crew).
5. Add 50 piL of the pink antibody solution to each well using the multi-channel pipettor and a
reagent reservoir. Use dedicated reagent reservoirs for each reagent to avoid contamination
from one reagent to another.
6. Place the sealing Parafilm over the wells.
7. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
for 30 seconds. Be careful not to spill the contents.
8. Place the plate in an area away from light for 90 minutes.
9. After 90 minutes, carefully remove the Parafilm.
oo
£ 10. Empty the contents of the plate into the sink, pat inverted plate dry on a stack of paper towels,
q and then wash the wells of the plate three times with 250 piL of washing solution using the
u multi-channel pipette. After adding the washing solution each time, empty the solution into the
§ sink and use the paper towels as before.
CL
< 11. Add 100 piL of enzyme conjugate solution to all wells using the multi-channel pipettor.
i/i
l/l
< 12. Cover the wells with Parafilm.
O
3 13. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
^ for 30 seconds. Be careful not to spill the contents.
5" 14. Place the strip holder in an area away from light for 30 minutes.
I—
> 15. After 30 minutes, remove the Parafilm, decant, and rinse the wells three times again with 250
O HL of washing solution as described in step 10.
QC
^ 16. Add 100 piL of color solution to the wells using the multi-channel pipette and reagent reservoir.
This color solution will make the contents have a blue hue.
O 17. Cover the wells with Parafilm.
i—
< 18. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
o
for 30 seconds. Be careful not to spill the contents.
54
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19. Place the plate in an area away from light for 20 minutes.
20. After 20 minutes, remove the Parafilm and add 50 piL of stopping solution to the wells in the
same sequence as for the color solution. This will turn the contents a bright yellow color. After
adding the stopping solution, read the plate within 15 minutes.
21. Within 15 minutes of adding the stopping solution, use the microplate ELISA photometer (plate
reader) to determine the absorbance at 450 nm. The software (i.e., commercial ELISA evaluation
program) calculates the absorbance and concentration values of the samples from the
calibration curve and the average values for each pair. Use a 4-parameter standard curve fit to
determine the concentrations.
22. Dispose of solution in plates in a lab sink. Rinse plates and sink with water to dilute the weak
acid present.
23. Perform QC evaluations of the data as follows:
a. If the following failures occur, then the laboratory must reanalyze all samples in the
analytical run:
i. Standard curve with a correlation coefficient of less than 0.99 (i.e., R<0.99)
ii. Standards S0-S5 must have decreasing absorbance values. First, calculate the average
values for each standard. That is, if A, is the absorbance average for S,, then the
absorbance averages must be:
iii. A0> Ai> A2> A3> A4>A5
iv. The average absorbance of the standard SO less than 0.8 (i.e., Ao< 0.8).
v. Two or more negative control samples with detectable concentrations of microcystins
(i.e., values > 0.1 ng/L). If this occurs, then evaluate possible causes (e.g., cross-
contamination between samples), and if appropriate, modify laboratory processes
before the next analytical run.
vi. Results for control samples of outside the acceptable range of 0.75 +/- 0.185 ppb. That
is, results must be between 0.565 and 0.935.
b. If either, or both, of the following failures occur, then the sample must be reanalyzed
(maximum of two analyses, consisting of the original analysis and, if necessary, one
reanalysis):
i. The concentration value registers as HIGH (exceeds the calibration range). Dilute the
sample for the reanalysis per Section 7.6.5
ii. The %CV > 15% between the duplicate absorbance values for a sample.
24. Record the results, even if the data failed the quality control requirements in #23b, for each well
in EPA's data template (see Table 7-2 for required elements). The required entries are for the
following columns:
a. TYPE should be one of the following codes: S0-S5 for standards; KC, NC, or SC for controls; U
for unknown sample.
b. CONC contains the numeric concentration value. Two special cases:
i. Non-detected concentrations: If the sample is non-detected, then provide the sample-
specific detection limit which is 0.1 ng/L if the sample is undiluted. See Section 7.3.1 for
calculating the sample-specific detection limit for a diluted sample.
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ii. If the result shows that it is "HI," this indicates that the sample value is outside of the
calibration range and must be diluted and re-run using another analytical run. Leave the
CONC column blank and record 'HI' in the DATA FLAG column.
c. DATA FLAGS have codes for the following special cases:
i. ND if the sample was non-detected;
ii. J if the value is detected but at a level below the reporting limit of 0.15 ng/L (for
undiluted samples);
iii. HI if the concentration value registers as HIGH (exceeds the calibration range).
d. QUALITY FLAGS have codes for the following special cases:
i. QCF if there is a QC failure per step 23 above. The QCF code must be used for all failures
to facilitate data analysis.
ii. Qfor any other quality issue (describe in COMMENTS)
e. DILUTION FACTOR is only required if the sample was diluted.
f. DUP AVG and DUP CV are required for duplicate samples and control samples (use all three
values if the controls are used in triplicate).
Table 7-2 Microcystin: required data elements - data submission
oo
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QC
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56
STAGE
FIELD
FORMAT
DESCRIPTION
LOGIN
LAB ID
text
Name or abbreviation for QC laboratory
DATE RECEIVED
text
Date sample was received by lab
SITE ID
text
NWCA site ID code as recorded on sample label or tracking form
(blank if standard or control)
VISIT NUMBER
numeric
Sequential visits to site (1 or 2) (blank if standard or control)
SAMPLE ID
numeric
6-digit Sample ID number as recorded on sample jar or tracking
form (blank if standard or control)
DATE COLLECTED
MMDDYY
Date sample was collected (blank if standard or control)
CONDITION CODE
text
Sample condition upon arrival at the laboratory (blank if standard
or control)
Flag Definition
Blank or N Not a sample (blank, standard, or control)
OK Sample is in good condition
C Sample container is cracked
L Sample or container is leaking
ML Sample label is missing
W Sample is warm (>8 °C)
Q Other quality concerns, not identified above
CONDITION
text
Comments about the condition of the sample. If the condition
COMMENT
code='W' then provide the temperature
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STAGE
FIELD
FORMAT
DESCRIPTION
ANALYSIS
TECHNICIAN
text
Name or initials of technician performing the procedure
KIT EXPIRE DATE
MMDDYY
Expiration date on kit box
KIT ID
text
Kit identification code. If one does not exist, assign a unique code
to each kit.
R2
numeric
R2 from curve fit to the average absorbance values for the
standards. Value is between 0 and 1.
TYPE
text
Type of solution being tested in the well
Code
Definition
KC
Kit Control
NC
Negative Control
SO,SI, S2,S3, S4, S5 Standard
U
Sample of unknown concentration
LOCATION
text
Location of well in the kit (e.g., B5 would be the fifth well from the
left in the second row B)
SALINITY
numeric
If the sample vial has the salinity marked on the vial, record the
value in units of parts per thousand. Otherwise, leave blank.
CONC
numeric
Concentration or sample-specific detection limit of contents of
well in ng/L. Sample-specific detection limit should be 0.1 ng/L if
the sample hasn't been diluted.
ABSORBANCE
numeric
Absorbance value
DILUTION FACTOR
numeric
10, 100, etc for number of times the sample was diluted. If not
diluted, leave blank or record 1
CV_ABSORB
numeric
Calculated %CV of duplicate values of absorbance for a sample.
Only calculated for TYPE=U, KC, or NC. Enter %CV. Value is
between 0 and 100%.
AVG_ABSORB
numeric
Calculated average of absorbance values for a sample. Only
provided forTYPE=U, KC, NC, or SC. Average value of the original
sample and its duplicate (or replicates for KC and NC).
AVG_CONC
numeric
Calculated average of concentration values for a sample.
Substitute 0.15 ng/Lfor any result recorded as <0.15 ng/L
DATA FLAG (if
text
Data qualifier codes associated with specific identifications of
appropriate)
voucher samples. These codes provide more information that
those used when reporting receipt of samples. A technician may
use alternative or additional qualifiers if definitions are provided
as part of the submitted data package (e.g., as a separate
worksheet page of the data submission file).
Flag
Definition
ND
Concentration below detection. Unless the sample was
diluted, the concentration will be 0.1 ng/L
HI
Result indicated that a high concentration (i.e., outside
calibration range)
J
Concentration above detection but below reporting limit.
Without dilution, these values are between 0.1 and 0.15
M-g/L
QUAL_FLAG
QCF/Q
QCF
QC failure
Q
Other quality concerns, not identified above
COMMENTS
text
Explanation for data flag(s) (if needed) or other comments.
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7.6.5 Dilutions (if needed)
Dilutions if needed are prepared as follows (using clean glass tubes):
• 1:10 dilution
a. Add 900 piL of distilled water to a clean vial. (Note: Dilutions may also be made using the
kit's diluent rather than distilled water.)
b. Pipette 100 piL from the sample into the vial. (To provide more accurate dilutions and less
chance of contaminating the diluent, the diluent should be added to the vial before the
sample.)
c. Mix by vortexing.
d. Multiply final concentration and Abraxis' detection limit of 0.1 ng/L by 10 to obtain the
sample-specific detection limit of 1.0 ng/L.
• 1:100 dilution
a. Add 3.96 mL of distilled water to a clean, appropriately labeled glass vial. (Note: Dilutions
may also be made using the kit's diluent rather than distilled water.)
b. Vortex the sample to mix thoroughly, then pipette 40 piL from the sample and add to the
water (or diluent) in the appropriate labeled vial. Vortex.
c. Multiply the final concentration and Abraxis' detection limit of 0.1 ng/L by 100 to obtain the
sample-specific detection limit of 10 ng/L.
• Other dilutions can be calculated in the same manner as #1 and #2 if needed.
7.7 Quality Measures
oo
a: This section describes the quality assurance and quality control measures used to ensure that the data
q will meet NWCA requirements.
LU
U
§ 7.7.1 Assistance Visits
O.
>-
Assistance visits are intended to familiarize EPA with actual procedures being implemented by different
< laboratories; and to ensure a clear and consistent understanding of procedures and activities by both
z EPA and the laboratories. If EPA decides to conduct an assistance visit, a qualified EPA scientist or
^ contractor will administer a checklist based upon the steps described in this chapter.
5" 7.7.2 QC Samples
I—
LD
^3 During the course of the survey, the External QC Coordinator will instruct the QC contractor to provide
O
QC
u
§ one or two identical sets of QC samples to all participating laboratories. Each set will contain up to five
QC samples. As determined by the External QC Coordinator, the QC samples may be synthetic; aliquots
^ of additional samples collected at NRSA reference sites; or reference samples obtained from an
>< organization such as the National Institute of Standards. Each laboratory will run the QC samples
h following the same procedures used for the other samples. The QC contractor will compare the results
< and assess patterns in the data (e.g., one laboratory being consistently higher or lower than all others).
o
^ Based upon the evaluation, the External QC Coordinator may request additional information from one
58
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or more laboratories about any deviations from the Method or unique laboratory practices that might
account for differences between the laboratory and others. With this additional information, the
External QC Coordinator will determine an appropriate course of action, including no action, flagging the
data, or excluding some or all of the laboratory's data.
7.7.3 Summary of QA/QC Requirements
Table 7-3 provides a summary of the quality control requirements for procedures described in Section
7.6.
Table 7-3 Microcystin: quality control - sample analysis
Quality Control Description and Requirements Corrective Action
Activity
Kit - Shelf Life
Is within its expiration date listed on kit box.
If kit has expired, then discard or set
aside for training activities.
Kit - Contents
All required contents must be present and in
acceptable condition. This is important because
Abraxis has calibrated the standards and reagents
separately for each kit.
If any bottles are missing or damaged,
discard the kit.
Calibration
All of the following must be met:
o Standard curve must have a correlation
coefficient of >0.99;
o Average absorbance value, Ao, for SO must
be >0.80; and
o Standards S0-S5 must have decreasing
average absorbance values. That is, if Ai is
the average of the absorbance values for
Si, then the absorbance average values
must be: Ao > Ai > A2 > A3 > A4 >As
If any requirement fails:
• Results from the analytical run
are not reported.
• All samples in the analytical run
are reanalyzed until calibtration
provides acceptable results.
Kit Control
The average concentration value of the duplicates
(or triplicate) must be within the range of 0.75 +/-
0.185 ng/L. That is, results must be between 0.565
and 0.935.
If either requirement fails:
• Results from the analytical run
are not reported
• The lab evaluates its processes,
and if appropriate, modifies its
processes to correct possible
contamination or other problems.
• The lab reanalyzes all samples in
the analytical run until the
controls meet the requirements.
Negative Control
The values for the negative control replicates must
meet the following requirements:
0 All concentration values must be < 0.15
Hg/L (i.e., the reporting limit); and
0 One or more concentration results must
be nondetectable (i.e., <0.10 ng/L)
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Quality Control
Activity
Description and Requirements
Corrective Action
Sample
Evaluations
All samples are run in duplicate. Each duplicate pair
must have %CV<15% between its absorbance
values.
If %CV of the absorbances for the
sample>15%, then:
• Record the results for both
duplicates.
• Report the data for both duplicate
results as Quality Control Failure
"QCF"; and
• Re-analyze the sample in a new
analytical run. No samples are to
be run more than twice.
If the second run passes, then the
data analyst will exclude the data
from the first run. If both runs fail, the
data analyst will determine if either
value should be used in the analysis
(e.g., it might be acceptable to use
data if the CV is just slightly over
15%).
ResuIts Within
Calibration Range
All samples are run in duplicate. If both of the
values are less than the upper calibration range
(i.e., 5.0 ng/Lfor undiluted samples), then the
requirement is met.
If one or both duplicates register as
'HIGH/ then the sample must be
diluted and re-run until both results
are within the calibration range. No
samples are to be run more than
twice.
External Quality
Control Sample
External QC Coordinator, supported by QC
contractor, provides 1-2 sets of identical samples
to all laboratories and compares results.
Based upon the evaluation, the
External QC Coordinator may request
additional information from one or
more laboratories about any
deviations from the Method or unique
laboratory practices that might
account for differences between the
laboratory and others. With this
additional information, the External
QC Coordinator will determine an
appropriate course of action,
including no action, flagging the data,
or excluding some or all of the
laboratory's data.
l/l
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QC
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7.8 Sample and Record Retention
The laboratory shall retain:
x
o
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1. The sample materials, including vials, for a minimum of 3 years from the date the EPA publishes
the final report. During this time, the laboratory shall freeze the materials. The laboratory shall
periodically check the sample materials for degradation.
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2. Original records, including laboratory notebooks and the reference library, for a minimum of 10
years from the date that EPA publishes the final report.
After the stated time periods, the laboratory shall follow its internal protocols for disposal.
7.9 References
Abraxis, "Microcystins-ADDA ELISA (Microtiter Plate)," Product 520011, R021412, Undated. Retrieved
January 2014 from
http://www.abraxiskits.com/uploads/products/docfiles/278_Microcystin%20PL%20ADDA%20users%20
R120214.pdf.
Abraxis, "Microcystin-ADDA ELISA Kit, Detailed Procedure," Undated. Retrieved January 2014 from
http://www.abraxiskits.com/uploads/products/docfiles/253_PN520011FLOW.pdf.
James, R., et al., "Environmental Technology Verification Report: Abraxis Microcystin Test Kits: ADDA
ELISA Test Kit; DM ELISA Test Kit; Strip Test Kit," in Environmental Technology Verification System Center
2010. Retrieved March 2013 from http://nepis.epa.gov/Adobe/PDF/P100EL6B.pdf
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8.0 RESEARCH INDICATOR: SOIL ISOTOPES
Soil isotopes laboratory procedures are not included in this manual. EPA's Office of Research and
Development will process and analyze these samples under a cooperative agreement with Michigan
State University and Kenyon College.
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APPENDIX A: CONTACT INFORMATION
Title
Name
Contact Information
EPA HQ NWCA Project
Manager
Gregg Serenbetz, OW
serenbetz.gregg(® eoa.gov
202-566-1253
EPA HQ NWCA Alternate
Project Manager
Chris Faulkner, OW
Faulkner.chris(® eoa.gov
202-566-1185
EPA HQ NARS QA Lead
Sarah Lehmann, OW
lehmann.sarah(® eoa.gov
202-566-1379
EPA HQ Logistics Lead
Colleen Mason, OW
Mason.colleenPeoa.gov
202-343-9641
EPA HQ NWCA Laboratory
Review Coordinator
Kendra Forde, OW
kendra.forde(® eoa.gov
202-564-0417
Information Management
Center Coordinator
Marlys Cappaert, SRA
International Inc.
caooaert.marlysPeoa.gov
541-754-4467
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APPENDIX B: LABORATORY REMOTE EVALUATION FORMS
Contents
NWCA 2016 Document Request Form - Chemistry Laboratories
Laboratory Signature Form - Chemistry Laboratories
NWCA 2016: Vegetation Laboratory Quality Assurance Evaluation
Laboratory Signature Form - Vegetation Laboratory/Herbarium
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NWCA 2016 Document Request Form -
Chemistry Laboratories
The U.S. EPA, states and other partners are planning the second National Wetland Condition
Assessment (NWCA) for 2016. The survey uses a probability-based sampling design to represent
the condition of wetlands across the Continental United States by sampling at approximately
1200 sites. Consistent sampling and analytical procedures ensure that EPA can compare the
results across the country and over time.
As part of the 2016 NWCA, the Quality Assurance Team has been requested to conduct a
technical assessment to verify quality control practices in your laboratory and its ability to
perform chemistry analyses under this project. Our review will be assessing your laboratory's
ability to receive, store, prepare, analyze, and report sample data generated under EPA's 2016
NWCA.
The first step of this assessment process will involve the review of your laboratory's
certification and/or documentation. Subsequent actions may include (if needed): reconciliation
exercises and/or an on-site visit. All labs will need to complete the following forms:
All laboratories will be required to complete the following forms and check the specific
parameter in which your laboratory will be conducting an analysis for the 2016 NWCA:
~ Water Chemistry and Chlorophyll-o (all of the analytes identified in the LOM and QAPP)
~ Microcystin
If your lab has been previously approved within the last 5 years for the water chemistry
indicator:
~ A signature on the attached Laboratory Signature Form indicates that your laboratory
will follow the quality assurance protocols required for chemistry labs conducting
analyses for the 2016 NWCA.
~ A signature on the Quality Assurance Project Plan (QAPP) and the Laboratory Operations
Manual (LOM) Signature Form indicates that you will follow both the QAPP and the
LOM.
If you have not been approved within the last 5 years through the laboratory verification
process for the water chemistry indicator, in order for us to determine your ability to
participate as a laboratory in the NWCA, we are requesting that you submit the following
documents (if available) for review:
~ Documentation of a successful quality assurance audit from a prior National Aquatic
Resource Survey (NARS) that occurred within the last 5 years.
~ Documentation showing participation in a previous NARS for Water Chemistry for the
same parameters/methods.
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Additionally, we request that all labs provide the following information in support of your
capabilities, (these materials are required if neither of the two items above are provided):
~ A copy of your laboratory's accreditations and certifications if applicable (i.e. NELAC,
ISO, state certifications, NABS, etc.).
~ An updated copy of your laboratory's QAPP and Laboratory Quality Assurance Manuals
~ Standard Operating Procedures (SOPs) for your laboratory for each analysis to be
performed (if not covered in 2016 NWCA LOM).
~ Documentation attesting to experience running all analytes for the 2016 NWCA,
including Chlorophyll a.
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Laboratory Signature Form - Chemistry Laboratories
performing the following data analysis and reporting for the 2016 National Wetland
Condition Assessment (NWCA).
1.) Use procedures identified in the 2016 NWCA Laboratory Operations Manual
(or equivalent). If using equivalent procedures, please provide the
procedures and obtain approval from EPA.
2.) Read and abide by the 2016 NWCA Quality Assurance Project Plan (QAPP)
and related Standard Operating Procedures (SOPs).
3.) Have an organized IT tracking system in place for recording sample tracking
and analysis data.
4.) Provide Quality Control (QC) data for internal QC check, on a quarterly basis.
5.) Provide data using the template provided on the NARS Sharefile.
6.) Provide data results in a timely manner. This will vary with the type of
analysis and the number of samples to be processed. Sample data must be
received no later than May 1, 2017 or as otherwise negotiated with EPA.
7.) Participate in a laboratory technical assessment or audit if requested by EPA
NWCA staff (this may be a conference call or on-site audit).
8.) Agree to analyze for all parameters specified in the LOM for the appropriate
indicator(s) identified above, including Chlorophyll-o for Water Chemistry.
located in
certify that the laboratory,
, will abide by the following standards in
This applies to the
chemistry indicator(s).
Signature
Date
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National Wetland Condition Assessment 2016: Vegetation
Laboratory Quality Assurance Evaluation
The National Wetland Condition Assessment (NWCA) is designed to provide statistically valid regional
and national estimates of the condition of wetlands in the 48 conterminous states of the U.S. Plant
samples collected in the field are sent to a designated laboratory/herbarium for identification using
standard laboratory protocols outlined in the NWCA 2016 Laboratory Operations Manual (LOM).
As specified in the NWCA Quality Assurance Project Plan (QAPP), a NWCA Evaluator will evaluate each
laboratory/herbarium to ensure the NWCA data quality objectives are satisfied. Each
laboratory/herbarium must participate in an evaluation and sign the laboratory signature form and
acknowledgement and commitment to implement page of the QAPP to satisfy the terms of the NWCA
QAPP.
It is essential that each laboratory/herbarium accurately implement standardized protocols for
vegetation identification and storage to ensure comparability of data among NWCA sites and minimize
data loss that could result from damaged or degraded specimens, errors in data recording, sample
processing, data storage, plant identification, or misinterpretation of guidance for laboratory operations.
These quality assurance evaluations are designed to:
1. Confirm the 2016 NWCA Laboratory Operations Manual (LOM) protocols are implemented
as intended.
2. Assist with questions the laboratory/herbarium may have.
3. Suggest corrections if any errors have been made by a laboratory/herbarium in
implementing methods described in the LOM.
i/i
This evaluation will include a discussion of the attached checklist between the NWCA Evaluator and the g
laboratory/herbarium over the phone rather than an actual laboratory visit. The checklist includes ^
descriptions of sample handling and other requirements to which each laboratory/herbarium must O
comply. The discussions will be scheduled with Chris Faulkner (EPA HQ NWCA Project Manager-
LU
Background: For all NWCA field work, whenever the identity of a species cannot be confirmed in the
field, a sample is collected for later identification in the office by the field botanist/ecologist or by °
another botanist at a designated laboratory/herbarium. All unknown species located in one of five ^
Vegetation Plots arrayed across a site's Assessment Area that are mature and have key structures ^
needed for identification are collected (unknown species voucher). Unknown species that are immature O
or senescent comprising more than 5% cover are also collected. The field botanist/ecologist will ship ^
unknown samples they cannot identify to the botanist (also called plant ID specialist or taxonomist in §
NWCA) at the laboratory/herbarium for initial identification. fj
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In addition to all unknown specimens, field crews collect five known plant voucher samples (randomly ><
selected from species identified by the Vegetation Team) for quality assurance (NWCA 2016 QAPP). §
These QA vouchers are sent to a QA "verifying botanist" for re-identification/verification. Collecting £
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voucher specimens of known species both provides a quality assurance check on species identity data,
and a permanent record of the occurrence of a particular species at a given location.
The QA verifying botanist is responsible for re-identification/verification of the QA vouchers as well as a
random selection of 10% of the unknown specimens that were initially determined by the "identifying
botanist" at the laboratory/herbarium.
If the unknown species specimens and QA voucher samples are planned to be sent to the same
institution, it is important that all quality assurance activities be completed by a taxonomist that did not
participate in the identification of unknown specimens. .
All laboratory methods and quality assurance requirements are fully described in the NWCA 2016 LOM
and QAPP.
For the purposes of the Vegetation Laboratory Quality Assurance Evaluations, the Vegetation Checklist
will focus on the lab's competence to receive and properly store specimens and to track and manage the
vegetation data.
Definitions:
Voucher Sample - A pressed and dried plant sample, ideally comprised of leaves, stems, flowers, fruits
and roots. An integral component of each voucher sample is written data describing the location, date
of collection, habitat, plant habit, characteristic features and other information. Vouchers provide
physical evidence that confirms the presence of plant species at specific locations.
Identifying Botanist - The person identifying and processing unknown samples. This could be a field
botanist/ecologist; university, state, national or regional herbarium botanist; or an EPA contractor that
has qualifying credentials in plant taxonomy. The identifying botanist is responsible for ensuring all
plant identification and processing tasks outlined in the LOM are completed. In some cases this may
require the identifying botanist to identify partners to assist with the work.
QA Verifying Botanist-The person re-identifying and verifying QA voucher identifications and a 10%
subset of unknown species identifications by the laboratory/herbarium. This could be a botanist,
ecologist, taxonomist, and/or plant ID specialist that is an expert in the identification of wetland plants.
The verifying botanist agrees to use the NWCA prescribed methods, as described in chapter 4 of the
LOM, to ensure that all QA vouchers are correctly verified.
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VEGETATION LABORATORY QUALITY EVALUATIONS
NWCA 2016 VEGETATION LABORATORY ASSISTANCE CHECKLIST
Lab ID: Individuals Performing IDs:
Individuals on Conference Call:
Vegetation Lab Evaluator:
Date:
Instructions:
1. All vegetation laboratories should be adhering to methods and QA requirements described in the
NWCA 2016 Laboratory Operations Manual (LOM) and the NWCA Quality Assurance Project Plan
(QAPP).
2. The Vegetation Laboratory Evaluator will discuss the following subjects with the vegetation
laboratories/herbarium and fill in the data bubble that reflects the results of their observation.
3. Yes = the task is being correctly completed, No = the task is not being completed as described, or
Not Applicable = the task was not needed at this lab.
Supplies and Equipment for Sample Handling
Does the herbarium have the following supplies and equipment?
• Plant dryer
Y
N
N/A
• Dissecting microscope
Y
N
N/A
• Storage cabinet or sealable plastic boxes for storing dried plant
samples prior to identification
Y
N
N/A
• Regional floras and plant lists
Indicate all floras used by lab:
Y
N
N/A
• Access to USDA PLANTS taxonomic standard
(http://plants.usda.gov/java/)
Y
N
N/A
• Plant sample folders
Y
N
N/A
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• OPTIONAL: Freezer for freeze treating plant specimens to kill
pests.
If "N", indicate lab method for killing pests on
specimens:
Y
N
N/A
• OPTIONAL: Mounting materials (herbarium sheets, mounting
glue, forceps, weights for holding samples with wet glue to the
herb Receiving Voucher Samples)
Y
N
N/A
Notes:
Processing and Managing Plant Samples
Plant samples may arrive at the Herbarium as: 1) dried, pressed samples, or 2) pressed but still wet
plant material enclosed in a plant press.
Drying Samples:
• If samples arrive in a press, but still wet, are the samples placed
on a plant dryer to complete drying, and then be treated for
pests?
Y
N
N/A
• Does the lab place the full presses on an electric plant dryer that
provides steady bottom heat (95°F to 113°F), for plants to dry in
12 to 48 hours?
Y
N
N/A
• If a plant dryer is not available does the lab place the presses in a
warm dry place to allow drying? Does the lab provide a place for
low ambient humidity and good airflow around and through the
presses to ensure rapid and thorough drying of plant material?
Y
N
N/A
• If plants arrive in a press, does the lab periodically tighten the
straps on the press to maintain pressure on the samples and
minimize shrinkage and wrinkling until plant identification?
Y
N
N/A
Treating Samples for Detritivores, Molds, and Pests: Dried plant material is highly susceptible to
contamination by detritivores, molds, and pests that can destroy herbaria collections; therefore, it is
important to treat all incoming samples to kill potential contaminants.
• Does the lab have standard treatment procedures for pests and
are those procedures being implemented for the NWCA
specimens?
Y
N
N/A
• After the samples are pressed and dried, does the lab treat
samples for detritivores, molds, and pests?
Y
N
N/A
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• If freezing, is the lab freezing the samples at (-20°C or below) for
at least three days for loosely stacked samples and seven days
for tightly packed samples?
Y
N
N/A
Storing Samples:
• Are plant samples being stored in herbarium cabinets or sealable
plastic container when not in use?
Y
N
N/A
• Does the lab ensure that samples are not left out in the
herbarium room overnight? If samples are found that have been
left out overnight or if a cabinet/plastic container has been left
open, does the lab decontaminate all samples again?
Y
N
N/A
Notes:
Tracking Specimens - Tracking Form T-2 and T-3, Plant Sample Specimen Label
Tracking Forms: In the field, each voucher sample collected is assigned a set of tracking information,
which is recorded on the Plant Sample Tracking Forms (Form T-2: NWCA 2016 Unknown Plant Sample
Tracking and T-3: NWCA 2016 QA Plant Sample Tracking). It is important that every specimen sent to
and received by the lab is tracked following the protocols described in the appropriate section of the
LOM Vegetation Chapter.
• Does the lab review all the Tracking Forms to ensure that all
samples listed are received by the lab?
Y
N
N/A
• If a sample listed on the tracking form is not part of the shipment
received, does the lab contact the Information Management
Coordinator (541-754-4663) as soon as possible?
Y
N
N/A
Plant Specimen Label: Every sample will arrive at the Herbarium with a Plant Specimen Label. This
label includes diagnostic information for known and unknown species collected.
• Does the lab review the information provided on the Plant
Specimen Label included with the sample?
o Plant Sample ID Number: NWCA Site Number-Plant
collection number
o Sampling Date
o Visit Number
o County and State of Site
o Scientific name for QA Voucher Specimen
o Pseudonym for Unknown Species
o Collector(s) name(s)
o Abundance of Plant
o Habitat
o Growth Habit
Y
N
N/A
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Notes:
Identification of Vegetation Samples
Names for all NWCA plants specimens identified (unknowns) or verified (QA vouchers) need to be
reconciled to the standard found in USDA Plants (http://plants.usda.aov/).
• Is the Lab reconciling names for all species that they identify to
the standard found in USDA Plants?
Y
N
N/A
• Is the lab a Heritage program or coordinating with a Heritage
program or university herbarium?
Y
N
N/A
• Is the lab using a reference herbarium and is it the state's
reference herbarium?
Y
N
N/A
• How many unknown plant samples does the lab identify in a
year?
# of samples:
Notes:
Mounting and Storing Herbarium Sheets
Once the samples are dried, pressed, and identifiedthey are to be stored at the Herbarium for at least
five years.
• Does the lab have the storage capabilities and facilities to
properly store dried, pressed, and identified samples for at least
five years?
Y
N
N/A
• Will vouchers be kept in sealable plastic containers in a cool dry
climate and will they be accessible to the EPA?
Y
N
N/A
• Will the lab incorporate the NWCA vouchers into their
permanent collections?
Y
N
N/A
• Will vouchers from the national survey be mounted on
herbarium sheets and labeled to indicate that they were
collected as part of the NWCA?
Y
N
N/A
Notes:
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Sending Resultant Data Forms
Data must be reported to EPA electronically using the 2016_NWCA Plant ID_Lab Spreadsheets.xlsx
data template. The template includes separate spreadsheets for recording names of identified
unknowns and reconciling to PLANTS nomenclature and for recording re-identification and verification
ofQA voucher specimens.
• The lab is aware of these reporting requirements and is sending
in the resultant data per the instructions in 2016_NWCA Plant
ID_Lab Spreadsheets.xlsx data templates.
N
N/A
• The species identifications are regularly entered into the
appropriate spreadsheets, and these forms are transmitted at
appropriate intervals to the EPA Project Management Team.
N
N/A
Notes:
Quality Assurance/Quality Control
A subset of plant samples collected as unknowns and later identified by the lab will need to be verified
by a verifying botanist for additional quality assurance. The lab will randomly select 10% of the
identified unknown samples to be sent to the verifying botanist, another experienced botanist,
taxonomist, and/or plant ID specialist who did not participate in the original identifications. A chain-
of-custody form (Tracking Form T-3) needs to be completed and sent with the specimens. Additionally,
five randomly selected species (from each AA) of known identity will be reassessed by an independent
botanist/taxonomist. (QAPP section 5.1.5).
• For QA, if the field botanist/ecologist is acting as the
laboratory/herbarium, does the lab ensure that another qualified
botanist, or a state or EPA identified laboratory/herbarium is
used for QA?
N
N/A
• Does the lab ensure that the person who made the first
identification of the unknown sample is not the same person
making the second identification of the sample (i.e., ten percent
of all "unknown species")?
N
N/A
• Does the lab record all identifications in the 2016 NWCA Plant ID
Lab Spreadsheet for each sample, and email to the EPA Project
Management Team?
N
N/A
Notes:
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Laboratory Signature Form-Vegetation Laboratory/Herbarium
performing the following data analysis and reporting for the 2016 National Wetland Condition
Assessment (NWCA).
1.) Use procedures identified in the 2016 NWCA Laboratory Operations Manual (or
equivalent). If using equivalent procedures, please provide the procedures and
obtain approval from EPA.
2.) Read and abide by the 2016 NWCA Quality Assurance Project Plan (QAPP) and any
related Standard Operating Procedures (SOPs).
3.) Have an organized IT tracking system in place for recording sample tracking and
analysis data.
4.) Notify EPA Project Management Team of any substantial differences in taxonomic
identifications between the identifying botanist(s) and the verifying botanist(s).
5.) Provide data using the template provided on the NARS Sharefile.
6.) Provide data results in a timely manner. This will vary with the type of analysis and
the number of samples to be processed. Sample data must be received no later
than May 1, 2017 or as otherwise negotiated with EPA.
7.) Participate in a laboratory technical assessment by EPA NWCA staff (this may be a
conference call or on-site audit).
located in
certify that the laboratory/herbarium,
, will abide by the following standards in
This applies to the
vegetation indicator.
Signature
Date
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.-...v-'v. . "-iplates
Electronic reporting templates will be provided on EPA's NARS Sharepoint site.
2016_NWCA Plant ID_Lab Spreadsheets.xlsx
2016_NWCA Water Chem-CHLA_Lab Spreadsheet.xlsx
2016_NWCA Microcystin Lab Spreadsheet.xlsx
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APPENDIX D: SUPPLEMENTARY MATERIAL FOR VEGETATION - LISTS OF
FLORISTIC RESOURCES
Recording Citations for Floristic Resources
The nomenclatural standard for the NWCA is the PLANTS Database (USDA, NRCS 2016,
http://plants.usda.gov/). Ideally, plant species names based on PLANTS nomenclature would be
recorded during data collection. However, for plant identification in the field or lab it is often necessary
to use local or regional floras and field guides, which may represent different nomenclature.
Partial List of Regional, State, and Local Floras and Field Guides
A list of floras and field guides is provided below. This table does not represent a complete listing of
floras and field guides available for the conterminous US, but is largely based on the list of floras and
field guides that the Botanist/Ecologists on the Field Crews selected and reported using in the NWCA
2011. In addition, several other floristic resources are included. Additonal printed and online floras, not
included in this list, are likely to also be useful (particularly those recently published).
Resources are alphabetized by author. The states in which they were used in the NWCA are listed. Use
of a floristic reference in a particular state does not necessarily mean that its utility is limited to that
state. Often floras may have regional applicability. To help consider regional utility, the EPA Regions
that include the states where the floras and field guides were used in 2011 are also listed. However, it is
important to note that EPA Regions do not necessarliy represent ecological boundaries and may exceed
the area to which a flora applies, or conversely may not include adjacent area that may be covered by a
particular flora.
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Potentially Useful Floras and Field Guides
EPA Region(s)
Where Potentially
Applicable
Used by States
in NWCA 2011
Allen, C. M., D. A. Newman, and H. W inters. 2004. Grasses of
Louisiana. 3rd ed. Allen's Native Adventures, Pitkin, LA
Region 6
LA, MS
Allen, Charles M., Dawn Allen Newman, and Harry H. Winters. 2002.
Trees, shrubs, and woody vines of Louisiana. Allen's Native Ventures,
LLC, Pitkin, LA.
Region 6
LA, MS
Allred, Kelly. 2005. A Field Guide to the Grasses of New Mexico. Third
Edition. New Mexico State University. Las Cruces, New Mexico.
Region 6
NM
Allred, K.W. and R.D. Ivey. DRAFT. Flora Neomexicana. Volume III: An
illustrated identification guide to the vascular plants of New Mexico.
New Mexico State University.
Region 6
NM
Allred, K.W. and R.D. Ivey. 2010. Flora Neomexicana (DRAFT).
Published by the authors.
Region 9
AZ
Anderton, L.K., and M.E. Barkworth. 2009. Grasses of the
Intermountain Region. Intermountain Herbarium, Utah State
University, Logan, UT 84322
Region 9
NV
Barkely, T.M. 2006. Senecio. In: Flora of North America Editorial
Committee, eds. 1993+.Flora of North America North of Mexico. 16+
vols. New York and Oxford. Vol. 20: Magnoliophyta: Asteridae:
Asteraceae, part 2. Accessed via www.efloras.org. Summer 2011.
All
ID
Barkley, T.M., L. Brouillet, J.L Strother. 2006. Asteraceae. In: Flora of
North America. Editorial Committee, eds. 1993+. Flora of North
America North of Mexico. 16+ vols. New York and Oxford. Vols. 19, 20,
and 21: Magnoilophyta: Asteridae: Asteraceae, part 1, part 2, part 3.
Accessed via www.efloras.org. Summer 2011.
All
ID
Barneby, R.C. 1989. Intermountain Flora: Vascular Plants of the
Intermountain West, U.S.A. Volume 3, Part B: Fabales. New York
Botanical Garden Press, New York, 292 pp.
Regions 8, 9,10
ID
Beidleman, L.H., R.G. Beidleman, and B.E. Willard. 2000. Plants of
Rocky Mountain National Park. Falcon Press, Helena, Montana, 266
pp.
Region 9
AZ
Beidleman, LH. and E. Kozloff. 2003. Plants of the San Francisco Bay
Region. University of California Press, Berkeley, California, 514 pp.
Region 9
CA
Bell, C.R. and B.J. Taylor. 1982. Florida wild flowers and roadside
plants. Laurel Hill Press, Chapel Hill, North Carolina, 308 pp.
Region 4
FL
Belliston, N.D., J. Merritt, R. Whitesides, and S.A. Dewey. 2004.
Noxious Weed Guide for Utah. Utah State University Extension. 50 pp.
Region 8
UT
Black, M.R. and E.J. Judziewiez. 2009. Wildflowers of Wisconsin and
the Great Lakes Region: A Comprehensive Field Guide. Second
Edition. The University of Wisconsin Press. Madison, Wisconsin. Til
pp.
Region 5
Wl
Braun, LE. 1967. The Vascular Flora of Ohio. Part 1: The
Monocotyledoneae: Cat-tails to Orchids. The Ohio State University
Press. Columbus, Ohio, 464 pp.
Region 5
OH
Brown, C. L, and K. Kirkman. 1990. Trees of Georgia and Adjacent
States. Timber Press. Portland, Oregon.
Region 4
FL
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EPA Region(s)
Where Potentially
Applicable
Used by States
in NWCA 2011
Brunsfeld, S. J., and F.D. Johnson. 1985. Field guide to the willows of
east-central Idaho. Bulletin Number 39, Forest, Wildlife, and Range
Experiment Station, University of Idaho, Moscow, Idaho.
Region 10
ID, MT
Center for Aquatic and Invasive Plants, University of Florida, IFAS
website address: http://plants.ifas.ufl.edu/
FL
Chadde, S.W. 2002. A Great Lakes Wetland Flora. Second Edition.
Pocketflora Press. Laurium, Michigan, 648pp.
Region 5
IL, IN, Ml, OH,
Wl
Clements, Steven. 1992. Chenopodiaceae and Amaranthacae of New
York State. Bulletin No. 485. New York State Museum. Albany, New
York.
Region 2
NJ
Clewell, A.F. 1985. Guide to the Vascular Plants of the Florida
Panhandle. Florida State University Press.
Region 4
FL
Colvin, et al. 2004. Weeds of Southern Turfgrass. University of Florida,
IFAS Extension.
Region 4
FL
Cooke, S.S. 1997. A Field Guide to the Common Wetland Plants of
Western Washington & Northwestern Oregon. Seattle Audubon
Society 403 pp.
Region 10
WA
Cooperrider, T. S. 1995. The Dicotyledonae of Ohio Part 2: Linaceae
through Campanulaceae. The Ohio State University Press, Columbus,
Ohio.
Regions 3 and 5
OH
Cope, E.A. 2001. Muenscher's Keys to Woody Plants: An Expanded
Guide to Native and Cultivated Species. Cornell University Press.
Ithaca, New York, 321 pp.
Region 1
NH
Cronquist A. and others. 1977+. Intermountain Flora. Columbia
University Press or New York Botanical Garden Press, New York.
Regions 8, 9,10
AZ, NV, UT
Cronquist, A. 1994. Intermoutain Flora: Vascular Plants of the
Intermountain West, U.S.A. Volume 5: Asterales. The New York
Botanical Garden Press, New York, 506 pp.
Regions 8, 9,10
ID
Cronquist, A. N.H. Holmgren, and P. K. Holmgren. 1997. Intermoutain
Flora: Vascular Plants of the Intermountain West, U.S.A. Volume 3,
Part A: Subclass Rosidae (except Fabales). New York Botanical
Garden Press, New York, 456 pp.
Regions 8, 9,10
ID
Crow, G.E., C.B. Hellquist, and N.C. Fasset. 2006. Aquatic and Wetland
plants of Northeastern North America. Vol. 1. Pteridophytes,
Gymnosperms, and Angiosperms Dicotyledons. The University of
Wisconsin Press, 448 pp.
Regions 1, 2, 3, 6
MN
Crow, G.E. and C.B. Hellquist. 2000. Aquatic and Wetland plants of
Northeastern North America. Vol. 2. Angiosperms: Monocotyledons.
The University of Wisconsin Press, 464 pp.
Regions 1, 2, and 3
NH
Culver, D.R. and J.M. Lemly. 2013. Field Guide to Colorado's Wetland
Plants: Identification, Ecology, and Conservation. Colorado Natural
Heritage Program, Colorado State University, Fort Collins, CO
CO
DiTomaso, J.M. and E.A. Healy. 2006. Weeds of California and Other
Western States. University of California Division of Agriculture and
Natural Resources, Publication 3488.
Region 9
CA
Dorn, R.D. and J.L Dorn. 1984. Vascular Plants of Montana. Mountain
West Publishing, Cheyenne, Wyoming, 276 pp.
Region 8
MT, WY
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Potentially Useful Floras and Field Guides
EPA Region(s)
Where Potentially
Applicable
Used by States
in NWCA 2011
Dorn, R.D. 2001. Vascular Plants of Wyoming. Third Edition. Mountain
West Publishing. Cheyenne, Wyoming.
Region 8
UT, WY
Duncan, W. H., and M. B. Duncan. 1988. Trees of the Southeastern
United States. University of Georgia Press. Athens, Georgia.
Region 4
FL
Duncan, W.H. and LE. Foote. 1999. Wildflowers of the southeastern
United States. University of Georgia Press, Athens, Georgia.
Region 4
GA
Eilers, L.J. and D.M. Roosa. 1994. The Vascular Plants of Iowa.
University of Iowa Press, Iowa City, Iowa, 319 pp.
Region 7
IA
Farnsworth, A., B. Cobb, and C. Lowe. 2005. Peterson Field Guide to
Ferns, Second Edition: Northeastern and Central North America.
Houghton Mifflin Harcourt. 440 pp.
Region 5
Wl
Fassett, N. C. 1957. A Manual of Aquatic Plants. Madison: The
University of Wisconsin Press. Madison, Wisconsin.
NE, PA
Fleenor, S.B, and S.W. Tabor. 2009. Plants of Central Texas Wetlands.
Texas Tech University Press. Lubbock, Texas, 275 pp.
Region 6
TX
Flora of North America Editorial Committee, eds. 1993+. Flora of
North America North of Mexico. 16+ vols. New York and Oxford.
All
AZ, MA, Ml,
MN, NC, NH,
NM, NV, Rl, SD,
Wl, WV
Flora of North America Editorial Committee, eds,1993+. Flora of North
America North of Mexico. Vol. 3: Magnoliidae and Hamamelidae. New
York and Oxford.
All
ID
Flora of North America. Editorial Committee, eds. 1993+. Flora of
North America North of Mexico. 16+ vols. New York and Oxford.
Brooks, R.E. and S.E. Clemants. 2000. Juncaeae In: Vol. 22:
Magnoliophyta: Alismatidae, Arecidae, Commelinidae (in part), and
Zingiberidae. Accessed via www.efloras.org. Summer 2011.
All
ID
Flora of North America Editorial Committee, eds. 1993+.Flora of North
America North of Mexico.16+ vols. New York and Oxford. Wolf, S.J.
2006 Arnica. In: Vol. 21: Mgnoiophyta: Asteridae: Asteraceae, part 3.
Accessed via www.efloras.org. Summer 2011.
All
ID
Flora of North America. Editorial Committee, eds. 1993+. Flora of
North America North of Mexico. 16+ vols. New York and Oxford.
Brouillet, L, J.C. Semple, G.A. Allen, K.L. Chambers, S.D. Sundberg.
2006. Symphyotrichum. In: Vol. 20: Mgnoiophyta: Asteridae:
Asteraceae, part 2. Accessed via www.efloras.org. Summer 2011.
All
ID
Flora of North America Editorial Committee, eds. 1993+. Flora of
North America North of Mexico. 16+ vols. New York and Oxford.
Morin, N.R. 2009. Ribes. In: Vol. 8: Magnoliophyta: Paeoniaceae to
Ericaceae. Accessed via www.efloras.org. Summer 2011.
All
ID
Flora of North America Editorial Committee, eds. 1993+. Flora of
North America North of Mexico. 16+ vols. New York and Oxford.
Romero-Gonzalez, G.A., G.C. Fernadez-Concha, R.L Dressier, L.K.
Magrath, and G.W. Argus. 2002. Orchidaceae. In: Vol. 26:
Magnoliophyta: Liliidae: Liliales and Orchdiales. Accessed via
www.efloras.org. Summer 2011.
All
ID
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Potentially Useful Floras and Field Guides
EPA Region(s)
Where Potentially
Applicable
Used by States
in NWCA 2011
Flora of North America Editorial Committee, eds. 1993+.Flora of North
America North of Mexico. 16+ vols. New York and Oxford. Landolt, E.
2000. Lemnaceae. In Vol. 22: Magnoliophyta: Alismatidae, Arecidae,
Commelinidae (in part), and Zingiberidae.
All
MA
Flora of North America Editorial Committee, eds. 1993+. Flora of
North America North of Mexico. 16+ vols. New York and Oxford.
Wiersema, J.H. and B. Hellquist. 1997. Nymphaceae. In: Vol. 3:
Magnoliophyta: Magnoliidae and Hmamelidae.
All
MA
Flora of North America Editorial Committee, eds. 1993+. Flora of North
America North of Mexico. 16+ vols. New York and Oxford. Vol. 7:
Magnoliophyta: Salicaceae to Brassicaceae.
All
Ml, MN
Flora of North America Editorial Committee, eds. 1993+. Flora of North
America North of Mexico. 16+ vols. New York and Oxford. Vol. 22:
Magnoliophyta: Alismatidae, Arecidae, Commelinidae (in part), and
Zingiberidae, 2000.
All
WV
Flora of North America Editorial Committee, eds. 1993+. Flora of
North America North of Mexico. 16+ vols. New York and Oxford. Vol.
23: Magnoliophyta: Commelinidae (in part): Cyperaceae. 2002.
All
WV
Flora of North America Editorial Committee, eds. 1993+. Flora of
North America North of Mexico. 16+ vols. New York and Oxford. Vol.
24: Magnoliophyta: Commelinidae (in part): Poaceae (part 1), 2007.
All
WV
Flora of North America Editorial Committee, eds. 1993+. Flora of
North America North of Mexico. 16+ vols. New York and Oxford. Vol.
25: Magnoliophyta: Commelinidae (in part): Poaceae (part 2), 2003.
All
WV
Foote, L.E. and S.B. Jones, Jr. 2005. Native shrubs and woody vines of
the south-east. Timber Press, Portland, Oregon.
All
GA
Gleason and Cronquist. 1963. Manual of the Vascular Plants of
Northeastern United States and Adjacent Canada. Van Nostrand,
Princeton NJ. 910 pp.
Regions 1, 2, 3, 4
and 5
MD, Ml, MN,
ND?, PA, Wl
Gleason, Henry A. and Arthur Cronquist. 1991. Manual of Vascular
Plants of the NE U.S. and Adjacent Canada. Second Edition. New York
Botanical Garden. Bronx, NY.
Regions 1, 2, 3, 4
and 5
CT, IL, IN, GA?
KY?, MA, ME,
NH, NY, OH, PA,
Rl, TN, VA, Wl,
WV
Godfrey, R.K. 1988. Trees, shrubs, and Woody vines of Northern
Florida and adjacent Georgia and Alabama. University of Georgia
Press, Athens, Georgia.
Region 4
FL NC, VA,
Godfrey, R. K. and J. W. Wooten. 1981. Aquatic and wetland plants of
the southeastern United States: dicotyledons. University of Georgia
Press, Athens. 933 pp
Regions 4 and 6
AL, AR, GA, LA,
MS, NC, TX, VA
Godfrey, R. K. and J. W. Wooten. 1979. Aquatic and wetland plants of
the southeastern United States: monocotyledons. University of
Georgia Press, Athens. 712 pp.
Regions 4 and 6
AL, AR, FL, GA,
LA, MS, NC, SC,
TX, VA
Great Plains Flora Association. 1977. Atlas of the Flora of the Great
Plains. Iowa State Press. Ames, IA.
Regions 7 and 8
KS, ND, SD
Great Plains Floras Association. 1986. The Flora of the Great Plains.
Coordinator, R.L McGregor. Editor, T.M. Barkely. University Press of
Kansas. Lawrence, Kansas, 1402 pp.
Regions 7 and 8
IA, KS, MO, ND,
NE, OK, SD
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Potentially Useful Floras and Field Guides
EPA Region(s)
Where Potentially
Applicable
Used by States
in NWCA 2011
Haines, A. and T.F. Vining. 1998. Flora of Maine. V.F. Thomas
Company, Bar Harbor, Maine. 847 pp.
Region 1
ME
Haines. 2011. Flora Novae Angliae. New England Wildflower Society.
(Due for publication in September 2011).
Region 1
ME
Hatch, S.L, Schuster, J.L, and D.L. Drawe, 2003. Grasses of the Texas
Gulf Prairies And Marshes, Texas A&M University Press, College
Station, Texas. 355 pp.
Region 6
TX
Hickman, James C., Editor. 1993. The Jepson Manual of Higher Plants
of California. University of California Press, Berkeley, California. Third
printing with corrections 1996.
Region 9
CA
Hipp, A.L. 2008 Field Guide to Wisconsin Sedges: An Introduction to
the Genus Carex (Cyperaceae). The University of Wisconsin Press. 280
pp.
Region 5
Wl
Hitchcock, A.S. 1971. Manual of the grasses of the United States.
Volume 1. Dover Publications, New York.
All
ID
Hitchcock, C.L. and A. Cronquist. 1973. Flora of the Pacific Northwest:
An Illustrated Manual. University of Washington Press, Seattle,
Washington, 730 pp.
Regions 8 and 10
ID, MT, OR,WA
Hitchcock, C.L., A. Cronquist, and M. Ownbey. 1969. Vascular Plants of
the Pacific Northwest. Part 1: Vascular Cryptogams, Gymnosperms,
and Monocotyledons. University of Washington Press, Seattle,
Washington, 914 pp.
Region 10
ID
Hitchcock, C.L, A. Cronquist, M. Ownbey, and J.W. Thompson. 1971.
Vascular Plants of the Pacific Northwest. Part 3: Saxifragaceae to
Ericaceae. University of Washington Press, Seattle, Washington, 614
pp.
Region 10
ID
Hitchcock, C.L, A. Cronquist, M. Ownbey, and J.W. Thompson. 1959.
Vascular Plants of the Pacific Northwest. Part 4: Ericaceae to
Campanulaceae. University of Washington Press, Seattle,
Washington, 510 pp.
Region 10
ID
Hitchcock, C.L, A. Cronquist, M. Ownbey, and J.W. Thompson. 1955.
Vascular Plants of the Pacific Northwest. Part 5: Compositae.
University of Washington Press, Seattle, Washington, 343 pp.
Region 10
ID
Holmgren, Noel H. et al. 1998. Illustrated Companion to Gleason and
Cronquist's Manual. NY Botanical Garden. Bronx, NY.
Regions 1, 2, 3, and
5
Ml, NH, OH, PA
Hurd, E.G., N.L. Shaw, J. Matrogiuseppe, LC. Smithman, and Sherel
Goodrich. 1998. Field Guide to Intermountain Sedges. General
Technical Report. RMRS-GTR-10. U.S. Department of Agriculture, U.S.
Forest Service, Rocky Mountain Research Station, 282 pp.
Regions 8, 9, and 10
CA, ID, MT, NV,
UT
Hurd, E.G., S. Goodrich, and N.L Shaw. 1997 - Revised. Field guide to
Intermountain Rushes. General Technical Report INT-306. U.S.
Department of Agriculture, Forest Service, Intermountain Research
Station. Ogden, Utah.
Regions 8, 9, and 10
MT
Ivey, R.D. 2003. Flowering Plants of New Mexico. 4th ed. Published by
the author.
Region 6 and 8
NM
The Jepson Online Interchange: California Floristics. University of
California, Berkley, http://ucjeps.berkeley.edu/interchange/.
Accessed 2011
Region 9
CA
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Potentially Useful Floras and Field Guides
EPA Region(s)
Where Potentially
Applicable
Used by States
in NWCA 2011
Jones, R.L 2005. Plant Life of Kentucky, An Illustrated Guide to the
Vascular Flora. University Press of Kentucky. Lexington, Kentucky.
Region 4
KY
Kartesz, J. T. 1988. A Flora of Nevada. Doctoral dissertation, University
of Nevada, Reno. ~vii + 1729 pgs
Region 9
NV
Kershner, B., C. Tufts, G. Nelson, D. Mathews, R. Spellenberg, and T.
Purinton. 2008. National Wildlife Federation Field Guide to Trees of
North America, Sterling Press. 528 pages
All
MS, NC, VA
Kershner, Mathews, Nelson, and Spellenberg. 2008. National Wildlife
Federation Field Guide to Trees of North America, Chanticleer Press,
Inc. p. 229
All
Al, AR, FL, GA,
LA
Larson, Gary E. 1993. Aquatic and wetland vascular plants of the
northern Great Plains. Gen. Tech. Rep. RM-238. U.S. Department of
Agriculture, Forest Service, Rocky Mountain Forest and Range
Experiment Station. Fort Collins, Colorado, 681pp.
Region 8
ND, SD
Lavin, M. and C. Seibert. 2011. Grasses of Montana. Montana State
Univestiy Herbarium.
http://www.montana.edu/mlavin/herb/mtgrass.pdf
Region 8
MT
Lazarine, P. 1981. Common Wetland Plants of Southeast Texas. U.S.
Army Corps of Engineers. Galveston District. Galveston, Texas. 154
pp.
Region 6
TX
Lehman, R.L, R. O'brien and T. White. 2005. Plants of The Texas
Coastal Bend. Texas A and M University Press, College Station, Texas,
352 Pp.
Region 6
TX
Leppig, G. and A.J. Pickart (compiled by). 2005 A Photographic Guide
to Plants of Humboldt Bay Dunes and Wetlands. Sponsored by U.S.
Fish and Wildlife Service, California Department of Fish and Game,
Friends of the Dunes, and National Fish and Wildlife Foundation.
http://www.fws.gov/HUMBOLDTBAY/PLANTGUIDE/. Accessed 2011.
Region 9
CA
Lesica, P. and P. Husby. 2006. Field Guide to Montana's Wetland
Vascular Plants: A non-technical key to the genera with keys to the
species of sedges and rushes. 2nd printing. Montana Wetlands Trust.
96 pp.
Region 8
MT
Lesica. 2011. Draft Flora of Montana. Unpublished.
Region 8
MT
Martin, W.C. and C.R. Hutchins. 1980,1981. A Flora of New Mexico.
Vols. 1,2. J. Cramer.Vaduz, Germany,
Region 8
NM
McDougall, W.B. 1973. Seed Plants of Northern Arizona. Museum of
Northern Arizona, Flagstaff, Arizona, 594 pp.
Region 9
AZ
Milburn, S. A., M. Bourdaghs, and J. J. Husveth. Floristic Quality
Assessment for Minnesota Wetlands. Minnesota Pollution Control
Agency, St. Paul, Minn.
www. pea.state, mn.us/water/biomonitoring/bio-wetlands. html
Region 5
MN
Missouriplants.com: Photographs and descriptions of the flowering
and non-flowering plants of Missouri, USA.
http://www.missouriplants.com/
Region 7
MS
Mississippi Trees (2011) Published by The Mississippi Forestry
Commission. 337pp.
Regions 4 and 6
MS
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Potentially Useful Floras and Field Guides
EPA Region(s)
Where Potentially
Applicable
Used by States
in NWCA 2011
Mittelhauser, G.H., LL Gregory, S.C. Rooney, J.E. Weber. 2010. The
Plants of Acadia National Park. University of Maine Press. Orono,
Maine, 594 pp.
Region 1
ME
Mohlenbrock, R. 2002. Vascular Flora of Illinois. Southern Illinois
University Press, Carbondale & Edwardsville, Illinois.
Region 5
IL, IN
Mohlenbrock, R .H. 1986. Guide To The Vascular Flora Of Illinois.
Southern Illinois University Press, Carbondale and Edwardsville,
Illinois. 507 pp.
Region 5
IL
Moore, D.M. and J.E. Grant. 2007. Trees of Arkansas. Arkansas
Forestry Commission.
Region 6
AR, LA
Natural Resources Database. 2011. Checklist of Flora in China Camp
SP. http://www.nrdb.org/checklistsearchresults.asp
Region 9
CA
Nelson, G. 1996. The shrubs and woody vines of Florida. Pineapple
Press, Inc., Sarasota, Florida.
Region 4
FL, MS
Nelson, G. 1994. The trees of Florida. Pineapple Press, Inc., Sarasota,
Florida.
Region 4
FL
Newcomb, Lawrence. 1977. Newcomb's Wildflower Guide. Little,
Brown, and Company. New York.
Regions 1, 2, 3, and
5
IL, IN, KY, Ml,
NY, OH, TN, Wl
Newcomb, Lawrence. 1989. Newcomb's Wildflower Guide. Little,
Brown, and Company. New York, 490 pp.
Regions 1, 2, 3, and
5
MN, NH , PA
Neyland, R. 2009. Wildflowers of the Coastal Plain: A Field Guide.
Louisiana State University Press, 352 pp.
Regions 4 and 6
LA, MS
Neyland, R. 2011. A Field Guide to the Ferns and Lycophytes of
Louisiana. Louisiana State University Press, 104 pp.
Region 6
LA
NOAA. 2010. Selected Plants of Coastal Mississippi and Alabama
Grand Bay and Weeks Bay Nerr. 160 pp.
Region 4
MS
Pojar, J., and A. MacKinnon. 1994. Plants of the Pacific Northwest
Coast: Washington, Oregon, British Columbia & Alaska. Lone Pine
Publishing. Redmond, Washington. 528 pp.
Region 10
OR, WA
Radford, A.E., H.A. Ahles, and C.R. Bell. 1968. Manual of the Vascular
Flora of the Carolinas. University of North Carolina Press. Chapel Hill,
North Carolina.
Regions 4 and 6
AL, AR, GA, LA,
NC, SC, TX
Rothrock, P.E. 2009. Sedges of Indiana and the Adjacent States: The
Non-Carex Species. Indiana Academy of Science. 271 pp.
Region 5
OH
San Luis National Wildlife Refuge Plant Species List
Region 9
CA
San Pablo National Wildlife Refuge Plant Species List
Region 9
CA
Sacramento Regional County Sanitation District Plant List:
http://www.srcsd.com/buffer-plant.php. Accessed 2011
Region 9
CA
Shaw, R.J. 1989. Vascular Plants of Northern Utah: An Identification
Manual. Utah State University Press, Logan, Utah, (note field crew
indicated 1982 copyright, but 1 could not find a citation with that
date).
Region 8
UT
Springer, J.D., M.D. Daniels, and M. Nazaire. 2009. Field Guide to
Forest and Mountain Plants of Northern Arizona: From the Mogollon
Rim and White Mountains North. Ecological Restoration Institution
Northern Arizona University, Flagstaff, AZ, 649 pp.
Region 9
AZ
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Potentially Useful Floras and Field Guides
EPA Region(s)
Where Potentially
Applicable
Used by States
in NWCA 2011
Standley. 2011. Field Guide to Carex of New England. Special
Publication of the New England Botanical Club. 182.pp.
Region 1
NH
Strausbaugh, P. D. and E. L Core. 1978. Flora of 1/l/est Virginia, Second
Edition. Seneca Books, Morgantown, West Virginia. 1079 pp.
Region 2
WV
Stutzenbaker, C.D. 1999. Aquatic and Wetland Plants of the Western
Gulf Coast. Texas Parks and Wildlife Press (Distributed by University of
Texas Press). 465 pp.
Regions 4 and 6
MS
Stutzenbaker, C.D. 2010. Aquatic and Wetland Plants of the Western
Gulf Coast. Texas A&M University Press. College Station, Texas. 468
pp.
Regions 4 and 6
LA, MS, TX
Swink, Floyd and Gerould Wilhelm. 1994. Plants of the Chicago
Region. 4th Ed. Indiana Academy of Science. Indianapolis.
Region 5
IL, IN, Wl
Taylor, W.K. 1998. Florida wildflowers in their natural communities.
University Press of Florida, Gainesville, Florida.
Region 4
FL, Ga
Tiner, R. W. 2009. Field Guide to Tidal Wetland Plants of the
Northeastern United States and Neighboring Canada. The University
of Massachusetts Press. Amherst, Massachusetts.
Regions 1 and 2
NH
Tiner, R. W. 1993. Field Guide to Coastal Wetland Plants of the
Southeastern United States. The University of Massachusetts Press.
Amherst, Massachusetts.
Region 4 and 5
AL, LA, MS, TX
Tobe, J.D., K.C. Burks, R.W. Cantrell, M.A. Garland, M.E. Sweeney,
D.W. Hall, P.Wallace, G. Anglin, G. Nelson, J.R. Cooper, B. Bickner, K.
Gilbert, N. Aymond, K. Greenwood, N. Raymond. 1998. Florida
wetland plants: an identification manual. Department of
Environmental Protection, Tallahassee, Florida.
Region 4
FL, GA, MS
Tueten, J. and G. Tueten. 1993. Wildflowers of Houston. Rice
University Press, Houston, Texas, 309 Pp.
Region 6
TX
Turner, M. and P. Gustafson. 2002. Wildflowers of the Pacific
Northwest. Timber Press, Portland, Oregon, 511 pp.
Region 10
ID
USDA, NRCS (U.S. Department of Agriculture, Natural Resources
Conservation Service). 2011. The PLANTS Database
(httoV/olants.usda.gov). National Plant Data Center, Baton Rouge, LA
70874-4490 USA.
All
CA, MD, NC, VA
Van Bruggen, T. 1985. The Vascular Plants of South Dakota. Iowa State
University Press. Ames, Iowa, 476 pp.
Region 8
SD
Voss, Edward G. 1972, 1985,1996. Michigan Flora: A Guide to the
Identification and Occurrence of the Native and Naturalized Seed-
plants of the State. Parti: Gymnosperms and Monocots. Part II:
Dicots (Saururaceae-Cornaceae). Part III: Dicots (Pyrolaceae-
Compositae). Cranbrook Institute of Science and University of
Michigan Herbarium.
Region 5
IN, Ml, MN, Wl
Washington Department of Ecology. 2001. An Aquatic Plant
Identification Manual for Washington's Freshwater Plants. Ecology
Publication # 01-10-032.
Region 10
WA
Weakley A. S. 2007. Flora of the Carolinas, Virginia, Georgia, and
Surrounding Areas. Working Draft of 11 Jan 2007, University of North
Carolina Herbarium. Chapel Hill, North Carolina.
Regions 3 and 4
NC
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Potentially Useful Floras and Field Guides
EPA Region(s)
Where Potentially
Applicable
Used by States
in NWCA 2011
Weakley, A. S. 2011. Flora of the Southern and Mid-Atlantic States.
Working Draft of 15 May 2011. University Of North Carolina
Herbarium, North Carolina Botanical Garden. Chapel Hill, North
Carolina. 1072 pp.
Regions 3 and 4
NC, WV
Weber, W.A. and R.C. Wittmann. 2001. Colorado Flora, Eastern Slope,
Third Edition. University Press of Colorado, Boulder, Colorado, 521 pp.
Region 8
CO
Weber, W.A. and R.C. Wittmann. 2001. Colorado Flora Western Slope,
Third Edition. University Press of Colorado, Boulder, Colorado.
Region 8
CO
Weishaupt, C.G. 1971. Vascular Plants of Ohio: A Manual for Use in
Field and Laboratory. 3rd ed. Kendall Hunt Publishing Co., Dubuque,
IA. 292 pp.
Region 5
OH
Welsh, S.L., N.D. Atwood, S. Goodrich, L.C. Higgins, eds. 1993. A Utah
Flora. Second Edition. Brigham Young University Press. Provo, Utah.
986 pp. (Is this the correct citation?)
Region 8
UT
Whitson, T.D., LC. Burrill, S.A. Dewey, D.W. Cudney, B.E. Nelson, R.D.
Lee, and R. Parker. 1992. Weeds of the l/l/est. University of Wyoming.
Regions 8, 9, and 10
UT, WA
Wilson, B.L., R. Brainerd, D. Lytjen, B. Newhouse, and N. Otting. 2008.
Field Guide to the Sedges of the Pacific Northwest. Oregon State
University Press. Corvallis, Oregon, 432 pp.
Region 10
ID, OR, WA
Wunderlin, R.P. 1998. Guide to the vascular plants of Florida.
University Presses of Florida, Gainesville, Florida.
Region 4
FL
Yatskeivych, George. 1999. Steyermark's Flora of Missouri, Volume 1.
Missouri Department of Consevation Missouri Botanical Garden Press.
Jefferson City and St. Louis, Missouri, 991 pp.
Region 7
MO
Yatskeivych, George. 2006. Steyermark's Flora of Missouri, Volume 2.
Missouri Botanical Garden Press. St. Louis, Missouri, 1200 pp.
Region 7
MO
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APPENDIX E; SUPPLEMENTARY MATERIAL FOR VEGETATION - PLANT
, . •: . ing
Plant specimens are pressed and dried in a standard plant press (30 X 45 cm, 12 X 18 inches) composed
of a breathable wooden frame, corrugated cardboard ventilators, blotters, folded newsprint, and a set
of adjustable straps.
• The wooden frame and straps bound the press.
• Newsprint specimen folders, each containing plant material, are sandwiched between two
moisture-absorbing blotters.
• The "blotter-newsprint sandwiches" are placed between corrugated cardboards.
• The corrugations of the cardboard should run parallel to the shorter dimension (30 cm) of the
press for best air circulation. Bulky specimens may require extra blotters and cardboard.
Protocol for Pressing Plant Specimens:
4) Carefully arrange the plant material to display diagnostic features.
a) Lay the specimen flat and avoid overlapping plant parts.
b) Spread leaves, flowers, and fruits so they can be easily observed from different perspectives.
c) Show upper and lower surfaces of leaves and flowers.
7) Add another blotter, then a cardboard on top of the newsprint folder.
To begin pressing the next specimen, place a blotter over the
steps 2-8 until the press is full or all specimens are included.
1) To begin pressing a specimen, place a cardboard on the bottom wooden frame of the press, then q
add a blotter. ^
Q
2) Lay a newsprint folder on top of the blotter. The newsprint folder should be affixed with a <
completed adhesive Plant Specimen Label. This label includes Site ID, the Plant Sample ID Number, ^
and other critical data about the specimen you are pressing (see below for a complete list). j/i
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3) Clean as much dirt as possible off the plant material before placing it in the newsprint folder. Place ^
the plant material inside the sheet of folded newsprint so that it lies entirely within the dimensions ^
of the plant press.
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d) If possible, arrange material so some flowers have the blossom open, and some flowers and ^
fruits appear in longitudinal and transverse views. 2
e) Multiples of smaller plants of the same species should be pressed together on one sheet. <
f) For large specimens, bend stems sharply into a V or N shape so they fit within the press frame. ^
Avoid curving or twisting stems.
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9) Use two adjustable straps to tighten and firmly compress the plant press and its contents.
Plant Specimen Label Information
Specimen Type: Samples collected for QA purposes will have QA Voucher filled in, while unknown
samples will have Unknown Species filled in.
Plant Sample ID Number: NWCA Site Number-Plant collection number. Plant collection numbers for
samples are assigned consecutive numbers depending on the specimen type (unknown specimens are
prefaced with the letter U and QA specimens are prefaced with the letter Q) for each site beginning with
one. For example, the sample number for the 14th unknown specimen collected at NWCA16-9999
would be NWCA16-9999-U14.
Visit Number: Indicates whether it was the first visit (1) or a repeat visit (2). Most sites are only visited
once.
^ Collection Date: Date is numerical: month, day, year, e.g. 06/14/2016.
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3 County and State: Information on county and State where specimen was collected.
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Q Species Name or Pseudonym: Species name from data form if known or descriptive name used on data
^ forms (e.g., Carex sp. 1) if unknown.
Zn Collector(s) name: Lists the first name, middle initial and surname of the person or persons who
collected the sample.
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Abundance of Plant: Indicates whether the species is dominant, common, sparse or uncommon at the
site.
O Habitat: The type of plant community or setting where the plant is growing, (e.g., such as wetland type
< Drying Plant Specimens
Pressed plant specimens should be thoroughly dried before removing them from the presses. Once dry,
remove specimens from the presses.
^ • To encourage drying, keep full presses in a warm, dry, well-ventilated location in the vehicle during
ili the day and in a well-ventilated warm location at the lodging location at night,
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g • As the specimens dry they will lose volume, so periodically tighten the straps on the press to
£ maintain pressure on the specimens and minimize shrinkage and wrinkling.
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• Rapid and thorough drying is enhanced by low humidity and ample airflow around and through the
presses. The best preservation of color and morphology is obtained with rapid drying over low heat.
Also, dry air circulating through the press may kill many insects and insect eggs, potentially
protecting the specimens from damage.
• The easiest way to achieve these conditions is by using an electric plant dryer that provides steady
bottom heat (95°F to 113°F), where plants usually dry in 12 to 48 hours. Plant dryers are typically
constructed as a simple box with a heat source (often light bulbs) and a fan for air circulation, on
which plant presses can be placed to accelerate drying.
• Periodically tighten the straps on the press as the specimens dry and shrink to maintain pressure on
the press.
• Once plant specimens are dry, remove them from the presses with individual specimens kept in
their newsprint folders with attached Plant Specimen Labels.
Mounting Plant Samples
Vascular plants should:
• Be mounted on archival-quality paper measuring 11.5 X 16.5 inches
• Be mounted using commercially available acid free adhesive, such as polyvinyl acetate (PVA)
• Allow for placement of a properly filled out label
• Have an acid free fragment envelop (where necessary for seeds and flowers)
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APPENDIX F: U8G8 PROCEDURE FOR ANALYSIS OF ALGAL TOXINS (OGRL-
SOP-5400)
Attached as separate PDF file
END OF DOC
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